Synthesis and Cytotoxicity of Amino-seco-DSA: An Amino Analogue of the DNA Alkylating Agent Duocarmycin SA

Article abstract of DOI:10.1021/jo990464j

This paper describes the synthesis of methyl 5-amino-1-(chloromethyl)-3-[(5,6,7-trimethoxyindol-2-yl)carbonyl]-1,2-dihydro- 3H-pyrrolo[3,2-e]indole-7-carboxylate 8, an amino analogue of the anticancer antibiotic and potent DNA minor groove alkylating agen

Full text of DOI:10.1021/jo990464j

5946
J . Org. Chem. 1999, 64, 5946-5953
Syn th esis a n d Cytotoxicity of Am in o-seco-DSA: An Am in o
An a logu e of th e DNA Alk yla tin g Agen t Du oca r m ycin SA
Moana Tercel,* Michael A. Gieseg, William A. Denny, and William R. Wilson
Auckland Cancer Society Research Centre, Faculty of Medicine and Health Science, The University of
Auckland, Private Bag 92019, Auckland, New Zealand
Received March 15, 1999
This paper describes the synthesis of methyl 5-amino-1-(chloromethyl)-3-[(5,6,7-trimethoxyindol-
2-yl)carbonyl]-1,2-dihydro-3H-pyrrolo[3,2-e]indole-7-carboxylate 8, an amino analogue of the
anticancer antibiotic and potent DNA minor groove alkylating agent seco-duocarmycin SA. Key
points in the synthesis are sequential radical cyclization and Hemetsberger reaction steps to
construct the indoline and indole rings of the target compound from a 1,2,3-trisubstituted benzene
precursor. An intermediate has been resolved by chiral chromatography to provide the separate
enantiomers of 8. Racemic 8 alkylates DNA at adenine in AT rich sequences, similar to
seco-duocarmycin SA and the previously reported amino-seco-CBI 7, but is 15-60 times less potent
than 7 in an in vitro cytotoxicity test. Derivatives of 8 in which the amino group is replaced by an
electron-withdrawing nitro or nitrobenzylcarbamate substituent are considerably less toxic and
may have application as prodrugs to be activated selectively in a tumor environment.
In tr od u ction
CC-1065 and the duocarmycins constitute a group of
exceptionally potent antitumor antibiotics which bind to
DNA in the minor groove and alkylate at N-3 of adenine.1
The natural products and related synthetic derivatives
have been extensively investigated to understand the
mechanism of the alkylation step and the basis for their
sequence selectivity.2 These studies have identified struc-
ture 2 (CI) as the minimum potent pharmacophore, which
can be formed by ring closure of a seco-CI parent 1.3 In
fact, the toxicity of many seco phenolic derivatives is
identical to that of the corresponding ring-closed form,
suggesting that cyclization is rapid under physiological
conditions.3,4 This observation has led to the preparation
of prodrugs in which the seco phenol is protected with
an electron-withdrawing group such as a carbamate.
Examples of this type are the drug candidates carzelesin5
and KW-2189,6 currently in clinical trial as anticancer
agents. Although in these cases the prodrug is cleaved
nonselectively by plasma esterases to give systemic
release of the cytotoxin, the compounds clearly exhibit
(1) Boger, D. L.; J ohnson, D. S. Angew. Chem., Int. Ed. Engl. 1996,
35, 1438, and references therein.
(2) For recent contributions, see: (a) Boger, D. L.; Santilla´n, A., J r.;
Searcey, M.; J in, Q. J . Am. Chem. Soc. 1998, 120, 11554. (b) Boger, D.
L.; Turnbull, P. J . Org. Chem. 1998, 63, 8004.
(3) Boger, D. L.; Ishizaki, T.; Zarrinmayeh, H.; Munk, S. A.; Kitos,
P. A.; Suntornwat, O. J . Am. Chem. Soc. 1990, 112, 8961.
(4) Boger, D. L.; J ohnson, D. S.; Yun, W. J . Am. Chem. Soc. 1994,
116, 1635.
(5) (a) Li, L. H.; DeKoning, T. F.; Kelly, R. C.; Krueger, W. C.;
McGovren, J . P.; Padbury, G. E.; Petzold, G. L.; Wallace, T. L.; Ouding,
R. J .; Prairie, M. D.; Gebhard, I. Cancer Res. 1992, 52, 4904. (b) Wolff,
I.; Bench, K.; Beijnen, J . H.; Bruntsch, U.; Cavalli, F.; de J ong, J .;
Groot, Y.; van Tellingen, O.; Wanders, J .; Sessa, C. Clin. Cancer Res.
1996, 2, 1717. (c) van Tellingen, O.; Punt, C. J . A.; Awada, A.; Wagener,
D. J . T.; Piccart, M. J .; Groot, Y.; Schaaf, L. J .; Henrar, R. E. C.;
Nooijen, W. J .; Beijnen, J . H. Cancer Chemother. Pharmacol. 1998,
41, 377.
improved therapeutic efficacy compared to their non-
prodrug analogues.5a,6c
With this background we have been investigating a
series of compounds, in which the seco phenol is replaced
by an amino group, and have earlier reported the
synthesis of amino-seco-CI 67 and amino-seco-CBI 7.8
These compounds appear to act by the same mechanism
as the known phenols 33 and 4:9 alkylation of DNA occurs
(6) (a) Kobayashi, E.; Okamoto, A.; Asada, M.; Okabe, M.; Naga-
mura, S.; Asai, A.; Saito, H.; Gomi, K.; Hirata, T. Cancer Res. 1994,
54, 2404. (b) Nagamura, S.; Asai, A.; Kanda, Y.; Kobayashi, E.; Gomi,
K.; Saito, H. Chem. Pharm. Bull. 1996, 44, 1723. (c) Nagamura, S.;
Kobayashi, E.; Gomi, K.; Saito, H. Bioorg. Med. Chem. 1996, 4, 1379.
(7) (a) Tercel, M.; Denny, W. A. J . Chem. Soc., Perkin Trans. 1 1998,
509. (b) Tercel, M.; Denny, W. A.; Wilson, W. R. Bioorg. Med. Chem.
Lett. 1996, 6, 2735.
10.1021/jo990464j CCC: $18.00 © 1999 American Chemical Society
Published on Web 07/16/1999

Synthesis and Cytotoxicity of Amino-seco-DSA
J . Org. Chem., Vol. 64, No. 16, 1999 5947
Sch em e 1
introduce the 5-substituent at a late stage allows for a
more concise synthetic plan than many of the previously
reported DSA syntheses.12 The indoline ring of 13 was
envisioned as being constructed via Boger’s radical
cyclization of an aryl radical onto a tethered alkene,13
while the indole could be prepared using a Hemetsberger
cyclization (see below). Although the indole and indoline
rings could be constructed in either order, the more
logical sequence places the generally lower-yielding
Hemetsberger reaction first. The strategy is thus reduced
to the preparation of a suitably functionalized trisubsti-
tuted benzene 14. Such a compound should be readily
prepared from 2-iodo-3-nitrobenzoic acid 15, itself easily
available from 3-nitrophthalic acid.14
Proceeding with this plan clearly requires reduction
of the acid and nitro groups of 15 without reductive
removal of the iodine (Scheme 2). The acid was first
reduced with BH3‚DMS and the alcohol protected as the
acetate 17.15 The nitro group was then reduced with Fe/
HOAc, which fortunately gave no significant deiodina-
tion.16 Protecting 18 proved surprisingly difficult, pre-
sumably due to the bulky ortho iodo substituent. Reaction
with BOC2O in dioxane proceeded cleanly but only to 50%
completion, even in the presence of excess BOC2O and
following solvent evaporation to remove t-BuOH from the
reaction mixture. More forcing conditions (e.g., DMAP)
gave several products, while NaHMDS17 led to acyl group
migration (or competing formation of the NBOC2 deriva-
tive when the alcohol protecting group was TBDMS). The
best procedure was simply to recycle recovered 18 which
eventually provided 19 in ca. 90% yield. This was then
converted to the allyl compound 20, the acetate was
cleaved, and the alcohol was oxidized to provide 14, the
substrate for the first of the proposed cyclization steps.
