Synthesis of 1-substituted 3-(chloromethyl)-6-aminoindoline (6-amino- seco-CI) DNA minor groove alkylating agents and structure-activity relationships for their cytotoxicity

Article abstract of DOI:10.1021/jm980545s

A series of racemic 6-amino-seco-cyclopropylindole (seco-CI)-compounds was prepared by coupling 1-(tert-butyloxycarbonyl)-3-(chloromethyl)-6- nitroindoline with appropriate acids, followed by nitro group reduction, and evaluated for cytotoxicity in AA8, U

Full text of DOI:10.1021/jm980545s

J . Med. Chem. 1999, 42, 649-658
649
Syn th esis of 1-Su bstitu ted 3-(Ch lor om eth yl)-6-a m in oin d olin e (6-Am in o-seco-CI)
DNA Min or Gr oove Alk yla tin g Agen ts a n d Str u ctu r e-Activity Rela tion sh ip s for
Th eir Cytotoxicity
J ared B. J . Milbank,† Moana Tercel,† Graham J . Atwell,† William R. Wilson,‡ Alison Hogg,‡ and
William A. Denny*,†
Auckland Cancer Society Research Centre, and Section of Oncology, Department of Pathology, Faculty of Medicine and Health
Science, The University of Auckland, Private Bag 92019, Auckland, New Zealand
Received September 28, 1998
A series of racemic 6-amino-seco-cyclopropylindole (seco-CI) compounds was prepared by
coupling 1-(tert-butyloxycarbonyl)-3-(chloromethyl)-6-nitroindoline with appropriate acids,
followed by nitro group reduction, and evaluated for cytotoxicity in AA8, UV4, EMT6, and
SKOV3 cell lines. These compounds are of interest due to their close structural relationship to
known AT-specific alkylating agents and cytotoxins and also for the possible construction of
stable amine-based prodrugs designed for tumor-specific release. Variations included indole
or furan side chains with different substituents, sulfonamide or carboxamide linkers, extension
of the minor groove binding side chain to two subunits, and the use of a pyrroylacryloyl unit
previously reported to give extremely potent analogues. The parent compound, with a
trimethoxyindole side chain, was a moderately potent cytotoxin (IC50 ) 0.34 µM in AA8 cells,
4 h exposure). A single 5-methoxy group on the indole minor groove binding unit was sufficient
to maintain potency, and a series of dimethylaminoethoxy-substituted analogues retained the
cytotoxicity of the parent compound, while providing increased aqueous solubility.
The cyclopropylindole class of antitumor antibiotics,
exemplified by CC-1065 (1)1 (Chart 1) and duocarmycin
SA (2)2 are extremely potent cytotoxins that have
engendered great interest both as potential anticancer
drugs1-4 and as targets for synthesis.5,6 These com-
pounds alkylate DNA in a sequence-selective manner
at adenine N-3 sites in the minor groove,5-7 a selectivity
that has been attributed in part to a binding-induced
change in reactivity.8 Simpler synthetic analogues such
as 3 retain the high cytotoxicity of the natural products,9
and open-chain seco precursors of these (e.g., 4) are as
cytotoxic as the corresponding ring-closed forms (to
which they are presumably converted rapidly in cells).6
While CC-1065 itself shows delayed toxicity in ani-
mals,10 many simpler analogues such as adozelesin (5)
do not. Adozelesin is in clinical trial as an anticancer
drug,11 as are the seco compounds KW-2189 (6) and
carzelesin (7). Both 6 and 7 are prodrugs of the corre-
sponding seco phenols, and are rapidly and nonspecifi-
cally hydrolyzed by plasma esterases.12,13
Many other synthetic analogues of this class have
been prepared, bearing different types of cyclopropylin-
dole alkylating subunits and modified DNA minor
groove binding side chains.5,6,14,15 These studies have
identified structure 8 (and the corresponding seco form
9) as representing the “minimum potent pharmaco-
phore”16 of this class (illustrated here with a tri-
methoxyindole minor groove binding side chain). Less
work has focused on the nature of the 6-substituent in
the alkylating subunit of 9, and research has generally
been limited to modification of the phenol by the
formation of ethers, esters, and carbamates. The limited
series of compounds described has, however, suggested
a positive relationship between the electron-donating
capability of this substituent and high cytotoxicity.17,18
We have recently reported the synthesis19 and pre-
liminary biological evaluation of (racemic) 6-amino-seco-
cyclopropylindole (seco-CI) compounds such as 10a , both
as potential cytotoxins20 (bearing an electron-donating
amino group) and as effectors to be derived from stable
amine-based prodrugs designed for tumor-specific re-
lease (e.g., by endogenous reductases, radiation, or gene
therapy).21 Compound 10a appears to function similarly
to the corresponding phenol analogue 9, by alkylation
of DNA at the N-3 position of adenine, with similar
sequence specificity.22 However, it is considerably less
cytotoxic than 9 (IC50s, AA8 cells, 4 h exposure; 320 and
6 nM, respectively).20
In this paper we report structure-activity relation-
ships for amino-seco-CI analogues 10a -10j, with vary-
ing minor groove binding side chains, seeking to improve
both cytotoxicity and solubility for this class of com-
pounds as cytotoxins and as components of tumor-
specific prodrugs.
Ch em istr y
The amino-seco-CI compounds (10a -10j) of Table 1
were prepared by reduction of the corresponding nitro
compounds (12) with hydrogen over platinum oxide or
with iron dust (Scheme 1). Nitro compounds 12 were in
turn prepared by deprotection of the BOC-protected
seco-CI 11,19 followed by either coupling with acids in
the presence of 1-(3-dimethylaminopropyl)-3-ethylcar-
bodiimide hydrochloride (EDCI‚HCl) to prepare the
amides 12a -12f, 12i, 12j) or reaction with sulfonyl
chlorides and N-methylimidazole to give the sulfona-
mides 12g and 12h .
† Auckland Cancer Society Research Centre.
‡ Section of Oncology.
10.1021/jm980545s CCC: $18.00 © 1999 American Chemical Society
Published on Web 02/05/1999

650 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 4
Milbank et al.
Ch a r t 1
Ta ble 1. Structures, Solubility, and Cytotoxicity of 1-Substituted 3-(Chloromethyl)-6-aminoindoline (6-Amino-seco-CI) Alkylating
Agents
b
IC50 (µM)
sol.a
no.
form.
X
Y
R
(µM)
AA8
UV4
EMT6
SKOV3
10a
10b
10c
10d
10e
10f
10g
10h
10i
A
A
A
A
A
B
B
B
C
D
5,6,7-triOMe
5-OMe
5-O(CH2)2NMe2
5-OMe, 6-O(CH2)2NMe2
5-OMe, 7-O(CH2)2NMe2
32
23
700
0.35 ( 0.02
0.31 ( 0.07
0.16 ( 0.01
0.22 ( 0.04
0.14 ( 0.01
0.88 ( 0.05
8.2 ( 2.2
0.055 ( 0.005
0.047 ( 0.001
0.044 ( 0.001
0.039 ( 0.007
0.029 ( 0.002
0.12 ( 0.01
4.3c
0.27 ( 0.04
0.23 ( 0.01
0.12 ( 0.01
0.11 ( 0.02
0.09 ( 0.01
0.64 ( 0.20
4.9 ( 1.1
2.6 ( 0.40
0.19 ( 0.02
0.91 ( 0.03
0.63 ( 0.04
0.67 ( 0.04
0.26 ( 0.04
0.15 ( 0.02
0.16 ( 0.01
1.1 ( 0.01
2.7 ( 0.27
4.4 ( 0.4
>1200
47
CO
SO2
SO2
O
NH
O
6.6c
2.8c
0.16 ( 0.04
2.1 ( 0.4
0.087 ( 0.003
0.31 ( 0.08
0.60 ( 0.02
1.9 ( 0.3
10j
a
b
Solubility in 0.1 M phosphate buffer (pH 7.0) at room temperature. IC50; concentration of drug to reduce cell numbers to 50% of
controls following a 4 h drug exposure. Average of 2-5 determinations ( SEM. c One determination only.
The acid used to prepare 12b was commercially
available, and those for 12f,23 12h ,24 and 12i25 were
prepared by reported methods. Acid 15, containing a
5-O(CH2)2NMe2 side chain designed to provide a more
water-soluble analogue, was prepared by Fischer indole
synthesis from known26 aniline 13, followed by hydroly-
sis of the ester (Scheme 2). The related 6-O(CH2)2NMe2
substituted acid 22 was prepared by a Hemetsberger

Synthesis of 6-Amino-seco-CI
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 4 651
Sch em e 1a
Sch em e 5a
a
Reagents and conditions: (i) HCl/dioxane/1 h, then R′CO2H/
EDCI/DMA/2-24 h, or R′SO2Cl/N-methylimidazole/CH2Cl2; (ii)
PtO2/H2/THF or Fe/AcOH/H2O/reflux.
Sch em e 2a
a
Reagents and conditions: (i) Ph3PdCHCO2Me/benzene/reflux/
24 h; (ii) CH2(CO2H)2/pyridine/cat. piperidine/20 h, then 100 °C/4
h, then 30% aq H2SO4; (iii) Me2SO4/NaHCO3/MeOH/H2O; (iv) Fe/
(n-PrCO)2O/MeOH/H2O/reflux/45 min; (v) NaOH/MeOH/H2O/re-
flux/50 min.
a
Sch em e 6a
Reagents and conditions: (i) NaNO2/HCl, then ethyl 2-me-
thylacetoacetate/NaOAc, then HCl/EtOH/reflux; (ii) Cs2CO3/MeOH/
H2O/reflux/2 h.
Sch em e 3a
a
Reagents and conditions: (i) BuLi/THF/-78 °C, then SO2; (ii)
N-chlorosuccinimide/CH2Cl2/0 °C; (iii) 11/HCl/dioxane, then 34/
N-methylimidazole/CH2Cl2; (iv) TFA.
malonic acid to give 29 and methylation), followed by
reduction with iron dust and in situ acylation to give
30, which was hydrolyzed to the desired acid 31 (Scheme
5).
The indolesulfonyl chloride 34 was prepared (Scheme
6) by lithiation of N-BOC-5-methoxyindole (32) and
treatment with sulfur dioxide to give the lithium sul-
finate 33. This was chlorinated with N-chlorosuccinim-
ide to give 34, which was reacted without isolation with
deprotected 11 to give the N-BOC derivative 35. Depro-
tection of the indole nitrogen with trifluoroacetic acid
gave the nitroindoline 12g.
a
Reagents and conditions: (i) (CH2Cl)2/K2CO3/DMF/70 °C/16
h; (ii) N3CH2CO2Me/NaOMe/MeOH; (iii) Xylene/reflux; (iv) Cs2CO3/
EtOH/H2O/reflux/6 h; (v)Me2NH/H2O/100 °C/1.25 h.
Sch em e 4a
Resu lts a n d Discu ssion
The cytotoxicities of the amino-seco-CI analogues
10a -10j were determined (as IC50 values, for a 4 h drug
exposure, using a growth inhibition assay30,31) in a panel
of tumor cell lines, including the Chinese hamster ovary
lines AA8 and UV4, the murine mammary carcinoma
line EMT6, and the human ovarian line SKOV3 (Table
1). The UV4 cell line is a repair-defective ERCC-1
mutant and is hypersensitive to agents whose cytotox-
icity is due to bulky DNA adducts32 and particularly so
to cross-links.33 Overall, there were no large differences
in the sensitivity of the wild-type cell lines AA8, EMT6,
and SKOV3, but the compounds were 2- to 8-fold more
cytotoxic in the UV4 line. This moderate hypersensitiv-
ity is at the low end of the range expected for monoalky-
lation.33
a
Reagents and conditions: (i) Cl(CH2)2NMe2‚HCl/K2CO3/bu-
tanone/reflux; (ii) KOEt/(CO2Et)2/5 days, then Pd/C/H2 in AcOH/
EtOH; (iii) NaOH/EtOH/H2O/reflux/1.5 h.
synthesis27 (Scheme 3). Pyrolysis of azidocinnamate 18,
available in two steps from vanillin 16, via diether 17,
gave a 3.8:1 mixture of isomers 19 and 20 which could
be separated chromatographically. After hydrolysis of
the ester group of 19 to give 21, the chloro group was
displaced with dimethylamine to provide 22. The
7-O(CH2)2NMe2 substituted acid 26 was prepared
(Scheme 4) by alkylation of the nitrophenol 23 to give
24 and by annulation of this by a Reissert synthesis.28
The resulting ester 25 was hydrolyzed to give the
desired acid.
The pyrroleacrylic acid 31 was prepared in three steps
from nitroaldehyde 27.29 Wittig olefination gave 28 (this
was also prepared from 27 by Doebner reaction with
As reported previously,20 the parent racemic com-
pound 10a , bearing a 5,6,7-trimethoxyindole (TMI) side
chain, is a moderately potent cytotoxin (IC50, AA8 cells,

652 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 4
Milbank et al.
0.35 µM). Because the two enantiomers of 10a show only
a 3-fold difference in cytotoxicity,22 racemic mixtures of
all compounds were used to explore structure-activity
relationships for side chain variations in the present
work.
0.76 fg/L (ca. 0.002 fM) against KB nasopharyngeal
tumor cells. Details of the preparation of the required
pyrroleacrylic acid side chain 31 for this analogue were
not provided, so the route shown in Scheme 5 was
developed, and the corresponding amino-seco-CI ana-
logue 10j was prepared. However, this showed slightly
lower potency than the parent TMI compound 10a ,
suggesting that while this side chain is acceptable, it
confers no potency advantage in the amino-seco-CI
series.
Boger has reported that only the 5-methoxy group of
the three on the TMI unit of duocarmycin SA (2) is
required for high potency.34 This appears to be true in
the amino-seco-CI-TMI series also, with the 5-OMe
analogue 10b showing an essentially identical cytotoxic
profile to 10a . This fact was exploited in the design of
more water-soluble analogues, of interest since both 10a
and 10b show poor aqueous solubility (32 and 23 µM,
respectively, in 0.1 M phosphate buffer, pH 7.0; see
Table 1). Three new substituted indole-2-carboxylic
acids (15, 22, and 26) were therefore designed as
alternative minor groove binding subunits. Each of
these retains a 5-alkoxy substituent for enhanced
potency but also bears a solubilizing dimethylaminoeth-
yl group in either the 5-, 6-, or 7-position. The data in
Table 1 shows this design to be successful, as the amino-
seco-CI products 10c, 10d , and 10e are generally more
cytotoxic than 10a and 10b across the cell line panel,
while showing increased aqueous solubility (although
the 7-isomer 10e showed a surprisingly small increase).
The effects on cytotoxic potency of a number of other
changes in the molecule were explored with compounds
10f-10h , in which the 5-OMe group was also retained.
A previous report35 suggested that benzofuran ana-
logues (in the CC-1065 series) can be more potent than
the corresponding indoles. However, replacing the indole
side chain of 10b with a benzofuran in 10f lowered
potency slightly (about 2-fold). A further study36a on
analogues of 3 in which the TMI group was replaced
with simple substituents showed that cytotoxicity cor-
related with the electron-withdrawing ability of this
substituent. Thus, of the four analogues investigated,
potency decreased in the order SO2Et > COEt > CO2-
Me > CONHMe. As these simple substituents have no
intrinsic interaction with the minor groove, this cor-
relation presumably reflects the influence of the sub-
stituent on the ease of carbonyl protonation (and
subsequent cyclopropyl ring cleavage)36a or, as recently
proposed,36b the extent of analogous amide stabilization.
We have investigated the influence on amino-seco-CI
cytotoxicity of a sulfonamide versus carboxamide link
to a minor groove binding subunit. Table 1 shows that
in this system the sulfonamide link significantly reduces
potency, both in the case of a 5-methoxyindole (10g
compared to 10b) and a 5-methoxybenzofuran (10h
compared to 10f).
Several studies with various cyclopropylindoline alky-
lating units6 (and with CI agents in particular17,18) have
shown that the potency conferred by a single suitably
substituted minor groove binding unit such as TMI may
be slightly increased (by a factor of between 1 and 3)
by using two linked subunits. The reason for this is
presumed to be increased noncovalent binding. Consis-
tent with this, the analogue 10i, bearing the adozelesin
side chain, was found to be marginally more potent than
either 10a or 10b.
Con clu sion s
This study significantly expands the range of 6-amino-
seco-CI cytotoxins known. For the series of (racemic)
analogues of 10a studied, some structure-activity
relationships were similar to those observed previously
for the phenol 9 and its analogues (the retention of
potency by a single 5-OMe group on the indole minor
groove binding unit, and the slight enhancement in
potency with a two-unit side chain). However, a pyr-
roleacryloyl side chain did not give the large increase
in potency reported in another series. Replacing the
usual carboxamide link group between the alkylator and
minor groove binding subunits by a sulfonamide greatly
lowered cytotoxic potency (cf. 10a and 10g, 10h ). The
most important observation was that analogues pos-
sessing both a 5-alkoxy group and a solubilizing di-
methylaminoethyl group, either as part of the 5-alkoxy
substituent (10c), or at other indole positions (10d and
10e), retained or enhanced the cytotoxicity of the parent
compound together with increased aqueous solubility.
This strategy has potential application to other classes
of these cyclopropylindole DNA alkylating agents.
Exp er im en ta l Section
Ch em istr y. Analyses were carried out in the Microchemical
Laboratory, University of Otago, Dunedin, New Zealand, and
are within 0.4% of the theoretical values. Melting points were
recorded on an electrothermal melting point apparatus. NMR
spectra were obtained on a Bruker DRX-400 spectrometer (400
MHz for 1H and 100 MHz for 13C), and are referenced to Me4-
Si. Carbon signals were assigned by a DEPT pulse sequence.
Column chromatography was carried out on Merck 300-400
mesh silica gel (flash) or Merck 24-40 µm silica gel (dry flash).
Petroleum ether refers to the fraction boiling at 40-60 °C,
hexanes refers to the fraction boiling at 60-65 °C. N,N-
dimethylacetamide (DMA) and DMF were dried over molecular
sieves. THF and Et2O were dried over sodium/benzophenone.
Mass spectra were determined on a VG-75SE mass spectrom-
eter.
5-[2-(Dim et h yla m in o)et h oxy]-1H -in d ole-2-ca r b oxylic
Acid Hyd r och lor id e (15) (Sch em e 2). A stirred solution of
4-[2-(dimethylamino)ethoxy]aniline26 (13) (3.61 g, 20 mmol) in
water (34 mL) and concentrated HCl (10 mL) was diazotized
at 0 °C with a solution of NaNO2 (1.52 g, 22 mmol) in water
(4 mL). The cold solution was added in one portion to a
vigorously stirred ice-cold mixture of ethyl 2-methylaceto-
acetate (3.03 g, 21 mmol), anhydrous NaOAc (17 g), EtOH (25
mL), and freshly added ice (20 g). After being stirred at 20 °C
for 1 h, the mixture was cooled to 0 °C, basified by the slow
addition of solid Na2CO3, and extracted immediately with CH2-
Cl2 (×2). The combined organic layers were washed with water,
dried (Na2SO4), and evaporated. The residue was extracted
with hot hexanes in the presence of decolorizing charcoal, and
the clarified solution was evaporated. The resulting oil (4.55
g) was dissolved in absolute EtOH (6 mL), treated with
saturated ethanolic HCl (10 mL), and heated at reflux for 25
min. The solution was then concentrated, and the residue was
Finally, Fregeau et al., building on earlier work,37
reported14 that a 1-methyl-2-pyrroleacryloyl CPI ana-
logue had extraordinary potency, with an IC50 value of

Synthesis of 6-Amino-seco-CI
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 4 653
partitioned between dilute Na2CO3 and CH2Cl2. The organic
layer was washed with water and saturated NaCl solution,
dried, and evaporated. The residue was triturated with Pr2O/
Hz, 1 H, H-3), 6.91 (s, 1 H, H-7), 4.24 (t, J ) 5.2 Hz, 2 H,
OCH2), 3.98 (t, J ) 5.2 Hz, 2 H, CH2Cl), 3.84, 3.78 (2 × s, 3 H
each, OCH3);13C NMR δ 161.5 (CO2), 147.8, 145.8, 132.4, 125.5,
120.3 (C-2, 3a, 5, 6, 7a), 107.8 (C-3), 103.0 (C-4), 96.3 (C-7),
68.9 (OCH2), 55.7 (OCH3), 51.4 (CO2CH3), 42.9 (CH2Cl). Anal.
(C13H14ClNO4) C, H, N, Cl. Purification of the evaporated
mother liquors by flash chromatography (CH2Cl2) followed by
crystallization from 1,2-dichloroethane and then MeOH gave
further 19 (0.42 g, 9%).
i
petroleum ether, and the resulting solid was recrystallized
i
from Pr2O/petroleum ether to give ethyl 5-[2-(dimethylamino)-
ethoxy]-1H-indole-2-carboxylate (14) (1.32 g, 24% overall
1
yield): mp 110 °C; H NMR [(CD3)2SO] δ 11.72 (s, 1 H, NH),
7.34 (d, J ) 9.0 Hz, 1 H, H-7), 7.12 (d, J ) 2.4 Hz, 1 H, H-4),
7.03 (d, J ) 1.6 Hz, 1 H, H-3), 6.91 (dd, J ) 9.0, 2.4 Hz, 1 H,
H-6), 4.32 (q, J ) 7.1 Hz, 2 H, CH2CH3), 4.03 (t, J ) 5.9 Hz, 2
H, OCH2CH3), 2.63 (t, J ) 5.9 Hz, 2 H, OCH2CH2), 2.22 (s, 6
H, N(CH3)2), 1.33 (t, J ) 7.1 Hz, 3 H, CH2CH3). Anal.
(C15H20N2O3) C, H, N.
Purification of the preliminary fractions by dry flash chro-
matography (5-60% EtOAc/hexanes) and by crystallization
i
from Pr2O gave isomeric methyl 6-(2-chloroethoxy)-7-methoxy-
1H-indole-2-carboxylate (20) (1.20 g, 16%): mp 103-104 °C;
1H NMR (CDCl3) δ 9.16 (br s, 1 H, indole NH), 7.31 (dd, J )
8.7, 0.6 Hz, 1 H, H-4), 7.16 (d, J ) 2.3 Hz, 1 H, H-3), 6.85 (d,
J ) 8.7 Hz, 1 H, H-5), 4.33 (t, J ) 5.7 Hz, 2 H, OCH2), 4.03,
3.92 (2 × s, 3 H each, OCH3), 3.83 (t, J ) 5.7 Hz, 2 H, CH2Cl);
13C NMR δ 162.2 (CO2), 147.4, 135.3, 132.1, 127.3, 124.6 (C-2,
3a, 6, 7, 7a), 117.7 (C-4), 112.2 (C-5), 109.3 (C-3), 70.6 (OCH2),
61.2 (OCH3), 52.0 (CO2CH3), 42.4 (CH2Cl). Anal. (C13H14ClNO4)
C, H, N, Cl.