In the Hemetsberger reaction a benzaldehyde is con-
densed with the anion of methyl azidoacetate, and the
resulting azidocinnamate pyrolyzed to give a substituted
indole (Scheme 3).18 Normally the pyrolysis proceeds in
good yield, but the condensation step can be problematic,
particularly since the anion of methyl azidoacetate is
unstable with respect to loss of nitrogen. The usual
procedure is to add the aldehyde and excess methyl
azidoacetate to methoxide at 0 °C and collect the azido-
cinnamate which precipitates from solution. However, 14
at N-3 of adenine in AT rich sequences,10 with the
“natural” S enantiomer being the more potent of the
pair.8a,10a We have also observed that replacing the amino
group with, for example, nitro (97 and 108) or nitroben-
zylcarbamate groups significantly reduces cytotoxicity.
This raises the possibility that these relatively stable
derivatives could be used as prodrugs, for example, with
a localized nitroreductase, for tumor specific release of a
cytotoxin. In this regard we have recently shown that
nitro-seco-CI 9 is 400-fold more cytotoxic in vitro when
administered in the presence of an Escherichia coli
nitroreductase.11
Although the amino-seco-CI 6 was found to be 50-100
times less cytotoxic than 3,7b in the CBI series the amino
and phenol forms were equitoxic, with 7 inhibiting tumor
cell proliferation at subnanomolar concentrations.8a We
now report the extension of our studies to the duocar-
mycin SA (DSA) alkylating agent, which in the phenol
form 5 is the most potent known member of this class.1
(12) Previous syntheses of duocarmycin SA: (a) Boger, D. L.;
Machiya, K.; Hertzog, D. L.; Kitos, P. A.; Holmes, D. J . Am. Chem.
Soc. 1993, 115, 9025. (b) Muratake, H.; Abe, I.; Natsume, M. Chem.
Pharm. Bull. 1996, 44, 67. (c) Muratake, H.; Tonegawa, M.; Natsume,
M. Chem. Pharm. Bull. 1998, 46, 400. (d) Muratake, H.; Matsumura,
N.; Natsume, M. Chem. Pharm. Bull. 1998, 46, 559. (e) Fukuda, Y.;
Terashima, S. Tetrahedron Lett. 1997, 38, 7207. For a concise synthesis
of an oxaduocarmycin SA analogue, see: (f) Patel, V. F.; Andis, S. L.;
Enkema, J . K.; J ohnson, D. A.; Kennedy, J . H.; Mohamadi, F.; Schultz,
R. M.; Soose, D. J .; Spees, M. M. J . Org. Chem. 1997, 62, 8868. See
also: (g) Muratake, H.; Okabe, K.; Takahashi, M.; Tonegawa, M.;
Natsume, M. Chem. Pharm. Bull. 1997, 45, 799.
(13) Boger, D. L.; McKie, J . A. J . Org. Chem. 1995, 60, 1271. A recent
modification involving cyclization onto a vinyl chloride has been
described.12f
(14) Culhane, P. J . Organic Syntheses; Wiley & Sons: New York,
1967; Collect. Vol. 1, p 125.
Resu lts a n d Discu ssion
Syn th esis. Preparation of the BOC protected nitro-
seco-DSA 12 was planned on the basis of nitration of an
advanced intermediate 13 (Scheme 1). This option to
(8) (a) Atwell, G. J .; Tercel, M.; Boyd, M.; Wilson, W. R.; Denny, W.
A. J . Org. Chem, 1998, 63, 9414. (b) Atwell, G. J .; Wilson, W. R.; Denny,
W. A. Bioorg. Med. Chem. Lett. 1997, 7, 1493.
(9) Boger, D. L.; Yun, W. J . Am. Chem. Soc. 1994, 116, 7996.
(10) (a) For amino-seco-CI, see: Tercel, M.; Gieseg, M. A.; Milbank,
J . B.; Boyd, M.; Fan, J .-Y.; Tan, L. K.; Wilson, W. R.; Denny, W. A.
Chem. Res. Toxicol. 1999, 12, in press. (b) For amino-seco-CBI, Gieseg,
M. A.; Matejovic, J .; Denny, W. A. Anti-Cancer Drug Des. 1999, 14, in
press.
(15) Attempts to reduce the nitro group and protect with BOC2O
without prior alcohol protection led to selective formation of the
carbonate.
(16) Ni2B has been recommended as a reagent for the selective
reduction of iodonitroaromatics, but in this case gave lower yields and
more deiodination than the simple metal-acid reduction. Seltzman,
H. H.; Berrang, B. D.; Tetrahedron Lett. 1993, 34, 3083.
(17) Kelly, T. A.; McNeil, D. W. Tetrahedron Lett. 1994, 35, 9003.
(18) (a) Hemetsberger, H.; Knittel, D.; Weidmann, H. Monatsh.
Chem. 1969, 100, 1599. (b) Hemetsberger, H.; Knittel, D.; Weidmann,
H. Monatsh. Chem. 1970, 101, 161. (c) Knittel, D. Synthesis 1985, 186.
(11) Tercel, M.; Denny, W. A.; Wilson, W. R. Bioorg. Med. Chem.
Lett. 1996, 6, 2741.

5948 J . Org. Chem., Vol. 64, No. 16, 1999
Tercel et al.
reported procedure,13 this compound was reacted with
Bu3SnH/TEMPO and the TEMPO group cleaved with Zn/
HOAc, which provided indoline 13 in a straightforward
manner.