A mixture of ester 19 (1.00 g, 3.52 mmol), Cs2CO3(1.723 g,
5.29 mmol), 95% EtOH (20 mL), and water (10 mL) was stirred
at reflux for 6 h. Water (15 mL) was added, and the EtOH
was evaporated. The solution was filtered through Celite and
acidified with 2 M HCl. The precipitate that formed was
collected by filtration, washed with water, and dried to give
6-(2-chloroethoxy)-5-methoxy-1H-indole-2-carboxylic acid (21)
(0.95 g, 100%): mp (MeOH) 187-189 °C; 1H NMR [(CD3)2SO]
δ 12.63 (br s, 1 H, CO2H), 11.47 (s, 1 H, NH), 7.12 (s, 1 H,
H-4), 6.97 (d, J ) 1.6 Hz, 1 H, H-3), 6.91 (s, 1 H, H-7), 4.23 (t,
J ) 5.2 Hz, 2 H, OCH2), 3.97 (t, J ) 5.2 Hz, 2 H, CH2Cl), 3.78
(s, 3 H, OCH3); 13C NMR 162.5 (CO2), 147.5, 145.6, 132.1,
126.9, 120.4 (C-2, 3a, 5, 6, 7a), 107.4 (C-3), 103.1 (C-4), 96.5
A mixture of ester 14 (0.75 g, 2.7 mmol), Cs2CO3 (3.0 g, 9.2
mmol), MeOH (6 mL), and water (3 mL) was heated at reflux
for 2 h and then evaporated to dryness. The residue was
dissolved in water (5 mL), and the filtered solution was
adjusted to pH 6.5 with HCl and cooled to 0 °C for 24 h. The
resulting crystalline solid was collected, washed with ice-cold
water and Me2CO, and treated with dry HCl/dioxane/EtOAc.
The resulting solid was recrystallized from MeOH/EtOAc/trace
1
HCl to give 15 (0.69 g, 89%): mp 239-240 °C (dec); H NMR
[(CD3)2SO] δ 12.88 (br s, 1 H, CO2H), 11.69 (s, 1 H, indole
NH+), 10.56 (br s, 1 H, NH+), 7.37 (d, J ) 9.1 Hz, 1 H, H-7),
7.20 (d, J ) 2.4 Hz, 1 H, H-4), 7.01 (d, J ) 1.7 Hz, 1 H, H-3),
6.98 (dd, J ) 9.0, 2.4 Hz, 1 H, H-6), 4.34 (t, J ) 5.1 Hz, 2 H,
OCH2), 3.50 (t, J ) 5.1 Hz, 2 H, OCH2CH2), 2.85 (s, 6 H,
N(CH3)2). Anal. (C13H16N2O3.HCl) C, H, N, Cl.
6-[2-(Dim eth yla m in o)eth oxy]-5-m eth oxy-1H-in d ole-2-
ca r boxylic Acid Hyd r och lor id e (22) (Sch em e 3). A mix-
ture of vanillin (16) (10.00 g, 65.7 mmol), K2CO3 (45.4 g, 0.329
mol), 1,2-dichloroethane (104 mL, 1.31 mol), and DMF (300
mL) was stirred at 65-70 °C for 16 h. The dichloroethane was
evaporated, and the remaining slurry was poured onto ice. The
oil that separated was extracted with Et2O (×4) and EtOAc
(×3). The combined extracts were washed with water (×3) and
saturated NaCl solution, dried (Na2SO4/MgSO4), and evapo-
rated to give a clear oil that solidified upon trituration with
hexanes. Crystallization from Et2O gave 4-(2-chloroethoxy)-
3-methoxybenzaldehyde (17) (12.04 g, 85%): mp 60-61 °C;
1H NMR (CDCl3) δ 9.87 (s, 1 H, CHO), 7.46 (dd, J ) 8.0, 2.0
Hz, 1 H, H-6), 7.43 (d, J ) 2.0 Hz, 1 H, H-2), 6.99 (d, J ) 8.0
Hz, 1 H, H-5), 4.36 (t, J ) 6.1 Hz, 2 H, OCH2), 3.94 (s, 3 H,
OCH3), 3.89 (t, J ) 6.1 Hz, 2 H, CH2Cl); 13C NMR δ 190.8
(CHO), 153.0 (C-4), 150.0 (C-3), 130.8 (C-1), 126.3 (C-6), 112.3
(C-2), 109.8 (C-5), 68.9 (OCH2), 56.0 (OCH3), 41.2 (CH2Cl).
Anal. (C10H11ClO3) C, H, Cl.
A solution of 17 (4.50 g, 20.97 mmol) and methyl azido-
acetate38 (8.20 g, 71.3 mmol) in MeOH (18 mL) was added to
a cooled (ice/salt) solution of sodium methoxide (from 7.45 g
of sodium, 62.9 mmol) in MeOH (36 mL) over 1 h. The white
slurry was allowed to stand at 5 °C for 1.5 h and then at -15
°C for 18 h and diluted with ice-cold water (200 mL). The
precipitate was collected, washed with water, and taken up
in CH2Cl2. This solution was washed with water, dried
(MgSO4), and evaporated to give methyl R-azido-4-(2-chloro-
ethoxy)-3-methoxycinnamate (18) (5.08 g, 78%): mp 115-116
°C (dec), that was used without further purification; 1H NMR
(CDCl3) δ 7.52 (d, J ) 2.0 Hz, 1 H, H-2), 7.33 (dd, J ) 8.6, 2.0
Hz, 1 H, H-6), 6.89 (d, J ) 8.6 Hz, 1 H, H-5), 6.87 (s, 1 H,
H-â), 4.31 (t, J ) 6.2 Hz, 2 H, OCH2), 3.91, 3.90 (2 × s, 3 H
each, OCH3), 3.85 (t, J ) 6.2 Hz, 2 H, CH2Cl); 13C NMR δ 164.1
(CO2), 149.2, 148.7 (C-3, 4), 127.3 (C-1), 125.6, 124.7 (C-6, â),
123.7 (C-R), 113.9, 113.3 (C-2, 5), 69.0 (OCH2), 56.1 (OCH3),
52.8 (CO2CH3), 41.4 (CH2Cl).
(C-7), 68.9 (OCH2), 55.7 (OCH3), 43.0 (CH2Cl). Anal. (C12H12
-
ClNO4‚0.25MeOH) C, H, N, Cl.
A mixture of 21 (1.20 g, 4.45 mmol), 25% aqueous Me2NH
(16 mL, 89 mmol), Na2CO3 (1.18 g, 11.1 mmol), and water (80
mL) was heated at 100 °C for 1.25 h, then evaporated. The
residue was taken up in 0.4 M aqueous Na2CO3 (30 mL),
extracted with Et2O (×2), acidified to pH 1 with 2 M HCl, and
evaporated. The residue was extracted with hot CH3CN (×8),
and the extracts were concentrated. The precipitate that
formed was removed by filtration and washed with CH3CN
and Et2O to give 22 (1.06 g, 76%): mp 204-205 °C (dec),
hygroscopic; 1H NMR [(CD3)2SO] δ 12.7 (br s, 1 H, CO2H), 11.57
(d, J ) 1.9 Hz, 1 H, indole NH), 10.7 (br s, 1 H, NH+), 7.16 (s,
1 H, H-4), 6.98 (d, J ) 1.9 Hz, 1 H, H-3), 6.96 (s, 1 H, H-7),
4.38 (t, J ) 4.9 Hz, 2 H, OCH2), 3.80 (s, 3 H, OCH3), 3.53 (t, J
) 4.9 Hz, 2 H, NCH2), 2.88 (s, 6 H, N(CH3)2); 13C NMR δ 162.5
(CO2), 147.0, 145.6, 132.0, 127.1, 120.7 (C-2, 3a, 5, 6, 7a), 107.4
(C-3), 102.9 (C-4), 97.1 (C-7), 64.0 (OCH2), 55.7 (OCH3), 55.3
(NCH2), 42.9 (N(CH3)2). HRMS (EI) C14H18N2O4 requires M+
278.1267; found 278.1269.
7-[2-(Dim eth yla m in o)eth oxy]-5-m eth oxy-1H-in d ole-2-
ca r boxylic Acid Hyd r och lor id e (26) (Sch em e 4). A mix-
ture of 5-methoxy-3-methyl-2-nitrophenol39 (23) (5.00 g, 27.3
mmol), dimethylaminoethyl chloride hydrochloride (4.33 g, 30
mmol), K2CO3 (15.1 g, 109 mmol), NaI (0.41 g, 2.7 mmol), and
butanone (50 mL) was heated at reflux for 1 h and then cooled
to room temperature. A mixture of dimethylaminoethyl chlo-
ride hydrochloride (4.33 g, 30 mmol), K2CO3 (7.6 g, 54.6 mmol),
and butanone (15 mL) that had been shaken for 5 min was
added, and the mixture was heated at reflux for 1.5 h. The
mixture was concentrated, and the remaining slurry was
diluted with water and extracted with EtOAc (×4). The
combined extracts were washed with 1 M aqueous Na2CO3
(×10) and extracted with 2 M aqueous HCl (×5). The combined
extracts were washed with EtOAc, made basic with Na2CO3,
and extracted with EtOAc (×4). These extracts were washed
with saturated NaCl solution, dried (MgSO4), and evaporated
to give 2-(5-methoxy-3-methyl-2-nitrophenyloxy)-N,N-dimeth-
ylethanamine (24) (4.38 g, 63%): oil; 1H NMR (CDCl3) δ 6.38
(d, J ) 2.4 Hz, 1 H, H-6′), 6.32 (d, J ) 2.4 Hz, 1 H, H-4′), 4.12
A warmed (40 °C) solution of 18 (5.08 g, 16.3 mmol) in
xylenes (140 mL) was added to boiling xylenes (60 mL) over 1
h. After a further 15 min at reflux, most of the xylene was
removed by distillation. The precipitate that formed in the
cooled residue was isolated by filtration, washed with CHCl3
and hexanes, and crystallized from MeOH to give methyl 6-(2-
chloroethoxy)-5-methoxy-1H-indole-2-carboxylate (19) (2.42 g,
52%): mp 110 °C (subl), 164-164 °C (melt); 1H NMR [(CD3)2-
SO] δ 11.65 (br s, 1 H, NH), 7.13 (s, 1 H, H-4), 7.03 (d, J ) 1.6

654 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 4
Milbank et al.
(t, J ) 5.8 Hz, 2 H, OCH2), 3.81 (s, 3 H, OCH3), 2.73 (t, J )
5.8 Hz, 2 H, NCH2), 2.31 (s, 6 H, N(CH3)2), 2.28 (s, 3 H, 3′-
CH3); 13C NMR δ 161.0 (C-5′), 151.8 (C-1′), 136.1 (C-2′), 132.7
(C-3′), 106.7 (C-4′), 97.9 (C-6′), 68.1 (OCH2), 57.4 (NCH2), 55.5
(OCH3), 45.8 (N(CH3)2), 17.7 (3′-CH3). HRMS (EI) C12H18N2O4
requires M+ 254.1267; found 254.1276. Starting material
(1.178 g, 24%) was recovered from the basic washes by
acidification and extraction.
The precipitate that formed was removed by filtration and
washed with water to give (E)-1-methyl-4-nitro-2-pyrroleacrylic
acid (29) (0.23 g, 92%): mp 180 °C (subl), 242-244 °C (melt);
1H NMR [(CD3)2SO] δ 12.35 (br s, 1 H, CO2H), 8.13 (d, J ) 1.9
Hz, 1 H, H-5), 7.44 (d, J ) 15.9 Hz, 1 H, H-â), 7.41 (d, J ) 1.9
Hz, 1 H, H-3), 6.46 (d, J ) 15.9 Hz, 1 H, H-R), 3.79 (s, 3 H,
NCH3); 13C NMR δ 167.4 (CO2H), 135.3, 129.9 (C-2, 4), 130.6,
127.0, 118.6, 105.8 (C-3, 5, R, â), 34.8 (NCH3). Anal. (C8H8N2O4)
C, H, N.
A suspension of potassium (0.308 g, 7.87 mmol) in xylenes
(6 mL) was heated to 100 °C and stirred rapidly as it was
allowed to cool. The xylenes were removed, and the potassium
was washed with Et2O (×3) and covered with Et2O (10 mL).
The mixture was treated with absolute EtOH (1.30 mL, 2.2
mmol) and stirred at reflux until the potassium had dissolved
(3 h). The cooled mixture was treated with diethyl oxalate (1.07
mL, 7.87 mmol) and then with a solution of 24 (2.00 g, 7.87
mmol) in dry Et2O (5 mL). After 115 h, the dark red precipitate
was removed by filtration and washed with Et2O. The red solid
(1.189 g) was dissolved in absolute EtOH (45 mL), acidified
with HOAc (0.78 mL), and hydrogenated over Pd/C (10%, 0.44
g) at 1 atm H2 for 8 h. The mixture was filtered through Celite,
concentrated, diluted with 0.2 M aqueous Na2CO3 (100 mL),
and extracted with EtOAc (×4). The combined extracts were
washed with water (×2) and saturated NaCl solution, dried
(Na2SO4), evaporated, and purified by flash chromatography
(2% Et3N/EtOAc) to give ethyl 7-[2-(dimethylamino)ethoxy]-
5-methoxy-1H-indole-2-carboxylate (25): oil (0.44 g, 18%); 1H
NMR (CDCl3) δ 10.93 (br s, 1 H, NH), 7.09 (d, J ) 2.1 Hz, 1
H, H-3), 6.67 (d, J ) 2.0 Hz, 1 H, H-4), 6.43 (d, J ) 2.0 Hz, 1
H, H-6), 4.38 (q, J ) 7.0 Hz, 2 H, OCH2CH3), 4.16 (t, J ) Hz,
2 H, OCH2CH2N), 8.38 (s, 3 H, OCH3), 2.79 (t, J ) Hz, 2 H,
NCH2), 2.37 (s, 6 H, N(CH3)2), 1.40 (t, J ) Hz, 3 H, OCH2CH3);
13C NMR δ 161.9 (CO2), 155.0, 146.0, 128.1, 127.7, 124.9 (C-2,
3a, 5, 7, 7a), 108.1 (C-3), 99.1 (C-4), 94.6 (C-6), 65.9 (OCH2-
CH2N), 60.6 (OCH2CH3), 58.3 (NCH2), 55.6 (OCH3), 45.1
(N(CH3)2), 14.4 (OCH2CH3). HRMS (EI) C16H22N2O4 requires
M+ 306.1580; found 306.1577. Starting material (0.77 g, 39%)
was recovered from the ethereal washes by extraction into acid,
basification, and extraction with Et2O.
A mixture of 29 (0.10 g, 0.51 mmol), NaHCO3 (0.10 g, 0.61
mmol), MeOH (6 mL), and water (2 mL) at reflux was treated
with dimethyl sulfate (0.12 mL, 1.27 mmol), heated at reflux
for 1 h, diluted with water, and extracted with EtOAc (×3).
The combined extracts were washed with water (×2) and
saturated NaCl solution, dried (MgSO4), evaporated, and
purified by dry flash chromatography (0-5% Et2O/CH2Cl2) to
give 28 (63 mg, 59%), identical to the material prepared above.
A solution of 28 (50 mg, 0.24 mmol) in aqueous MeOH (1:
12.5, 5.4 mL) at reflux was treated with iron powder (70 mg,
1.25 mmol) and butyric anhydride (0.40 mL, 2.45 mmol). After
30 min further butyric anhydride (0.10 mL, 0.61 mmol) was
added, and 45 min after the addition of the iron the mixture
was allowed to cool. The solids were removed by filtration and
washed with MeOH and water. The combined filtrates were
diluted with water and extracted with EtOAc (×3). The
combined extracts were washed sequentially with water,
saturated aqueous NaHCO3, water, and saturated NaCl solu-
tion and then dried (MgSO4), evaporated, and purified by dry
flash chromatography (0-50% EtOAc/CH2Cl2) to give methyl
(E)-4-butyramido-1-methyl-2-pyrroleacrylate (30) (45 mg,
75%): mp 109-110 °C; 1H NMR (CDCl3) δ 7.41 (br s, 1 H,
NH), 7.51 (d, J ) 15.6 Hz, 1 H, H-â), 7.31 (d, J ) 1.8 Hz, 1 H,
H-5), 6.39 (d, J ) 1.8 Hz, 1 H, H-3), 6.03 (d, J ) 15.6 Hz, 1 H,
H-R), 3.72, 3.62 (2 × s, 3 H each, CO2CH3, NCH3), 2.27 (t, J )
7.4 Hz, 2 H, CH2CH2CH3), 1.71 (sx, J ) 7.4 Hz, 2 H, CH2CH2-
CH3), 0.96 (t, J ) 7.4 Hz, 3 H, CH2CH2CH3); 13C NMR δ 170.3,
168.1 (NHCO, CO2), 131.9, 118.5, 112.5, 102.0 (C-3, 5, R, â),
126.7, 123.5 (C-2, 4), 51.5 (CO2CH3), 38.8 (NCH3), 34.2 (CH2-
CH2CH3), 19.1 (CH2CH2CH3), 13.7 (CH2CH2CH3). Anal.
(C13H18N2O3) C, H, N.
A solution of 30 (0.167 g, 0.667 mmol) and 0.2 M aqueous
NaOH (5.7 mL, 1.13 mmol) in MeOH (10 mL) was heated
under reflux for 50 min. The mixture was cooled in ice,
acidified with 2 M aqueous HCl, and poured onto ice. The
precipitate that formed was collected by filtration and washed
with water to give acid 31 (0.133 g, 85%): mp 74-76 °C (dec);
1H NMR [(CD3)2SO] δ 12.02 (br s, 1 H, CO2H), 9.76 (br s, 1 H,
CONH), 7.44 (d, J ) 15.6 Hz, 1 H, H-â), 7.27 (d, J ) 1.6 Hz,
1 H, H-5), 6.53 (d, J ) 1.6 Hz, 1 H, H-3), 6.02 (d, J ) 15.6 Hz,
1 H, H-R), 3.66 (s, 3 H, NCH3), 2.19 (t, J ) 7.3 Hz, 2 H, CH2-
CH2CH3), 1.57 (sx, J ) 7.3 Hz, 2 H, CH2CH2CH3), 0.88 (t, J )
7.3 Hz, 3 H, CH2CH2CH3); 13C NMR δ 169.2, 168.0 (NHCO,
CO2), 131.9, 117.8, 113.6, 101.9 (C-3, 5, R, â), 125.8, 124.2 (C-
2, 4), 37.5 (CH2CH2CH3), 33.6 (NCH3), 18.7 (CH2CH2CH3), 13.6
(CH2CH2CH3). Anal. (C12H16N2O3) C, H, N.
A mixture of 25 (0.410 g, 1.34 mmol), 95% EtOH (21 mL),
and 1.0 M aqueous NaOH (2.7 mL, 2.7 mmol) was stirred at
reflux for 1.5 h. The EtOH was evaporated, 2 M aqueous HCl
(5 mL) was added, and the solution was evaporated. The
residue was extracted with hot CH3CN (×3), and the extracts
were concentrated and diluted with Et2O. The precipitate that
formed was collected by filtration and washed with Et2O to
1
give 26 (0.372 g, 88%): mp 175-178 °C; H NMR [(CD3)2SO]
δ 12.85 (br s, 1 H, CO2H), 11.52 (s, 1 H, indole NH), 10.48 (br
s, 1 H, NH+), 7.00 (d, J ) 2.1 Hz, 1 H, H-3), 6.72 (d, J ) 2.1
Hz, 1 H, H-4), 6.49 (d, J ) 2.1 Hz, 1 H, H-6), 4.41 (t, J ) 4.8
Hz, 2 H, OCH2), 3.76 (s, 3 H, OCH3), 3.58 (t, J ) 4.8 Hz, 2 H,
NCH2), 2.88 (s, 6 H, N(CH3)2); 13C NMR δ 162.5 (CO2H), 154.4
(C-5), 144.9 (C-7), 128.5, 127.6, 123.5 (C-2, 3a, 7a), 107.7 (C-
3), 97.6 (C-4), 94.4 (C-6), 61.3 (OCH2), 55.3 (OCH3), 54.8
(NCH2), 42.0 (N(CH3)2). HRMS (EI) C14H18N2O4 requires M+
278.1267; found 278.1263.
6-Am in o-3-(ch lor om eth yl)-1-[(5-m eth oxyin d ol-2-yl)ca r -
bon yl]in d olin e (10b): Gen er a l Exa m p le of Sch em e 1. A
suspension of 1-(tert-butyloxycarbonyl)-3-chloromethyl-6-ni-
troindoline19 (11) (470 mg, 1.60 mmol) in dioxane (30 mL) was
saturated with HCl, allowed to stand for 1 h, and evaporated.
A solution of 5-methoxy-1H-indole-2-carboxylic acid (0.31 g,
1.60 mmol) was added, followed by EDCI‚HCl (2.5-3.0 equiv),
and the mixture was stirred for 28 h and poured onto ice. The
product was recovered by filtration and crystallized from
acetone/water to give 3-(chloromethyl)-1-[(5-methoxyindol-2-
yl)carbonyl]-6-nitroindoline (12b) (0.37 g, 60%): mp > 235 °C;
1H NMR [(CD3)2SO] δ 11.70 (br s, 1 H, NH), 8.96 (d, J ) 2.2
Hz, 1 H, H-7), 8.02 (dd, J ) 8.3, 2.2 Hz, 1 H, H-5), 7.72 (d, J
) 8.3 Hz, 1 H, H-4), 7.40 (d, J ) 8.9 Hz, 1 H, H-7′), 7.15 (d, J
) 2.5 Hz, 1 H, H-4′), 7.13 (d, J ) 2.2 Hz, 1 H, H-3′), 6.93 (dd,
J ) 8.9, 2.5 Hz, 1 H, H-6′), 4.83 (dd, J ) 10.7, 9.7 Hz, 1 H,
H-2), 4.48 (dd, J ) 10.7, 5.0 Hz, 1 H, H-2), 4.03-4.16 (m, 3 H,
H-3, CH2Cl), 3.78 (s, 3 H, 5′-OCH3); 13C NMR δ 160.5 (NCO),
153.8 (C-5′), 147.6 (C-6), 144.8 (C-3a), 139.8 (C-7a), 131.8,
(E)-4-Bu tyr a m id o-1-m eth yl-2-p yr r olea cr ylic Acid (31)
(Sch em e 5). A mixture of 1-methyl-4-nitro-2-pyrrolecarbox-
aldehyde29 (27) (0.24 g, 1.56 mmol), methyl triphenylphospho-
rylideneacetate (0.57 g, 1.71 mmol), and benzene (25 mL) was
heated under reflux for 24 h. The still-warm solution was
purified directly by dry flash chromatography (0-5% Et2O/
CH2Cl2) to give methyl (E)-1-methyl-4-nitro-2-pyrroleacrylate
(28) (0.33 g, 100%): mp 146-147 °C; 1H NMR (CDCl3) δ 7.55
(d, J ) 1.8 Hz, 1 H, H-5), 7.47 (d, J ) 15.8 Hz, 1 H, H-â), 7.07
(d, J ) 1.8 Hz, 1 H, H-3), 6.27 (d, J ) 15.8 Hz, 1 H, H-R), 3.77,
3.75 (2 × s, 3 H each, CO2CH3, NCH3); 13C NMR δ 166.9 (CO2),
136.6, 129.7 (C-2, 4), 130.3, 125.4 (C-3, 5), 117.8, 106.0
(CHdCH), 51.8 (CO2CH3), 35.3 (NCH3). Anal. (C9H10N2O4) C,
H, N.