Sch em e 2
Nitration of 13 (HNO3/CH3NO2/0 °C) produced a 2:1
mixture of the 5- and 8-NO2 isomers, with the desired
product 12 isolated in 53-60% yield. The des(hydroxy-
methyl) analogue of 13 has been nitrated under identical
conditions to give a >8:1 mixture in favor of the 5-NO2
isomer,19 suggesting that the hydroxymethyl group of 13
is directing nitration to the 8-position. However, nitration
of the mesylate of 13 gave the same 2:1 isomer ratio,
while nitration of 23 (i.e., with the TEMPO group intact)
resulted in oxidation of the indoline ring as the predomi-
nant reaction. Other nitration reagents that are also
compatible with the acid-sensitive BOC group (NO2BF4,
AcONO2) did not improve the yield of 12. The nitration
products were also quite sensitive to the reaction condi-
tions: at 20 °C 12 was isolated in the same yield but with
the 5,8-dinitro compound as byproduct, while under
dilute conditions (<20 mM) at 0 °C no nitration took
place. The starting material recovered from this reaction
was found to contain 10% of the ring-expanded alcohol
31.20 Presumably this product is formed via a cyclopropyl
iminium intermediate 30, analogous to the mechanism
proposed for the alkylation of DNA by the prodrug KW-
2189.21
The alcohol 12 was next converted to the mesylate 25
and treated with LiCl, but conditions that were appropri-
ate in the CBI series (DMF, 80 °C) here gave elimination
and rearrangement of the initial exomethylene product
to the corresponding 3-methylindole. While milder condi-
tions (20 °C) did yield 26, this product was particularly
insoluble, and it was more convenient to reverse the order
of the next steps. Thus, 12 was deprotected and coupled
with 5,6,7-trimethoxyindole-2-carboxylic acid under the
standard conditions3,7a to give 24, and the alcohol con-
verted to chloride 11 (PPh3Cl2, pyridine) in excellent
yield. The synthesis was completed by hydrogenation of
the nitro group over PtO2, which proceeded without any
significant reductive cleavage of the chloro substituent,
and produced the target compound 8 in 14 steps and 8%
overall yield. Finally, in the same way as described for
the amino CI and CBI analogues,7,8 8 was converted to
the methyl, dimethyl, and nitrobenzylcarbamate deriva-
tives 27, 28, and 29.
Sch em e 3
Resolu tion of En a n tiom er s. Of the intermediates
examined, mesylate 25 gave the highest solubility and
best resolution on a Daicel Chiracel OD column (R ) 1.37,
1:1 i-PrOH/hexane). Although this compound is prone to
elimination, when the resolution, chromatography, and
gave incomplete reaction and poor recovery of material
by this method, the corresponding azidocinnamate being
produced in only 20-35% yield as an oil. After some
experimentation it was found that NaHMDS at -78 °C
allowed isolation of the intermediate azido alcohol (as a
1:1 mix of diastereoisomers) in good yield (64%) along
with recovered 14. Reaction with MsCl/Et3N then pro-
duced the azidocinnamate, which was heated in xylene
to give indole 22 in 46% yield from 14. Following the
(19) Boger, D. L.; Sakya, S. M. J . Org. Chem. 1992, 57, 1277.
(20) 31 was separated from the co-eluting 13 by selective acylation
of the primary alcohol. Particularly diagnostic features of the 1H NMR
spectrum of 31 were the OH doublet and large (17.5 Hz) geminal
coupling constant of the benzylic protons.
(21) Asai, A.; Nagamura, S.; Saito, H. J . Am. Chem. Soc. 1994, 116,
4171.

Synthesis and Cytotoxicity of Amino-seco-DSA
J . Org. Chem., Vol. 64, No. 16, 1999 5949
Ta ble 1. In Vitr o Cytotoxicity (IC50’s in n M, 4 h
Exp osu r e, (SEa )
compound
AA8
EMT6
SKOV3
CI
CBI
DSA
6
7
8
347 ( 23
0.46 ( 0.05
27 ( 7
269 ( 37
0.27 ( 0.03
7.2 ( 0.9
5.3 ( 1.6
15.6 ( 7.1
628 ( 40
1.04 ( 0.11
15.8 ( 1.3
10.7 ( 3.2
23 ( 1
(S)-8b
(R)-8b
11
14.9 ( 3.3
49 ( 2
13100 ( 8350 2480 ( 860
11300 ( 3100
1.98 ( 0.12
11.6 ( 1.4
27
28
29
2.4 ( 0.5
12.5 ( 0.9
3030 ( 420
0.88 ( 0.19
5.3 ( 0.7
10200 ( 4800 19600 ( 2500
a
b
Average of two or more determinations. Absolute configura-
tion assigned on the basis of the “natural” S-enantiomer being the
more potent.
scribed thermal cleavage and gel electrophoresis assay.10b,23
As shown in Figure 1, alkylation was confined to adenine
in AT rich sequences, with 8 alkylating the same sites,
at similar relative intensities and at the same low (10-8
M) concentration as previously observed for amino-seco-
CBI 7.10b However, this activity did not translate to
equivalent cytotoxicity: IC50’s determined in a cell line
panel (AA8 Chinese hamster ovary, EMT6 murine mam-
mary carcinoma, and SKOV3 human ovarian cancer cell
lines) showed 8 to be 15-60-fold less toxic than 7 (Table
1). The relative order of toxicity for phenol seco agents
CI < CBI < DSA,1 is thus altered in the amino series to
CI < DSA < CBI. Further IC50 values in Table 1 provide
information of relevance to the formation of amino-seco-
DSA prodrugs. Monomethylation to give 27 provides an
analogue of slightly enhanced potency (as observed
previously with amino-seco-CI and CBI derivatives).
Dimethylation to give 28 has a similar effect on toxicity,
a surprising result (also observed in the amino-seco-CI
series) since 28 clearly cannot ring close to a cyclic
intermediate. In contrast, derivatives bearing electron-
withdrawing groups, the nitro compound 11 and ni-
trobenzylcarbamate 29, show a significant loss of potency
of several 100-fold (up to 1200-fold for 29) when compared
to the parent compound 8.
Con clu sion s
F igu r e 1. Cleavage products from DNA alkylated with 7 and
8 resolved using 6% denaturing polyacrylamide gel electro-
phoresis. The compounds were incubated with 50 ng of end-
labeled DNA for 6 h at 37 °C and then heated at 100 °C for 30
min to generate the thermal cleavage products. The molar
concentration of each compound is given at the top of the lanes
as 1 × 10x where x is the value shown. Dideoxy sequencing
lanes are shown on the right with the position numbering
following the GenBank entry for the gpt gene (×00221). The
untreated control is labeled Con. The smear visible toward the
top of the control and alkylation lanes is an artifact of the
preparation of the end-labeled DNA and has no effect on the
alkylation specificity.10b
The synthesis described in this paper makes available
both enantiomers of a further amino-seco analogue of the
cyclopropylindole class of alkylating agents. Like the
previously described amino-seco-CI and CBI compounds,
amino-seco-DSA 8 has been found to alkylate DNA at
adenine in a sequence-selective manner. Although 8 is
unexpectedly less cytotoxic than the CBI analogue 7, it
remains a potent cytotoxin with IC50’s in the nanomolar
range. Further, the observation that analogues 11 and
29 are considerably less cytotoxic than 8 provides the
basis for the future investigation of these and similar
compounds as prodrugs of these potent DNA alkylating
agents.
solvent evaporation were carried out at room tempera-
ture, the separate enantiomers were obtained in 70%
recovery and 92 and 88% ee, respectively. Each enanti-
omer was converted to optically active 11 and 8 by the
route shown in Scheme 2. The slower eluting (+)-25 has
been tentatively assigned the natural S configuration on
the basis of the greater cytotoxicity of the derived
enantiomer of 8.22
DNA Alk yla tion a n d in Vitr o Cytotoxicity. A
preliminary examination of DNA alkylation by racemic
8 was carried out with a 5′ end-labeled 502 bp fragment
from the bacterial gpt gene, using the previously de-
Exp er im en ta l Section
2-Iod o-3-n itr oben zyl Alcoh ol (16). BH3‚DMS (34 mL,
0.34 mmol) was added to a solution of 2-iodo-3-nitrobenzoic
(22) Suitable crystals for a determination of absolute configuration
by X-ray crystallography have not been obtained. However this
assignment is consistent with the previous pattern of nitro-seco-CI and
CBI properties: in each case the slower eluting mesylate enantiomer
is dextrorotatory and gives rise to the more cytotoxic amino derivative.
(23) Boger, D. L.; Munk, S. A.; Zarrinmayeh, H.; Ishizaki, T.;
Haught, J .; Bina, M. Tetrahedron 1991, 47, 2661.

5950 J . Org. Chem., Vol. 64, No. 16, 1999
Tercel et al.
acid14 (82.3 g, 0.28 mol) and B(OMe)3 (64 mL, 0.56 mol) in dry
THF (400 mL) under nitrogen, and the mixture stirred at
reflux for 90 min. The solution was cooled, MeOH and then
H2O were added, and the mixture was evaporated. Aqueous
NaCl was added to the residue, and the mixture was extracted
with EtOAc (×3). The extracts were washed with aqueous
NaCl, dried (Na2SO4), and evaporated to give crude 16 as a
yellow-orange solid suitable for use in the next step. A sample
was filtered through a short column of silica, eluting with
CH2Cl2, and recrystallized from PhH as pale yellow needles:
(3%, M + H), 393 (100%, M - C4H8 + NH4), 376 (30%, M -
C4H8 + H). HRMS Calcd for C17H23INO4 432.0672. Found
432.0665.
3-[N-(ter t-Bu tyloxyca r bon yl)-N-(2-p r op en yl)]a m in o-2-
iod oben zyl Alcoh ol (21). Crude 20 from the previous reac-
tion was dissolved in MeOH (400 mL), and a solution of K2CO3
(12.4 g, 90 mmol) in H2O (80 mL) was added. The mixture
was stirred at 20 °C for 15 min, and the MeOH was evapo-
rated. The aqueous residue was extracted with EtOAc (×2),
and the extracts were dried (Na2SO4) and evaporated to give
crude 21 as a pale yellow oil, suitable for use in the next step.
A sample crystallized from petroleum ether as white prisms:
mp 91.5-92.5 °C; 1H NMR (CDCl3) δ 7.39-7.29 (m, 2 H), 7.16
(br d, J ) 6.3 Hz, ca. 0.4 H, minor rotamer), 7.06 (br d, J )
7.2 Hz, ca. 0.6 H, major rotamer), 6.00-5.89 (m, 1 H), 5.14-
5.03 (m, 2 H), 4.75-4.68 (m, 2 H), 4.57-4.42 (m, 1 H), 3.74-
3.59 (m, 1 H), 2.12 (br s, 1 H), 1.53 (s, ca. 3 H, minor rotamer),
1.34 (s, ca. 6 H, major rotamer). Anal. Calcd for C15H20INO3:
C, 46.29; H, 5.18; N, 3.60. Found: C, 46.51; H, 5.35; N, 3.58.
3-[N-(ter t-Bu tyloxyca r bon yl)-N-(2-p r op en yl)]a m in o-2-
iod oben za ld eh yd e (14). Crude 21 from the previous reaction
was dissolved in EtOAc (300 mL), MnO2 (40 g, 0.46 mol) was
added, and the mixture was stirred at reflux for 20 h. The
mixture was filtered through Celite, eluting with EtOAc. More
MnO2 (40 g, 0.46 mol) was added, and the mixture was stirred
at reflux for a further 6 h. The filtration and oxidation were
repeated once more with fresh MnO2 (40 g, 0.46 mol), and after
a further 6 h at reflux TLC (25% EtOAc/petroleum ether)
indicated that the oxidation was complete. The mixture was
filtered through Celite, eluting with EtOAc, and the filtrate
was evaporated to give 14 as a pale yellow oil (25.3 g, 88% for
three steps): 1H NMR (CDCl3) δ 10.17 (s, 1 H), 7.80-7.75 (m,
1 H), 7.49-7.34 (m, 2 H), 6.01-5.89 (m, 1 H), 5.18-5.03 (m, 2
H), 4.62-4.46 (m, 1 H), 3.77-3.64 (m, 1 H), 1.55 (s, ca. 4 H,
minor rotamer), 1.34 (s, ca. 5 H, major rotamer); MS (CI, NH3)
m/z 405 (4%, M + NH4), 388 (4%, M + H), 349 (100%, M -
C4H8 + NH4), 332 (40%, M - C4H8 + H). HRMS Calcd for
1
mp 91-91.5 °C; H NMR (CDCl3) δ 7.72 (dd, J ) 7.7, 1.1 Hz,
1 H), 7.59 (dd, J ) 8.1, 1.5 Hz, 1 H), 7.50 (t, J ) 7.8 Hz, 1 H),
4.78 (s, 2 H), 2.14 (br s, 1 H). Anal. Calcd for C7H6INO3: C,
30.13; H, 2.17; N, 5.02. Found: C, 30.42; H, 2.05; N, 4.91.
2-Iod o-3-n itr oben zyl Aceta te (17). Ac2O (37 mL, 0.39 mol)
was added to a solution of 16 from the previous reaction, Et3N
(59 mL, 0.42 mol) and DMAP (0.25 g, 2 mmol) in CH2Cl2 (400
mL) at 0 °C. The ice-bath was removed, and the yellow solution
was stirred for 10 min and then evaporated. The residue was
dissolved in EtOAc, washed with aqueous HCl (2 N, ×2) and
aqueous NaHCO3 (×2), dried (Na2SO4), and evaporated. The
resulting orange oil crystallized from aqueous MeOH to give
17 as a pale yellow solid (75.8 g, 84% for two steps): mp 64-
65 °C; 1H NMR (CDCl3) δ 7.60 (dd, J ) 7.9, 1.7 Hz, 1 H), 7.57
(dd, J ) 7.9, 1.7 Hz, 1 H), 7.48 (t, J ) 7.8 Hz, 1 H), 5.22 (s, 2
H), 2.18 (s, 3 H). Anal. Calcd for C9H8INO4: C, 33.67; H, 2.51;
N, 4.36. Found: C, 33.88; H, 2.43; N, 4.32.
3-Am in o-2-iod oben zyl Aceta te (18). 17 (8.00 g, 24.9
mmol) was dissolved in EtOH (100 mL) at reflux, and H2O
(100 mL) and AcOH (20 mL) were added. Fe powder (5.57 g,
97 mmol) was washed with HCl (2 N) and then H2O and added
to the hot solution. The mixture was stirred vigorously at
reflux for
a further 15 min and then cooled to 20 °C.
Concentrated NH3 (40 mL) was added and the mixture was
filtered through Celite, eluting with EtOAc. The EtOH was
evaporated, and the residue was diluted with aqueous NaCl
and extracted with EtOAc (×2). The extracts were dried
(Na2SO4) and evaporated, and the resulting solid was recrys-
tallized from MeOH to give 18 as a cream powder (3.72 g,
51%): mp 80-81 °C. The mother liquor was evaporated and
the residue purified by chromatography (30% EtOAc/petroleum
C
15H19INO3 388.0410. Found 388.0399.