Alternatively, a mixture of 27 (0.20 g, 1.30 mmol), malonic
acid (0.68 g, 6.5 mmol), piperidine (2 drops), and pyridine (2
mL) was stirred at room temperature at for 20 h and at 100
°C for 4 h, and then 30% aqueous H2SO4 (10 mL) was added.

Synthesis of 6-Amino-seco-CI
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 4 655
129.9, 127.4 (C-2′, 3a′, 7a′), 125.4 (C-4), 119.2 (C-5), 115.9,
113.2 (C-4′, 6′), 111.1 (C-7), 106.0 (C-7′), 102.0 (C-3′), 55.2 (5′-
125.4 (C-4), 119.0 (C-5), 111.0 (C-7), 106.6 (C-3′), 103.0 (C-4′),
96.8 (C-7′), 64.0 (OCH2), 55.8 (OCH3), 55.3 (NCH2), 53.9 (C-
OCH3), 54.0 (C-2), 47.0 (CH2Cl), 42.0 (C-3). Anal. (C19H16
ClN3O4) C, H, N.
-
2), 47.1 (CH2Cl), 43.0 (N(CH3)2), 42.0 (C-3). Anal. (C23H25-
ClN4O5‚HCl) C, H, N, Cl.
A solution of 12b (0.12 g, 0.31 mmol) in THF (60 mL) was
hydrogenated over platinum oxide (28 mg) at 50 psi H2 for 45
min, filtered through Celite, evaporated, and purified by dry
flash chromatography (0-60% EtOAc/hexanes) to give 10b (92
mg, 83%): mp (EtOAc/Et2O/hexanes) 205-206 °C; 1H NMR
[(CD3)2SO] δ 11.52 (br s, 1 H, NH), 7.50 (d, J ) 2.0 Hz, 1 H,
H-7), 7.37 (d, J ) 9.0 Hz, 1 H, H-7′), 7.14 (d, J ) 2.3 Hz, 1 H,
H-4′), 7.06 (d, J ) 8.1 Hz, 1 H, H-4), 7.00 (d, J ) 1.8 Hz, 1 H,
H-3′), 6.89 (dd, J ) 9.0, 2.3 Hz, 1 H, H-6′), 6.31 (dd, J ) 8.1,
2.0 Hz, 1 H, H-5), 5.19 (br s, 2 H, NH2), 4.61 (dd, J ) 10.7, 8.8
Hz, 1 H, H-2), 4.27 (dd, J ) 10.7, 4.3 Hz, 1 H, H-2), 3.87-3.95
(m, 1 H, CHHCl), 3.77 (s, 3 H, 5′-OCH3), 3.65-3.72 (m, 2 H,
CHHCl, H-3); 13C NMR δ 159.8 (NCO), 153.7 (C-5′), 149.0 (C-
6), 144.3 (C-7a), 131.3, 131.1, 127.4 (C-2′, 3a′, 7a′), 124.6 (C-
4), 118.8 (C-3a), 115.2, 113.0 (C-4′, 6′), 109.6, 104.9 (C-5, 7′),
103.1, 102.0 (C-7, 3′), 55.2 (5′-OCH3), 54.4 (C-2), 48.0 (CH2Cl),
41.9 (C-3). Anal. (C19H18ClN3O2) C, H, N.
The free base of 12d (46 mg, 0.086 mmol) was hydrogenated
at 50 psi H2 in THF (10 mL) over PtO2 (18 mg) for 2 h. The
mixture was filtered through Celite, evaporated, and purified
by preparative reverse-phase HPLC on C-18 silica gel, eluting
with a mixture of 0.05 M ammonium formate (pH 4.5)/80%
MeCN (59:41). Appropriate fractions were pooled, the CH3CN
was evaporated, and the aqueous layer was poured into
aqueous NaHCO3 and extracted with CH2Cl2 (×3). The ex-
tracts were dried (Na2SO4), evaporated, and treated with HCl
to give 10d as the hydrochloride salt (12 mg, 24%): mp 206-
208 °C; 1H NMR [(CD3)2SO] δ 11.57 (br s, 1 H, NH), 10.57 (br
s, 1 H, NH+), 9.6 (very br s, 3 H, NH3+), 8.11 (br s, 1 H, H-3′),
7.45 (d, J ) 8.0 Hz, 1 H, H-4), 7.22 (s, 1 H, H-4′), 7.09 (d, J )
1.9 Hz, 1 H, H-7), 7.05 (s, 1 H, H-7′), 6.96 (br d, J ) 8.0 Hz, 1
H, H-5), 4.72 (t, J ) 9.9 Hz, 1 H, H-2), 4.35-4.40 (m, 3 H,
H-2, OCH2), 3.88-4.06 (m, 3 H, CH2Cl, H-3), 3.82 (s, 3 H,
OCH3), 3.56 (t, J ) 4.6 Hz, 2 H, NCH2), 2.90 (s, 6 H, N(CH3)2).
HRMS (EI) C23H27ClN4O3 requires M+ 442.1772, 444.1742;
found 442.1782, 444.1758.
6-Am in o-3-(ch lor om et h yl)-1-[[5-[2-(d im et h yla m in o)-
eth oxy]-1H-in d ol-2-yl]ca r bon yl]in d olin e Hyd r och lor id e
(10c). Deprotected 11 (from 53 mg, 0.18 mmol) and the
hydrochloride of acid 15 (50 mg, 0.18 mmol) were reacted as
above for 3 h. The precipitate was recovered by filtration and
converted to the hydrochloride salt to give 3-(chloromethyl)-
1-[[5-[2-(dimethylamino)ethoxy]-1H-indol-2-yl]carbonyl]-6-ni-
troindoline hydrochloride (12c) (56 mg, 67%): mp (Me2CO/
6-Am in o-3-(ch lor om et h yl)-1-[[7-[2-(d im et h yla m in o)-
eth oxy]-5-m eth oxy-1H-in d ol-2-yl]ca r bon yl]in d olin e Hy-
d r och lor id e (10e). Deprotected 11 (from 188 mg, 0.64 mmol)
and the hydrochloride of acid 26 (0.20 g, 0.64 mmol) were
reacted as above for 3 h. The precipitate was recovered by
filtration and converted to the hydrochloride salt to give
3-(chloromethyl)-1-[[7-[2-(dimethylamino)ethoxy]-5-methoxy-
1H-indol-2-yl]carbonyl]-6-nitroindoline hydrochloride (12e) (0.24
1
water then MeOH/Et2O) 228-232 °C; H NMR [(CD3)2SO] δ
11.79 (d, J ) 1.8 Hz, 1 H, indole NH), 10.7 (br s, 1 H, NH+),
8.96 (d, J ) 2.2 Hz, 1 H, H-7), 8.03 (dd, J ) 8.3, 2.2 Hz, 1 H,
H-5), 7.74 (d, J ) 8.3 Hz, 1 H, H-4), 7.45 (d, J ) 8.9 Hz, 1 H,
H-7′), 7.26 (d, J ) 2.4 Hz, 1 H, H-4′), 7.16 (d, J ) 1.8 Hz, 1 H,
H-3′), 7.02 (dd, J ) 8.9, 2.4 Hz, 1 H, H-6′), 4.84 (dd, J ) 10.7,
9.7 Hz, 1 H, H-2), 4.46 (dd, J ) 10.7, 5.0 Hz, 1 H, H-2), 4.37 (t,
J ) 5.0 Hz, 2 H, OCH2), 4.04-4.18 (m, 3 H, CH2Cl, H-3), 3.50
(t, J ) 5.0 Hz, 2 H, NCH2), 2.83 (s, 6 H, N(CH3)2); 13C NMR δ
160.5 (NCO), 152.1, 147.6, 144.7, 139.8, 132.1, 130.2, 127.3
(C-3a, 6, 7a, 2′, 3a′, 5′, 7a′), 125.5, 119.3, 116.1, 113.3, 111.1,
106.0, 103.9 (C-4, 5, 7, 3′, 4′, 6′, 7′), 62.9 (OCH2), 55.3 (NCH2),
54.0 (C-2), 47.7 (CH2Cl), 42.7 (N(CH3)2), 42.0 (C-3). Anal.
(C22H23ClN4O4‚HCl) C, H, N, Cl.
1
g, 75%): mp (MeOH) 181-183 °C and 207-209 °C; H NMR
[(CD3)2SO] δ 11.61 (d, J ) 2.3 Hz, 1 H, indole NH), 10.39 (br
s, 1 H, NH+), 8.94 (d, J ) 2.2 Hz, 1 H, H-7), 8.02 (dd, J ) 8.3,
2.2 Hz, 1 H, H-5), 7.74 (d, J ) 8.3 Hz, 1 H, H-4), 7.13 (d, J )
2.3 Hz, 1 H, H-3′), 7.21 (d, J ) 1.9 Hz, 1 H, H-4′), 7.04 (d, J )
1.9 Hz, 1 H, H-6′), 4.83 (t, J ) 10.2 Hz, 1 H, H-2), 4.39-4.48
(m, 3 H, H-2, OCH2), 4.04-4.17 (m, 3 H, CH2Cl, H-3), 3.78 (s,
3 H, OCH3), 3.60 (t, J ) 4.8 Hz, 2 H, NCH2), 2.89 (s, 6 H,
N(CH3)2); 13C NMR δ 160.5 (NCO), 154.6, 147.6, 144.73, 144.65,
139.9, 129.9, 127.9, 122.7 (C-3a, 6, 7a, 2′, 3a′, 5′, 7′, 7a′), 125.5
(C-4), 119.3 (C-5), 111.0 (C-7), 106.5 (C-3′), 97.8 (C-4′), 94.5
(C-6′), 61.4 (OCH2), 55.3 (OCH3), 54.8 (NCH2), 54.0 (C-2), 47.1
(CH2Cl), 42.0 (N(CH3)2), 41.9 (C-3). Anal. (C23H25ClN4O5‚HCl‚
1.5H2O) C, H, N.
The free base of 12c (20 mg, 0.045 mmol) was hydrogenated
at 50 psi H2 in THF (10 mL) over PtO2 (10 mg) for 2 h. The
mixture was filtered through Celite and evaporated to give
10c (8 mg, 43%): mp 173-175 °C; 1H NMR [(CD3)2SO] δ 11.50
(br s, 1 H, NH), 7.49 (d, J ) 2.0 Hz, 1 H, H-7), 7.37 (d, J ) 8.8
Hz, 1 H, H-7′), 7.15 (d, J ) 1.7 Hz, 1 H, H-4′), 7.04 (d, J ) 8.0
Hz, 1 H, H-4), 6.98 (d, J ) 1.0 Hz, 1 H, H-3′), 6.88 (dd, J )
8.8, 1.7 Hz, 1 H, H-6′), 6.31 (dd, J ) 8.0, 2.0 Hz, 1 H, H-5),
5.17 (br s, 2 H, NH2), 4.61 (dd, J ) 11.0, 8.6 Hz, 1 H, H-2),
4.26 (dd, J ) 11.0, 3.5 Hz, 1 H, H-2), 4.05 (t, J ) 5.8 Hz, 2 H,
OCH2), 3.65-3.93 (m, 3 H, CH2Cl, H-3), 2.64 (t, J ) 5.8 Hz, 2
H, NCH2), 2.23 (s, 6 H, N(CH3)2). HRMS (EI) C22H25ClN4O3
requires M+ 412.1666, 414.1637; found 412.1653, 414.1634.