Meth yl 5-[N-(ter t-Bu tyloxyca r bon yl)-N-(2-p r op en yl)]-
a m in o-4-iod oin d ole-2-ca r boxyla te (22). NaHMDS (38.4 mL
of a 2 M solution in THF, 77 mmol) was added dropwise over
45 min to a solution of 14 (7.43 g, 19.2 mmol) and methyl
azidoacetate (11.0 g, 96 mmol) in THF (80 mL) under nitrogen
at -78 °C. The brown solution was stirred at this temperature
for 1 h and then was poured into H2O (300 mL) containing
aqueous HCl (2 N, 43 mL). The mixture was extracted with
EtOAc (×2), and the extracts were dried (Na2SO4) and
evaporated. Chromatography (10-20% EtOAc/petroleum ether)
gave recovered 14 (0.83 g, 11%) and crude azido alcohol as a
pale yellow foam (6.18 g, 64%).
This azido alcohol (6.34 g, 12.6 mmol) was dissolved in
CH2Cl2 (50 mL) at 0 °C, and Et3N (4.40 mL, 32 mmol) and
MsCl (1.2 mL, 15 mmol) were added. The mixture was stirred
at 0 °C for 30 min and then diluted with H2O and extracted
with CH2Cl2 (×2). The extracts were dried (Na2SO4) and
evaporated, and the residue was purified by chromatography
(8% EtOAc/petroleum ether) to give crude azidocinnamate as
a pale yellow oil (5.11 g, 84%).
1
ether) to give more 18 (2.93 g, 40%). H NMR (CDCl3) δ 7.12
(t, J ) 7.7 Hz, 1 H), 6.76 (dd, J ) 7.2, 1.3 Hz, 1 H), 6.72 (dd,
J ) 7.9, 1.4 Hz, 1 H), 5.11 (s, 2 H), 4.24 (br s, 2 H), 2.14 (s, 3
H). Anal. Calcd for C9H10INO2: C, 37.14; H, 3.46; N, 4.81.
Found: C, 37.42; H, 3.41; N, 4.78.
3-(ter t-Bu tyloxyca r bon yl)a m in o-2-iod oben zyl Aceta te
(19). A solution of 18 (26.9 g, 92 mmol) and BOC2O (40.3 g,
184 mmol) in dioxane (200 mL) was stirred at reflux for 2 days.
The solution was evaporated and the residue separated by
chromatography (10-20% EtOAc/petroleum ether) to give
recovered 18 (13.2 g, 49%) and 19 as a pale yellow oil (17.5 g,
48%). A sample crystallized from petroleum ether as a white
solid: mp 62-63 °C; 1H NMR (CDCl3) δ 8.00 (dd, J ) 8.2, 1.0
Hz, 1 H), 7.31 (t, J ) 7.9 Hz, 1 H), 7.08 (dd, J ) 7.7, 1.4 Hz,
1 H), 6.99 (br s, 1 H), 5.14 (s, 2 H), 2.15 (s, 3 H), 1.53 (s, 9 H).
Anal. Calcd for C14H18INO4: C, 42.98; H, 4.64; N, 3.58.
Found: C, 43.27; H, 4.70; N, 3.83.
3-[N-(ter t-Bu tyloxyca r bon yl)-N-(2-p r op en yl)]a m in o-2-
iod oben zyl Aceta te (20). NaH (4.48 g of a 60% dispersion
in oil, 113 mmol) was washed with petroleum ether (×3) and
suspended in DMF (80 mL) under nitrogen at 0 °C. A solution
of 19 (29.2 g, 75 mmol) and allyl bromide (19.4 mL, 225 mmol)
in DMF (100 mL) was added in a single portion. After 5 min
the mixture was allowed to warm to 20 °C and stirred for a
further 1 h. H2O was added, and the DMF was evaporated.
The residue was diluted with H2O and extracted with CH2Cl2
(×3), and the extracts were dried (Na2SO4) and evaporated.
This gave crude 20 as a colorless oil, suitable for use in the
next step: 1H NMR (CDCl3) δ 7.36-7.24 (m, 2 H), 7.19 (br d,
J ) 7.1 Hz, ca. 0.4 H, minor rotamer), 7.08 (br d, J ) 7.1 Hz,
ca. 0.6 H, major rotamer), 6.00-5.90 (m, 1 H), 5.18 (s, 2 H),
5.24-5.05 (m, 2 H), 4.59-4.43 (m, 1 H), 3.75-3.58 (m, 1 H),
2.16 (s, 3 H), 1.53 (s, ca. 3 H, minor rotamer), 1.34 (s, ca. 6 H,
major rotamer); MS (CI, NH3) m/z 449 (10%, M + NH4), 432
A solution of this azidocinnamate (5.11 g, 10.6 mmol) in
xylene (80 mL) was added dropwise over 40 min to xylene (70
mL) at reflux under nitrogen. The solution was stirred at reflux
for a further 10 min and then evaporated. Chromatography
(20% EtOAc/petroleum ether) gave 22 as a cream solid (4.09
g, 85%, 46% overall from 14): mp 178-179 °C (MeOH); 1H
NMR (CDCl3) δ 9.32 (br s, 1 H), 7.35-7.04 (m, 3 H), 6.03-
5.92 (m, 1 H), 5.13-5.02 (m, 2 H), 4.58-4.46 (m, 1 H), 3.97 (s,
3 H), 3.90 (dd, J ) 14.6, 5.9 Hz, ca. 0.7 H, major rotamer),
3.75 (dd, J ) 15.5, 6.5 Hz, ca. 0.3 H, minor rotamer), 1.56 (s,
ca. 3 H, minor rotamer), 1.33 (s, ca. 6 H, major rotamer). Anal.
Calcd for C18H21IN2O4: C, 47.38; H, 4.64; N, 6.14. Found: C,
47.32; H, 4.78; N, 6.30.
Meth yl 3-(ter t-Bu tyloxyca r bon yl)-1-[(2,2,6,6-tetr a m eth -
ylp ip er id in o)oxy]m eth yl-1,2-d ih yd r o-3H-p yr r olo[3,2-e]-
in d ole-7-ca r boxyla te (23). A solution of Bu3SnH (13.8 mL,
51 mmol) in toluene (150 mL) was added dropwise over 3 h to

Synthesis and Cytotoxicity of Amino-seco-DSA
J . Org. Chem., Vol. 64, No. 16, 1999 5951
a solution of 22 (5.86 g, 12.8 mmol) and TEMPO (2,2,6,6-
tetramethylpiperidinyloxy, 10.0 g, 64 mmol) in toluene (400
mL) under nitrogen at 70-80 °C, during which time the red
TEMPO color faded to pale yellow. TLC analysis (30% EtOAc/
petroleum ether) showed some unreacted starting material.
More TEMPO (3.0 g, 19 mmol) was added in a single portion,
followed by a solution of Bu3SnH (3.5 mL, 13 mmol) in toluene
(80 mL) added dropwise over 2 h. The mixture was cooled and
evaporated. Chromatography (CHCl3 and then 5-20% EtOAc/
petroleum ether) gave a pink solid (ca. 10 g) which was
recrystallized from MeOH to give 23 as a white solid (3.65 g,
59%): mp 191.5-193 °C. The mother liquor was evaporated
and purified by chromatography (5-20% EtOAc/petroleum
ether) followed by recrystallization (MeOH) to give a second
crop (1.12 g, 18%). 1H NMR (CDCl3) δ 8.84 (s, 1 H), 8.05 (br s,
ca. 0.7 H, major rotamer), 7.63 (br s, ca. 0.3 H, minor rotamer),
7.26 (d, J ) 8.8 Hz, 1 H), 7.13 (s, 1 H), 4.20-4.05 (m, 3 H),
3.94 (s, 3 H), 3.91-3.74 (m, 2 H), 1.58 (br s, 9 H), 1.48-1.27
(m, 6 H), 1.21 (s, 3 H), 1.11 (s, 3 H), 1.08 (s, 3 H), 1.06 (s, 3 H);
13C NMR (one peak not observed) δ 162.2, 152.7, 137.2, 134.0,
127.9, 124.4, 114.5, 110.9, 106.3, 80.1, 78.4, 59.9, 52.2, 52.0,
39.7, 33.1, 28.5, 20.2, 17.1. Anal. Calcd for C27H39N3O5: C,
66.78; H, 8.09; N, 8.65. Found: C, 66.79; H, 8.16; N, 8.66.
Meth yl 3-(ter t-Bu tyloxyca r bon yl)-1-(h yd r oxym eth yl)-
1,2-d ih yd r o-3H-p yr r olo[3,2-e]in d ole-7-ca r boxyla te (13).
Zinc powder (7.39 g, 113 mmol) was added to a solution of 23
(6.86 g, 14.1 mmol) in THF (150 mL), HOAc (150 mL), and
H2O (50 mL). The mixture was stirred at reflux for 40 min,
cooled, and filtered through Celite eluting with EtOAc. The
filtrate was evaporated, and the residue was diluted with H2O
and extracted with EtOAc (×2). The extracts were washed with
H2O and aqueous NaHCO3, dried (Na2SO4), and evaporated.
Recrystallization from MeOH gave 13 as a white solid (3.86
g, 79%): mp 189.5-191 °C dec. The mother liquor was purified
by chromatography (40% EtOAc/petroleum ether) to give more
chromatography (30% EtOAc/petroleum ether) gave, in addi-
tion to 12 (55%), the 5,8-dinitro product as an orange solid
1
(4%): mp 187-189 °C (MeOH); H NMR (d6-DMSO) δ 13.47
(s, 1 H), 8.84 (s, ca. 0.8 H, major rotamer), 8.54 (s, ca. 0.2 H,
minor rotamer), 4.95 (s, 1 H), 4.18-4.12 (m, 1 H), 4.10-4.04
(m, 2 H), 3.98 (s, 3 H), 3.48 (dd, J ) 10.4, 4.7 Hz, 1 H), 3.32
(dd, J ) 10.6, 6.5 Hz, 1 H), 1.55 (s, 9 H). Anal. Calcd for
C
18H20N4O9: C, 49.54; H, 4.62; N, 12.84. Found: C, 49.77; H,
4.61; N, 12.74.
Met h yl 6-(ter t-Bu t yloxyca r b on yl)-8-h yd r oxy-6,7,8,9-
tetr ah ydr o-3H-pyr r olo[3,2-f]-qu in olin e-2-car boxylate (31).
When the nitration was attempted at 0 °C under dilute
conditions [concentrated HNO3 (0.72 mL) added dropwise to
a solution of 13 (1.97 g) in CH3NO2 (290 mL)], TLC analysis
showed that no nitration occurred, but recovered 13 (1.90 g)
contained (1H NMR) ca. 10% of another co-eluting product.
Recrystallization from MeOH gave pure 13, and from the
mother liquor a 1:1 mixture of 13 and the second product. This
mixture was partially acetylated (Ac2O, pyridine, catalytic
DMAP, THF, 20 °C, 1 h), and the unreacted starting material
was recovered by chromatography (40% EtOAc/petroleum
ether) and recrystallized from PhH to give 31 as a cream
1
solid: mp 186-187 °C; H NMR (d6-DMSO) δ 11.90 (s, 1 H),
7.40 (d, J ) 9.0 Hz, 1 H), 7.21 (d, J ) 9.0 Hz, 1 H), 7.10 (d, J
) 1.2 Hz, 1 H), 5.19 (d, J ) 4.1 Hz, 1 H), 4.09-4.01 (m, 1 H),
3.82 (dd, J ) 12.5, 2.7 Hz, 1 H), 3.40 (dd, J ) 12.5, 7.6 Hz, 1
H), 3.38 (s, 3 H), 3.22 (dd, J ) 17.5, 6.4 Hz, 1 H), 2.75 (dd, J
) 17.5, 6.2 Hz, 1 H), 1.45 (s, 9 H). Anal. Calcd for C18H22N2O5‚1/
4PhH: C, 64.00; H, 6.47; N, 7.66. Found: C, 63.73; H, 6.62; N,
7.75.
Meth yl 1-(Hydr oxym eth yl)-5-n itr o-3-[(5,6,7-tr im eth oxy-
in dol-2-yl)car bon yl]-1,2-dih ydr o-3H-pyr r olo[3,2-e]in dole-7-
ca r boxyla te (24). 12 (1.52 g, 3.87 mmol) was stirred in HCl-
saturated dioxane (120 mL) for 100 min (until TLC indicated
complete reaction), and the suspension was evaporated. EDCI
(1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochlo-
ride, 1.48 g, 7.74 mmol) and 5,6,7-trimethoxyindole-2-carbox-
ylic acid (0.97 g, 3.87 mmol) in DMA (12 mL) were added, and
the mixture was stirred at 20 °C for 16 h. Dilute aqueous
NaHCO3 (100 mL) was added, and the precipitated solid was
filtered off, washed with H2O, and dried. Trituration with hot
MeOH gave 24 as an orange powder (1.45 g, 71%): mp 239-
1
13 (0.74 g, 15%). H NMR (d6-DMSO) δ 11.88 (s, 1 H), 7.87 (v
br s, 1 H), 7.29 (d, J ) 8.9 Hz, 1 H), 7.11 (d, J ) 1.4 Hz, 1 H),
4.95 (t, J ) 5.2 Hz, 1 H), 4.03 (t, J ) 10.5 Hz, 1 H), 3.92-3.86
(m, 1 H), 3.87 (s, 3 H), 3.82-3.75 (m, 1 H), 3.67 (br s, 1 H),
3.55-3.48 (m, 1 H), 1.51 (s, 9 H); 13C NMR δ 161.6, 151.8,
136.2, 134.6, 127.8, 123.7, 122.6, 113.3, 111.3, 105.5, 79.3, 63.1,
51.7, 51.2, 42.2, 28.1. Anal. Calcd for C18H22N2O5: C, 62.42;
H, 6.40; N, 8.09. Found: C, 62.31; H, 6.56; N, 8.32.
1
240.5 °C dec; H NMR (d6-DMSO) δ 11.46 (d, J ) 1.6 Hz, 1
Meth yl 3-(ter t-Bu tyloxyca r bon yl)-1-(h yd r oxym eth yl)-
5-n it r o-1,2-d ih yd r o-3H -p yr r olo[3,2-e]in d ole -7-ca r b ox-
yla te (12). 13 (447 mg, 1.29 mmol) was dissolved in CH3NO2
(50 mL) by heating, and the solution cooled in an ice bath. As
the starting material began to precipitate (internal tempera-
ture ca. 5 °C) concentrated HNO3 (0.16 mL, 2.6 mmol) was
added dropwise, giving an orange-red mixture. The suspension
was stirred at 0 °C for 40 min, then poured into cold H2O, and
extracted with CH2Cl2 (×3). The extracts were washed with
aqueous NaHCO3, dried (Na2SO4), and evaporated. The residue
was recrystallized from MeOH to give 12 as an orange powder
H), 11.30 (s, 1 H), 9.19 (s, 1 H), 7.54 (s, 1 H), 7.09 (s, 1 H),
6.95 (s, 1 H), 5.11 (t, J ) 5.3 Hz, 1 H), 4.71 (t, J ) 10.1 Hz, 1
H), 4.47 (dd, J ) 10.6, 4.1 Hz, 1 H), 4.02-3.95 (m, 1 H), 3.94
(s, 3 H), 3.93 (s, 3 H), 3.82 (s, 3 H), 3.80 (s, 3 H), 3.81-3.75
(m, 2 H). Anal. Calcd for C25H24N4O9‚1/2H2O: C, 56.28; H, 4.72;
N, 10.50. Found: C, 56.43; H, 4.39; N, 10.28.