The free base of 12e (93 mg, 0.18 mmol) was hydrogenated
at 50 psi H2 in THF (20 mL) over PtO2 (46 mg) for 2 h. The
mixture was filtered through Celite and evaporated to give
1
10e (66 mg, 76%): mp 189-191.5 °C; H NMR [(CD3)2SO] δ
11.34 (br s, 1 H, NH), 7.43 (d, J ) 2.0 Hz, 1 H, H-7), 7.04 (d,
J ) 8.0 Hz, 1 H, H-4), 6.93 (d, J ) 1.8 Hz, 1 H, H-3′), 6.71 (d,
J ) 1.8 Hz, 1 H, H-4′), 6.46 (d, J ) 1.8 Hz, 1 H, H-6′), 6.30
(dd, J ) 8.0, 2.0 Hz, 1 H, H-5), 5.17 (br s, 2 H, NH2), 4.53 (dd,
J ) 11.3, 9.1 Hz, 1 H, H-2), 4.15-4.24 (m, 1 H, H-2), 4.17 (t,
J ) 5.6 Hz, 2 H, OCH2), 3.83-3.94 (m, 1 H, CHHCl), 3.76 (s,
3 H, OCH3), 3.61-3.72 (m, 2 H, H-3, CHHCl), 2.73 (t, J ) 5.6
Hz, 2 H, NCH2), 2.27 (s, 6 H, N(CH3)2). HRMS (EI) C23H27
-
6-Am in o-3-(ch lor om eth yl)-1-[[6-[2-(d im eth yla m in o)et-
h oxy]-5-m et h oxy-1H -in d ol-2-yl]ca r b on yl]in d olin e H y-
d r och lor id e (10d ). Deprotected 11 (from 85 mg, 0.29 mmol)
and the hydrochloride of acid 22 (90 mg, 0.29 mmol) were
reacted as above for 3 h. The precipitate was recovered by
filtration and converted to the hydrochloride salt to give
3-(chloromethyl)-1-[6-[2-(dimethylamino)ethoxy]-5-methoxy-
1H-indol-2-yl]carbonyl-6-nitroindoline hydrochloride (12d ) (82
ClN4O3 requires M+ 442.1772, 444.1742; found 442.1759,
444.1752.
6-Am in o-3-(ch lor om eth yl)-1-[(5-m eth oxyben zofu r a n -2-
yl)ca r bon yl]in d olin e (10f). Deprotected 11 (from 0.415 g,
1.41 mmol) and methoxybenzofuran-2-carboxylic acid23 (0.27
g, 1.41 mmol) were reacted as above for 3.5 h. The precipitate
was recovered by filtration to give 3-(chloromethyl)-1-[(5-
methoxybenzofuran-2-yl)carbonyl]-6-nitroindoline (12f) (0.42
g, 75%): mp (Me2CO/water) 206-207 °C; 1H NMR [(CD3)2SO]
δ 8.90 (br s, 1 H, H-7), 8.05 (dd, J ) 8.3, 2.2 Hz, 1 H, H-5),
7.75 (d, J ) 8.3 Hz, 1 H, H-4), 7.73 (s, 1 H, H-3′), 7.65 (d, J )
9.1 Hz, 1 H, H-7′), 7.31 (d, J ) 2.6 Hz, 1 H, H-4′), 7.12 (dd, J
) 9.1, 2.6 Hz, 1 H, H-6′), 4.68 (dd, J ) 11.0, 9.8 Hz, 1 H, H-2),
4.52 (dd, J ) 11.0, 5.0 Hz, 1 H, H-2), 4.05-4.17 (m, 3 H, H-3,
CH2Cl), 3.82 (s, 3 H, 5′-OCH3); 13C NMR δ 157.6 (NCO), 156.2
(C-5′), 149.4 (C-7a′), 148.4 (C-6), 147.6 (C-2′), 144.3 (C-7a),
140.0 (C-3a), 127.2 (C-3a′), 125.6 (C-4), 119.8 (C-5), 117.0 (C-
1
mg, 57%): mp (MeOH/iPrOH) 218-222 °C; H NMR [(CD3)2-
SO] δ 11.65 (d, J ) 1.8 Hz, 1 H, indole NH), 10.80 (br s, 1 H,
NH+), 8.96 (d, J ) 2.2 Hz, 1 H, H-7), 8.00 (dd, J ) 8.3, 2.2 Hz,
1 H, H-5), 7.72 (d, J ) 8.3 Hz, 1 H, H-4), 7.21 (s, 1 H, H-4′),
7.13 (d, J ) 1.8 Hz, 1 H, H-3′), 7.04 (s, 1 H, H-7′), 4.81 (dd, J
) 10.6, 9.8 Hz, 1 H, H-2), 4.46 (dd, J ) 10.6, 5.0 Hz, 1 H, H-2),
4.41 (t, J ) 5.6 Hz, 2 H, OCH2), 4.02-4.19 (m, 3 H, CH2Cl,
H-3), 3.82 (s, 3 H, OCH3), 3.55 (t, J ) 4.6, 2 H, NCH2), 2.89 (s,
6 H, N(CH3)2); 13C NMR δ 160.3 (NCO), 147.6, 147.3, 145.7,
144.9, 139.7, 131.4, 128.5, 121.1 (C-3a, 6, 7a, 2′, 3a′, 5′, 6′, 7a′)

656 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 4
6′), 113.7 (C-3′), 112.7 (C-7′), 111.2 (C-7), 104.1 (C-4′), 55.6 (5′-
Milbank et al.
butyramido-1-methyl-2-pyrroleacryloyl]-3-(chloromethyl)-6-ni-
troindoline (12j) (62%): mp 205-207 °C; H NMR (CDCl3) δ
1
OCH3), 53.6 (C-2), 46.9 (CH2Cl), 41.9 (C-3). Anal. (C19H15
ClN2O5) C, H, N, Cl.
-
9.09 (br s, 1 H, H-7), 7.94 (dd, J ) 8.2, 2.2 Hz, 1 H, H-5), 7.77
(d, J ) 14.9 Hz, 1 H, H-â′), 7.36 (d, J ) 8.2 Hz, 1 H, H-4), 7.24
(d, J ) 1.6 Hz, 1 H, H-5′), 7.19 (br s, 1 H, NH), 6.66 (d, J ) 1.6
Hz, 1 H, H-3′), 6.47 (d, J ) 14.9 Hz, 1 H, H-R′), 4.46 (d, J )
10.7, 9.8 Hz, 1 H, H-2), 4.25 (d, J ) 10.7, 5.0 Hz, 1 H, H-2),
3.95-3.61 (m, 3 H, CH2Cl, H-3), 3.70 (s, 3 H, NCH3), 2.32 (t,
J ) 7.4 Hz, 2 H, CH2CH2CH3), 1.75 (sx, J ) 7.4 Hz, 2 H,
CH2CH2CH3), 1.00 (t, J ) 7.4 Hz, 3 H, CH2CH2CH3); 13C NMR
δ 170.4, 165.2 (2 × NHCO), 148.9, 144.6, 137.7, 127.5, 123.7
(C-3a, 5, 7a, 2′, 6′), 132.2, 124.3, 119.0, 118.5, 112.4, 112.0,
102.4 (C-4, 5, 7, 3′, 5′, R, â), 52.8 (C-2), 46.2 (CH2Cl), 42.5 (C-
3), 38.9 (CH2CH2CH3), 34.2 (NCH3), 19.1 (CH2CH2CH3), 13.8
(CH2CH2CH3). Anal. (C21H23ClN4O4) C, H, N.
A solution of 12f (0.10 g, 0.27 mmol) in THF (50 mL) was
hydrogenated over platinum oxide (20 mg) at 50 psi H2 for 30
min, filtered through Celite, and evaporated. The crude
material was purified by dry flash chromatography (0-55%
EtOAc/CH2Cl2) and precipitated from EtOAc/Et2O with hex-
anes to give 10f (0.06 g, 59%): mp 167-169 °C; 1H NMR
[(CD3)2SO] δ 7.62 (d, J ) 9.1 Hz, 1 H, H-7′), 7.57 (s, 1 H, H-3′),
7.45 (br s, 1 H, H-7), 7.28 (d, J ) 2.6 Hz, 1 H, H-4′), 7.08 (dd,
J ) 9.1, 2.6 Hz, 1 H, H-6′), 7.06 (d, J ) 8.1 Hz, 1 H, H-4), 6.33
(dd, J ) 8.1, 2.1 Hz, 1 H, H-5), 5.20 (br s, 2 H, NH2), 4.61 (dd,
J ) 11.1, 8.8 Hz, 1 H, H-2), 4.28 (dd, J ) 11.1, 4.4 Hz, 1 H,
H-2), 3.91 (dd, J ) 10.1, 3.6 Hz, 1 H, CHHCl), 3.82 (s, 3 H,
5′-OCH3), 3.74 (dd, J ) 10.1, 7.9 Hz, 1 H, CHHCl), 3.69 (dddd,
J ) 8.8, 7.9, 4.4, 3.6 Hz, 1 H, H-3); 13C NMR δ 156.9 (NCO),
156.0 (C-5′), 149.4, 149.1, 149.0 (C-6, 2′, 7a′), 143.8 (C-7a),
127.4 (C-3a′), 124.7 (C-4), 118.9 (C-3a), 116.3 (C-6′), 112.5 (C-
3′), 112.0 (C-7′), 110.0 (C-5), 104.0 (C-4′), 102.9 (C-7), 55.5 (5′-
A mixture of 12j (0.40 g, 0.93 mmol), AcOH (0.60 mL, 9.3
mmol), MeOH (90 mL), and water (16 mL) was treated at
reflux with iron powder (0.52 g, 9.28 mmol). After 15 min the
mixture was cooled in ice, diluted with saturated aqueous
NaHCO3 (20 mL), filtered through Celite, diluted with water,
and extracted with EtOAc (×4). The combined extracts were
washed with dilute aqueous NaHCO3 (×2) and saturated NaCl
solution, dried (MgSO4), evaporated, and purified by dry flash
chromatography (1-4.5% MeOH/CH2Cl2) to give 10j (0.26 g,
69%): mp 142-144 °C; 1H NMR (CDCl3) 7.76 (br s, 1 H, H
-7), 7.68 (d, J ) 14.9 Hz, 1 H, H-â), 7.35 (br s, 1 H, NH), 7.24
(d, J ) 1.7 Hz, 1 H, H-5′), 6.97 (d, J ) 8.0 Hz, 1 H, H-4), 6.59
(br s, 1 H, H-3′), 6.49 (br d, J ) 14.9 Hz, 1 H, H-R), 6.37 (dd,
J ) 8.0, 2.2 Hz, 1 H, H-5), 4.29 (dd, J ) 8.0, 2.2 Hz, 1 H, H-2),
4.11 (dd, J ) 10.8, 4.4 Hz, 1 H, H-2), 3.74 (dd, J ) 10.7, 4.3
Hz, 1 H, CHHCl), 3.64 (s, 3 H, NCH3), 3.71-3.59 (m, 1 H, H-3),
3.49 (dd, J ) 10.7, 9.7 Hz, 1 H, CHHCl), 2.29 (t, J ) 7.4 Hz,
2 H, CH2CH2CH3), 1.73 (sx, J ) 7.4 Hz, 2 H, CH2CH2CH3),
0.98 (t, J ) 7.4 Hz, 3 H, CH2CH2CH3); 13C NMR δ 169.2, 163.7
(2 × NHCO), 148.6, 144.2, 126.7, 124.1, 119.0 (C-3a, 6, 7a, 2′,
4′), 129.9, 124.6, 117.3, 113.9, 109.1, 102.7, 101.9 (C-4, 5, 7,
3′, 5′, R, â), 52.4 (C-2), 48.2 (CH2Cl), 41.1 (C-3), 37.5 (CH2CH2-
CH3), 33.6 (NCH3), 18.7 (CH2CH2CH3), 13.6 (CH2CH2CH3).