Meth yl 1-(Ch lor om eth yl)-5-n itr o-3-[(5,6,7-tr im eth oxy-
in d ol-2-yl)ca r bon yl]-1,2-d ih yd r o-3H-p yr r olo[3,2-e]in d ole-
7-ca r boxyla te (11). PPh3Cl2 (1.65 g, 5.1 mmol) was added to
a solution of 24 (1.34 g, 2.55 mmol) in pyridine (75 mL) and
the solution stirred at 20 °C. After 10 min more PPh3Cl2 (2.06
g, 6.4 mmol) was added, and after a further 10 min the solution
was poured into H2O and the mixture stirred for 5 min. The
precipitated solid was filtered off, washed with H2O, and
redissolved in CH2Cl2 (400 mL). This solution was filtered
through Celite, eluting with CH2Cl2, and the filtrate was dried
(Na2SO4) and evaporated. The resulting orange solid was
recrystallized from CH2Cl2 to give 11 as an orange powder
(0.88 g, 64%): mp 246-247.5 °C. The mother liquor was
evaporated onto silica and purified by chromatography (50%
EtOAc/petroleum ether) to give more 11 (0.50 g, 36%). 1H NMR
(CDCl3) δ 10.39 (s, 1 H), 9.42 (s, 1 H), 9.39 (s, 1 H), 7.31 (d, J
) 2.2 Hz, 1 H), 6.97 (d, J ) 2.4 Hz, 1 H), 6.86 (s, 1 H), 4.80 (t,
J ) 10.0 Hz, 1 H), 4.69 (dd, J ) 10.8, 4.4 Hz, 1 H), 4.32-4.23
(m, 1 H), 4.09 (s, 3 H), 4.05 (dd, J ) 11.4, 3.9 Hz, 1 H), 4.02 (s,
3 H), 3.95 (s, 3 H), 3.91 (s, 3 H), 3.77 (dd, J ) 11.4, 8.8 Hz, 1
H); 13C NMR (d6-DMSO) δ 160.5, 160.0, 149.2, 140.0, 139.0,
137.5, 133.6, 132.1, 131.3, 130.2, 127.7, 126.1, 125.5, 123.2,
111.3, 107.5, 106.4, 97.9, 61.1, 60.9, 55.9, 54.1, 52.3, 47.0, 42.0.
Anal. Calcd for C25H23ClN4O8: C, 55.31; H, 4.27; N, 10.32.
Found: C, 55.39; H, 4.12; N, 10.26.
1
(229 mg, 45%): mp 200 °C dec; H NMR (d6-DMSO) δ 11.22
(d, J ) 1.5 Hz, 1 H), 8.72 (br s, ca. 0.8 H, major rotamer), 8.42
(br s, ca. 0.2 H, minor rotamer), 7.47 (d, J ) 2.0 Hz, 1 H), 5.02
(t, J ) 5.2 Hz, 1 H), 4.14 (t, J ) 10.5 Hz, 1 H), 3.95 (dd, J )
11.2, 4.7 Hz, 1 H), 3.92 (s, 3 H), 3.89-3.80 (m, 1 H), 3.73 (t, J
) 5.2 Hz, 2 H), 1.54 (s, 9 H). Anal. Calcd for C18H21N3O7: C,
55.24; H, 5.41; N, 10.74. Found: C, 55.35; H, 5.35; N, 10.65.
The mother liquor was purified by chromatography (40%
EtOAc/petroleum ether) to give more 12 (70 mg, 14%) and
methyl 3-(tert-butyloxycarbonyl)-1-(hydroxymethyl)-8-nitro-
1,2-dihydro-3H-pyrrolo[3,2-e]indole-7-carboxylate as an orange
solid (139 mg, 28%): mp 151-155 °C dec, PhH/petroleum
1
ether; H NMR (d6-DMSO) δ 13.3 (v br s, 1 H), 8.04 (br s, 1
H), 7.46 (d, J ) 9.0 Hz, 1 H), 4.93 (s, 1 H), 4.05 (dd, J ) 11.2,
2.3 Hz, 1 H), 3.99-3.93 (m, 1 H), 3.95 (s, 3 H), 3.88-3.82 (m,
1 H), 3.48-3.40 (m, 1 H), 3.19-3.11 (m, 1 H), 1.53 (s, 9 H).
Anal. Calcd for C18H21N3O7: C, 55.24; H, 5.41; N, 10.74.
Found: C, 55.48; H, 5.10; N, 10.57.
Met h yl 3-(ter t-Bu t yloxyca r b on yl)-5,8-d in it r o-1-(h y-
d r oxym eth yl)-1,2-d ih yd r o-3H-p yr r olo[3,2-e]in d ole-7-ca r -
boxyla te. When the nitration was conducted at 20 °C,

5952 J . Org. Chem., Vol. 64, No. 16, 1999
Tercel et al.
Meth yl 5-Am in o-1-(ch lor om eth yl)-3-[(5,6,7-tr im eth oxy-
in d ol-2-yl)ca r bon yl]-1,2-d ih yd r o-3H-p yr r olo[3,2-e]in d ole-
7-ca r boxyla te (8). A solution of 11 (559 mg, 1.0 mmol) in THF
(100 mL) with PtO2 (0.10 g, 0.44 mmol) was hydrogenated at
50 psi for 30 min. The catalyst was filtered off, the filtrate
was evaporated, and the residue was triturated with EtOAc
to give 8 as a yellow-orange solid (404 mg, 76%): mp 190-
196 °C (dec). The mother liquor was evaporated to give more
3.79 (s, 3 H). Anal. Calcd for C33H30ClN5O10: C, 57.27; H, 4.37;
N, 10.12. Found: C, 57.43; H, 4.40; N, 10.09.