Anal. (C21H25ClN4O2‚0.5H2O) C, H, N.
6-Am in o-3-(ch lor om eth yl)-1-[(5-m eth oxyin d ol-2-yl)su l-
fon yl]in d olin e (10g): Meth od of Sch em e 6. n-Butyllithium
(2.5 M in hexanes, 2.7 mL, 6.8 mmol) was added to a cooled
(dry ice/acetone) solution of 5-methoxyindole (1.00 g, 6.8 mmol)
in THF (10 mL) at such a rate that the internal temperature
did not exceed -60 °C (15 min). The mixture was allowed to
warm to -15 °C over 30 min; it was then re-cooled to -70 °C,
and a solution of di-tert-butyl dicarbonate (2.6 g, 11.9 mmol)
in THF (5 mL) was added at such a rate that the internal
temperature did not exceed -60 °C (15 min). The mixture was
allowed to warm to room temperature over 30 min and was
stirred at that temperature for a further 30 min. Water was
added, and after 30 min the mixture was extracted with Et2O
(×3). The combined extracts were washed with water (×2) and
saturated NaCl solution, dried (MgSO4), and evaporated.
Crystallization of the residue from pentane gave tert-butyl
5-methoxyindole-1-carboxylate (32) (1.43 g, 85%): mp 76-77
°C (lit.40 74-76 °C).
n-Butyllithium (2.5 M in hexanes, 2.7 mL, 6.8 mmol) was
added to a cooled (dry ice/acetone) solution of 32 (1.00 g, 4.0
mmol) in THF (10 mL) over 15 min and the resulting bright
yellow solution stirred for a further 30 min. Sulfur dioxide was
passed through a needle just above the solution until an
aliquot was acidic to damp litmus. The mixture was allowed
to warm to room temperature over 30 min, and then pentane
(25 mL) was added. The mixture was concentrated to a volume
of 2 mL whereupon Et2O (2 mL) and pentane (25 mL) were
added to give a precipitate of the lithium salt of 1-tert-butyl
5-methoxy-2-sulfinoindole-1-carboxylate (33) (1.08 g, 84%),
which was used directly: 1H NMR (D2O) δ 7.72 (d, J ) 9.1
Hz, 1 H, H-7), 7.06 (d, J ) 2.6 Hz, 1 H, H-4), 6.94 (s, 1 H,
H-3), 6.82 (dd, J ) 9.1, 2.6 Hz, 1 H, H-6), 3.81 (s, 3 H, 5-OCH3),
1.67 (s, 9 H, C(CH3)3); 13C NMR δ 157.6, 155.9, 153.0 (C-2, 5,
CO), 134.4, 131.4 (C-3a, 7a), 118.7, 116.8, 110.0 (C-4, 6, 7),
107.0 (C-3), 89.0 (C(CH3)3), 58.3 (5-OCH3), 30.2 (C(CH3)3).
OCH3), 53.9 (C-2), 47.9 (CH2Cl), 41.7 (C-3). Anal. (C19H17
ClN2O3) C, H, N, Cl.
-
6-Am in o-1-[[5-[[(ben zofu r a n -2-yl)ca r bon yl]a m in o]-1H-
in dol-2-yl]car bon yl]-3-(ch lor om eth yl)in dolin e (10i). Depro-
tected 11 (from 0.39 g, 1.33 mmol) and 5-[[(benzofuran-2-
yl)carbonyl]amino]-1H-indole-2-carboxylic acid25 (0.425 g, 1.33
mmol) were reacted as above for 2.5 h. The precipitate was
recovered by filtration to give 1-[[5-[[(benzofuran-2-yl)carbonyl]-
amino]-1H-indol-2-yl]carbonyl]-3-(chloromethyl)-6-nitroindo-
1
line (12i) (0.45 g, 69%): mp (THF/water) > 230 °C; H NMR
[(CD3)2SO] δ 11.86 (d, J ) 1.1 Hz, 1 H, indole NH), 10.50 (s, 1
H, amide NH), 8.99 (d, J ) 2.1 Hz, 1 H, H-7), 8.25 (d, J ) 2.0
Hz, 1 H, H-4′), 8.05 (dd, J ) 8.2, 2.1 Hz, 1 H, H-5), 7.84 (d, J
) 7.7 Hz, 1 H, H-4′′), 7.77 (s, 1 H, H-3′′), 7.74 (d, J ) 8.3 Hz,
1 H, H-7′′), 7.73 (d, J ) 8.2 Hz, H-4), 7.64 (dd, J ) 9.0, 2.0 Hz,
1 H, H-6′), 7.52 (dd, J ) 8.3, 7.1 Hz, 1 H, H-6′′), 7.50 (d, J )
9.0 Hz, 1 H, H-7′), 7.37 (dd, J ) 7.7, 7.1 Hz, 1 H, H-5′′), 7.28
(s, 1 H, H-3′), 4.88 (dd, J ) 10.8, 9.5 Hz, 1 H, H-2), 4.52 (dd,
J ) 10.8, 5.1 Hz, 1 H, H-2), 4.16 (ddt, J ) 9.5, 5.1, 4.7 Hz, 1
H, H-3), 4.11 (d, J ) 4.6 Hz, 2 H, CH2Cl); 13C NMR δ 160.5,
156.4, 154.3, 149.1, 147.6, 144.8, 139.9, 133.7, 131.2, 130.3,
127.1, 126.94 (2 × CO, C-3a, 6, 7a, 2′, 3a′, 5′, 7a′, 2′′, 3a′′, 7a′′),
126.89, 125.5, 123.7, 122.8, 119.8, 119.3, 113.3, 112.3, 111.8,
111.1, 110.1, 106.5 (C-4, 5, 7, 3′, 4′, 6′, 7′, 3′′, 4′′, 5′′, 6′′, 7′′),
53.9 (C-2), 47.0 (CH2Cl), 41.9 (C-3). Anal. (C27H19ClN4O5) C,
H, N, Cl.
A solution of 12i (150 mg, 0.29 mmol) in THF/water (49:1,
70 mL) was hydrogenated over platinum oxide (60 mg) at 50
psi H2 for 2 h, filtered through Celite, and evaporated. The
residue was triturated with EtOAc/acetone (1:1, 8 mL) and
diluted with Et2O (20 mL), and the precipitate was collected
by filtration and washed with EtOAc to give 10i (113 mg,
1
80%): mp > 230 °C; H NMR [(CD3)2SO] δ 11.68 (br s, 1 H,
indole NH), 10.48 (s, 1 H, amide NH), 8.20 (d, J ) 1.7 Hz, 1
H, H-4′), 7.83 (d, J ) 7.7 Hz, 1 H, H-4′′), 7.77 (s, 1 H, H-3′′),
7.73 (dd, J ) 8.3, 0.7 Hz, 1 H, H-7′′), 7.61 (dd, J ) 8.9, 2.1 Hz,
1 H, H-6′), 7.51 (ddd, J ) 8.3, 7.8, 1.0 Hz, 1 H, H-6′′), 7.50 (d,
J ) 2.0 Hz, 1 H, H-7), 7.47 (d, J ) 8.9 Hz, 1 H, H-7′), 7.38
(ddd, J ) 7.8, 7.7, 0.7 Hz, 1 H, H-5′′), 7.13 (d, J ) 1.1 Hz, 1 H,
H-3′), 7.06 (d, J ) 8.0 Hz, H-4), 6.31 (dd, J ) 8.0, 2.0 Hz, 1 H,
H-5), 5.19 (br s, 2 H, NH2), 4.65 (dd, J ) 10.7, 8.9 Hz, 1 H,
H-2), 4.30 (dd, J ) 10.7, 4.1 Hz, 1 H, H-2), 3.90 (dd, J ) 10.0,
3.6 Hz, H, CHHCl), 4.16 (m, 2 H, CHHCl, H-3); 13C NMR δ
159.7, 156.4, 154.3, 149.1, 149.0, 144.3, 133.3, 131.6, 130.9,
127.2, 126.94, 118.9 (2 × CO, C-3a, 6, 7a, 2′, 3a′, 5′, 7a′, 2′′,
3a′′, 7a′′), 126.88, 124.7, 123.7, 122.8, 119.2, 113.3, 112.1, 111.9,
110.0, 109.7, 105.4, 103.1 (C-4, 5, 7, 3′, 4′, 6′, 7′, 3′′, 4′′, 5′′, 6′′,
7′′), 54.3 (C-2), 48.0 (CH2Cl), 41.8 (C-3). Anal. (C27H21ClN4O3‚
0.25EtOAc) C, H, N, Cl.
6-Am in o-1-[(E)-4-bu tyr a m id o-1-m eth yl-2-p yr r olea cr y-
loyl]-3-(ch lor om eth yl)in d olin e (10j). Deprotected 11 and
acid 31s were reacted as above for 20 h. The product was
recovered by extraction with EtOAc and purified by dry flash
chromatography (0-100% EtOAc/CH2Cl2) to give 1-[(E)-4-

Synthesis of 6-Amino-seco-CI
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 4 657
A chilled (ice/water) solution of the sulfinate 33 (0.48 g, 1.50
mmol) in CH2Cl2 (5 mL) was treated with N-chlorosuccinimide
(0.21 g, 1.55 mmol). The mixture was stirred for 20 min, the
cooling bath was removed, and the mixture was stirred for a
further 15 min. The mixture was filtered, and the resulting
solution of crude sulfonyl chloride 34 was added to 11 (0.31 g,
1.00 mmol; deprotected as described above) and N-methylimi-
dazole (0.40 mL, 5.0 mmol). This mixture was stirred for 22
h, diluted with 0.5 M aqueous HCl, and extracted with EtOAc
(×3). The combined extracts were washed with water (×2) and
saturated NaCl solution, dried (MgSO4), evaporated, and
purified by dry flash chromatography (5-60% EtOAc/hexanes)
to give 3-(chloromethyl)-1-[(1-tert-butyloxycarbonyl-5-meth-
oxyindol-2-yl)sulfonyl]-6-nitroindoline (35) (0.38 g, 73%): mp
diluted with 0.5 M aqueous HCl, and extracted with EtOAc
(×3). The combined organic phases were washed with 0.5 M
aqueous HCl (×1), water (×2), and saturated NaCl solution,
dried (MgSO4), and concentrated to a volume of 4 mL. Addition
of hexanes (12 mL) gave a precipitate that was crystallized
from MeOH to give 3-(chloromethyl)-1-[(5-methoxybenzofuran-
2-yl)sulfonyl]-6-nitroindoline (12h ) (0.354 g, 87%): mp 141.5-
142.5 °C; 1H NMR [(CD3)2SO] δ 8.17 (d, J ) 2.1 Hz, 1 H, H-7),
8.00 (dd, J ) 8.4, 2.1 Hz, 1 H, H-5), 7.84 (d, J ) 0.7 Hz, 1 H,
H-3′), 7.64 (d, J ) 8.4 Hz, 1 H, H-4), 7.56 (d, J ) 9.2 Hz, 1 H,
H-7′), 7.26 (d, J ) 2.6 Hz, 1 H, H-4′), 7.10 (dd, J ) 9.2, 2.6 Hz,
1 H, H-6′), 4.38 (dd, J ) 9.5, 9.5 Hz, 1 H, H-2), 4.07 (dd, J )
9.5, 5.9 Hz, 1 H, H-2), 4.04 (dddd, J ) 9.5, 5.9, 5.3, 3.7 Hz, 1
H, H-3), 3.96 (dd, J ) 11.1, 3.7 Hz, 1 H, CHHCl), 3.90 (dd, J
) 11.1, 5.3 Hz, 1 H, CHHCl), 3.77 (s, 3 H, 5′-OCH3); 13C NMR
δ 156.4 (C-5′), 150.4 (C-7a′), 148.0 (C-6), 146.6 (C-2′), 142.0
(C-7a), 139.5 (C-3a), 126.4 (C-4), 126.0 (C-3a′), 119.7 (C-5),
118.2 (C-6′), 115.1 (C-3′), 113.0 (C-7′), 107.8 (C-7), 104.4 (C-
4′), 55.6 (5′-OCH3), 53.6 (C-2), 46.3 (CH2Cl), 40.9 (C-3). Anal.
(C18H15N2O6S) C, H, N, Cl.
1
78-82 °C; H NMR (CDCl3) δ 8.14 (d, J ) 2.0 Hz, 1 H, H-7),
7.94 (d, J ) 9.3 Hz, 1 H, H-7′), 7.88 (dd, J ) 8.3, 2.0 Hz, 1 H,
H-5), 7.40 (d, J ) 8.3 Hz, 1 H, H-4), 7.26 (s, 1 H, H-3′), 7.09
(dd, J ) 9.3, 2.6 Hz, H-6′), 7.01 (d, J ) 2.6 Hz, 1 H, H-4′), 4.53
(dd, J ) 10.8, 9.2 Hz, 1 H, H-2), 4.40 (dd, J ) 10.8, 5.3 Hz, 1
H, H-2), 3.95-3.80 (m, 1 H, H-3), 3.83 (s, 3 H, 5′-OCH3), 3.76
(d, J ) 6.3 Hz, 2 H, CH2Cl), 1.70 (s, 9 H, C(CH3)3); 13C NMR
δ 156.4 (C-5′), 149.0 (C-6), 148.2 (CO), 143.5 (C-3a), 137.3 (C-
7a), 133.5, 133.4, 126.5 (C-2′, 3a′, 7a′), 125.3 (C-4), 118.5 (3 ×
C), 116.8 (C-5, 4′, 6′, 7′), 108.8 (C-7), 103.5 (C-3′), 86.5
(C(CH3)3), 55.6 (5′-OCH3), 55.0 (C-2), 45.5 (CH2Cl), 42.3 (C-3),
21.0 (C(CH3)3). HRMS (EI) C23H24ClN3O7S requires M+
421.0499, 423.0470; found 421.0494, 423.0478.
A solution of 12h (0.15 g, 0.355 mmol) in THF (30 mL) was
hydrogenated over platinum oxide (60 mg) at 50 psi H2 for 2
h, filtered through Celite, evaporated, and purified by dry flash
chromatography (20-50% EtOAc/hexanes) to give 10g (60 mg,
1
43%): mp (Et2O/pentane) 60-62 °C; H NMR (CDCl3) δ 7.39
(dd, J ) 9.9, 0.8 Hz, 1 H, H-7′), 7.46 (d, J ) 0.8 Hz, H-3′),
7.05-7.02 (m, 2 H, H-4′,6′), 6.93 (d, J ) 2.1 Hz, 1 H, H-7),
6.90 (d, J ) 8.1 Hz, 1 H, H-4), 6.33 (dd, J ) 8.1, 2.1 Hz, 1 H,
H-5), 4.28-4.15 (m, 2 H, H-2), 3.83 (s, 3 H, 5′-OCH3), 3.77 (br
s, 2 H, NH2), 3.57-3.47 (m, 2 H, CHHCl, H-3), 3.28-3.21 (m,
1 H, CHHCl); 13C NMR δ 156.8 (C-5′), 150.8 (C-7a′), 148.8 (C-
6), 147.7 (C-2′), 142.1 (C-7a), 126.2 (C-3a′), 125.5 (C-4), 120.9
(C-3a), 117.7 (C-6′), 113.6 (C-3′), 113.0 (C-7′), 110.8 (C-5), 103.9
(C-4′), 101.7 (C-7), 55.9 (5′-OCH3), 54.8 (C-2), 46.7 (CH2Cl),
42.3 (C-3). Anal. (C18H17ClN2O4S) C, H, N.
A solution of protected indole 35 (0.30 g, 0.59 mmol) in
trifluoroacetic acid (1.5 mL) was stirred for 70 min, and then
water (60 mL) was added. The resulting precipitate was
removed by filtration, dissolved in EtOAc, washed with water
(×2) and saturated NaCl solution, dried (MgSO4), evaporated,
and purified by dry flash chromatography (0-45% EtOAc/
hexanes) to give 3-(chloromethyl)-1-[(5-methoxyindol-2-yl)-
sulfonyl]-6-nitroindoline (12g) (0.22 g, 88%): mp (iPrOH/
hexanes) 163-164 °C; 1H NMR (CDCl3) δ 9.08 (br s, 1 H, NH),
8.47 (d, J ) 2.1 Hz, 1 H, H-7), 7.91 (dd, J ) 8.3, 2.1 Hz, 1 H,
H-5), 7.32 (d, J ) 8.5 Hz, 1 H, H-7′), 7.30 (d, J ) 8.3 Hz, 1 H,
H-4), 7.07 (d, J ) 2.4 Hz, 1 H, H-4′), 7.02 (s, 1 H, H-3′), 7.00
(dd, J ) 8.5, 2.4 Hz, 1 H, H-6′), 4.21 (dd, J ) 11.0, 9.2 Hz, 1
H, H-2), 4.10 (dd, J ) 11.0, 5.0 Hz, 1 H, H-2), 4.82 (s, 3 H,
5′-OCH3), 3.67 (dddd, J ) 9.0, 8.1, 5.1, 5.0 Hz, 1 H, H-3), 3.57
(dd, J ) 11.2, 5.2 Hz, 1 H, CHHCl), 3.34 (dd, J ) 11.2, 8.1 Hz,
1 H, CHHCl); 13C NMR δ 155.3 (C-5′), 149.1 (C-6), 142.9 (C-
3a), 138.2 (C-7a), 132.2, 128.7, 127.1 (C-2′, 3a′, 7a′), 125.5 (C-
4), 119.6 (C-5), 118.2, 113.3 (C-4′, 6′), 109.6 (C-7′), 108.7 (C-
7), 102.3 (C-3′), 55.6 (5′-OCH3), 54.1 (C-2), 45.4 (CH2Cl), 42.3
(C-3). Anal. (C18H16ClN3O5S) C, H, N.
Aqu eou s Solu bilities. Solids were added to 0.1 M phos-
phate buffer, pH 7.0, and the suspensions were sonicated for
5 min at room temperature and then centrifuged for 5 min.
The supernatants were analyzed by HPLC, and concentrations
were determined from a standard curve constructed from
HPLC of a series of known concentrations of the compounds
in acetonitrile. Determinations were repeated three times and
averaged.
Gr ow th In h ibition Assa y. Cell lines were maintained as
monolayers in exponential phase growth using R-MEM con-
taining fetal bovine serum (5% v/v) without antibiotics. Drug
stock solutions were stored frozen in DMSO and diluted into
culture medium (with adjustment of pH to 7.4 under 5% CO2
if necessary) immediately before use to give final DMSO
concentrations of <1%. Drug concentrations were checked
routinely by spectrophotometry in 0.1 N HCl. Growth inhibi-
tory potency under aerobic conditions was determined using
log-phase cultures in 96-well plates. Cultures were initiated
using 200 AA8 cells, 300 UV4 cells, 50 EMT6 cells, or 600
SKOV3 cells in 50 µL of medium per well. After 24 h, drugs
were added in culture medium adjusted to final pH values of
7.4, and cultures were incubated at 37 °C in a 5% CO2
incubator for 4 h. Cultures were then washed thoroughly with
fresh medium and grown for a further 4 days (5 days for
SKOV3) in 150 µL of medium, and cell density was then
determined by staining with methylene blue.30 The IC50 was
calculated as the drug concentration providing 50% inhibition
of growth relative to controls on the same multiwell plate.
A solution of 12g (0.11 g, 0.26 mmol) in THF (50 mL) was
hydrogenated over platinum oxide (50 mg) at 50 psi H2 for 2.5
h, filtered through Celite, evaporated, and purified by dry flash
chromatography (20-60% EtOAc/hexanes) to give 10g (75 mg,
1
73%): mp (Et2O/pentane) 88-90 °C; H NMR (CDCl3) δ 8.58
(br s, 1 H, NH), 7.27 (d, J ) 9.5 Hz, 1 H, H-7′), 7.08 (d, J ) 2.1
Hz, H-7), 6.97-7.03 (m, 3 H, H-3′, 4′, 6′), 6.88 (d, J ) 8.1 Hz,
1 H, H-4), 6.36 (dd, J ) 8.1, 2.1 Hz, 1 H, H-5), 3.96-4.06 (m,
2 H, H-2), 3.84 (br s, 2 H, NH2), 3.81 (s, 3 H, 5′-OCH3), 3.34-
3.46 (m, 2 H, CHHCl, H-3), 2.95 (dd, J ) 10.2, 9.1 Hz, CHHCl);
13C NMR δ 155.0 (C-5′), 147.6 (C-6), 142.6 (C-7a), 132.0, 129.5,
126.9 (C-2′, 3a′, 7a′), 125.6 (C-4), 121.3 (C-3a), 117.4, 113.2
(C-4′, 6′), 111.2, 107.9 (C-5, 7′), 102.26, 102.23 (C-7, 3′), 55.6
(5′-OCH3), 54.4 (C-2), 46.7 (CH2Cl), 42.3 (C-3). Anal. (C18H18
-
ClN3O3S) C, H, N, Cl.
Ack n ow led gm en t. We thank Dr. Bridget Sykes for
the solubility measurements. This work was supported
by the Auckland Division of the Cancer Society of New
Zealand, the Health Research Council of New Zealand,
Circadian Technologies Ltd., and the U.S. National
Cancer Institute (under contract CM 47019).
6-Am in o-3-(ch lor om eth yl)-1-[(5-m eth oxyben zofu r a n -2-
yl)su lfon yl]in d olin e (10h ). A cooled (ice/water) suspension
of lithium 5-methoxybenzofuran-2-sulfinate24 (0.315 g, 1.45
mmol) in dry CH2Cl2 (3 mL) was treated with N-chlorosuccin-
imide (0.20 g, 1.45 mmol). The mixture was stirred for 15 min,
the cooling bath was removed, and the mixture was stirred a
further 15 min. The resulting solution of crude sulfonyl
chloride was filtered through Celite, and the Celite was washed
with dry CH2Cl2 (4 mL). Deprotected 11 (as the free base, 0.205
g, 0.96 mmol) and N-methylimidazole (0.15 mL, 1.88 mmol)
were added to the filtrate. The mixture was stirred for 18 h,
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