Resolu tion of En a n tiom er s. (+)- a n d (-)-Meth yl 3-
(ter t-Bu tyloxyca r bon yl)-1-[(m eth a n esu lfon yloxy)m eth -
yl]-5-n itr o-1,2-d ih yd r o-3H-p yr r olo[3,2-e]in d ole-7-ca r box-
yla te (25). MsCl (0.21 mL, 2.8 mmol) was added to a solution
of 12 (540 mg, 1.38 mmol) and Et3N (0.58 mL, 4.2 mmol) in
CH2Cl2 (150 mL) at 0 °C, and the mixture was stirred at this
temperature for 10 min. The mixture was diluted with H2O
and extracted with CH2Cl2 (×2), and the extracts were dried
(Na2SO4) and evaporated at 20 °C onto silica. Chromatography
(50% EtOAc/petroleum ether) gave 25 as a yellow solid (627
1
8 (111 mg, 21%). H NMR (d6-DMSO) δ 11.62 (d, J ) 1.7 Hz,
1 H), 11.30 (d, J ) 1.6 Hz, 1 H), 7.51 (br s, 1 H), 7.21 (s, 1 H),
6.95 (s, 2 H), 5.63 (br s, 2 H), 4.62 (dd, J ) 10.7, 9.4 Hz, 1 H),
4.29 (dd, J ) 11.0, 4.0 Hz, 1 H), 4.05 (dd, J ) 10.8, 3.6 Hz, 1
H), 4.01-3.94 (m, 1 H), 3.93 (s, 3 H), 3.89 (s, 3 H), 3.83 (dd, J
) 10.8, 7.6 Hz, 1 H), 3.81 (s, 3 H), 3.79 (s, 3 H). Anal. Calcd
1
mg, 97%): mp 179-180 °C (dec, EtOAc/petroleum ether); H
NMR (d6-DMSO) δ 11.34 (s, 1 H), 8.74 (s, ca. 0.8 H, major
rotamer), 8.44 (s, ca. 0.2 H, minor rotamer), 7.59 (s, 1 H), 4.62-
4.55 (m, 2 H), 4.29-4.18 (m, 2 H), 4.00-3.94 (m, 1 H), 3.93 (s,
3 H), 3.13 (s, 3 H), 1.54 (s, 9 H). Anal. Calcd for C19H23N3O9S:
C, 48.61; H, 4.94; N, 8.95. Found: C, 48.46; H, 4.69; N, 8.75.
for
C
25H25ClN4O6‚1/2EtOAc: C, 58.22; H, 5.24; N, 10.06.
Found: C, 58.20; H, 5.09; N, 10.06.
Meth yl 1-(Ch lor om eth yl)-5-(m eth yla m in o)-3-[(5,6,7-tr i-
m eth oxyin d ol-2-yl)ca r bon yl]-1,2-d ih yd r o-3H-p yr r olo[3,2-
e]in d ole-7-ca r boxyla te (27). Freshly prepared AcOCHO [40
µL of a solution prepared from HCO2H (1.22 mL, 32 mmol)
and Ac2O (2.45 mL, 26 mmol)] was added to a solution of 8
(94 mg, 0.18 mmol) in THF (10 mL) under nitrogen at 20 °C.
The solution was stirred for 2 h and then evaporated. The
residue was redissolved in THF (8 mL) under nitrogen, BH3‚
DMS (45 µL, 0.45 mmol) added, and the yellow solution was
stirred at reflux for 30 min. The mixture was cooled, MeOH
and H2O were added, and the mixture was evaporated. The
residue was diluted with H2O and extracted with EtOAc (×2).
The extracts were washed with H2O, dried (Na2SO4), and
evaporated. Chromatography (50-60% EtOAc/petroleum ether)
followed by recrystallization from PhH gave 27 as a yellow
solid (11 mg, 11%): mp 201-205 °C dec; 1H NMR (d6-DMSO)
δ 11.61 (s, 1 H), 11.34 (s, 1 H), 7.42 (v br s, 1 H), 7.23 (s, 1 H),
6.96 (s, 2 H), 6.06 (s, 1 H), 4.64 (t, J ) 9.6 Hz, 1 H), 4.33 (dd,
J ) 11.0, 3.6 Hz, 1 H), 4.06 (dd, J ) 10.5, 3.4 Hz, 1 H), 4.05-
3.96 (m, 1 H), 3.91 (s, 3 H), 3.88 (s, 3 H), 3.88-3.83 (m, 1 H),
25 was resolved by HPLC on a Daicel Chiralcel OD semi-
preparative column (10 µm, 2 × 25 cm). Samples were
dissolved in CH3CN and eluted in i-PrOH/hexane (1:1) at a
flow rate of 6.75 mL/min, giving RT’s of 45.8 min for (-)-25
and 62.8 min for (+)-25 (R ) 1.37). The pooled samples of each
enantiomer were evaporated at room temperature and purified
by chromatography (50% EtOAc/petroleum ether), and a
sample of each was reinjected on the chiral column to establish
optical purity:
(-)-(R)-25: mp 180-181 °C; [R]D -21.2°
(c 0.259, THF); ee 92%
(+)-(S)-25: mp 180-181 °C; [R]D +18.9°
(c 0.233, THF); ee 88%
(+)- a n d (-)-Meth yl 3-(ter t-Bu tyloxyca r bon yl)-1-(ch lo-
r om eth yl)-5-n itr o-1,2-d ih yd r o-3H-p yr r olo[3,2-e]in d ole-7-
ca r boxyla te (26). (-)-(R)-25 (214 mg, 0.46 mmol) and LiCl
(0.10 g, 2.4 mmol) were dissolved in DMF (20 mL), and the
solution was stirred at 20 °C for 6 days. H2O was added, the
solid was filtered off and redissolved in CH2Cl2, and the
solution was dried (Na2SO4) and evaporated onto silica.
Chromatography (30% EtOAc/petroleum ether) and trituration
with hot EtOAc gave (-)-(R)-26 as a pale yellow solid (124 mg,
3.81 (s, 3 H), 3.78 (s, 3 H), 2.81 (s, 3 H). Anal. Calcd for C26H27
-
ClN4O6‚H2O: C, 57.30; H, 5.36; N, 10.28. Found: C, 57.16; H,
5.41; N, 10.35.
Meth yl 1-(Ch lor om eth yl)-5-(d im eth yla m in o)-3-[(5,6,7-
tr im eth oxyin d ol-2-yl)ca r bon yl]-1,2-d ih yd r o-3H-p yr r olo-
[3,2-e]in d ole-7-ca r boxyla te (28). NaBH3CN (40 mg, 0.6
mmol) and then aqueous HCl (2 N, 0.4 mL) were added to a
solution of 8 (111 mg, 0.22 mmol) and HCHO (0.17 mL of a
40% w/v aqueous solution, 2.3 mmol) in THF (15 mL), and the
pale orange solution was stirred at 20 °C for 100 min. The
THF was evaporated, and the residue was diluted with H2O
and extracted with CH2Cl2 (×2). The extracts were dried
(Na2SO4) and evaporated. Chromatography (1% MeOH/CHCl3)
followed by crystallization from PhH-petroleum ether gave
1
66%): mp 210 °C dec; [R]D -17.5° (c 0.292, CHCl3); H NMR
(CDCl3) δ 10.34 (s, 1 H), 8.98 (br s, ca. 0.7 H, major rotamer),
8.61 (br s, ca. 0.3 H, minor rotamer), 7.26 (s, 1 H coincident
with CHCl3, signal appears at δ 7.66 in d6-DMSO), 4.30-4.03
(m, 3 H), 4.00 (s, 3 H), 3.96 (dd, J ) 11.2, 3.7 Hz, 1 H), 3.72
(dd, J ) 10.9, 8.6 Hz, 1 H), 1.61 (s, 9 H). Anal. Calcd for C18H20
-
ClN3O6‚3/4H2O: C, 51.07; H, 5.12; N, 9.93. Found: C, 50.93;
1
28 as a cream powder (33 mg, 28%): mp 200-202 °C dec; H
H, 4.83; N, 9.64.
NMR (d6-DMSO) δ 11.64 (s, 1 H), 11.38 (s, 1 H), 7.92 (v br s,
1 H), 7.34 (s, 1 H), 6.99 (s, 1 H), 6.97 (s, 1 H), 4.67 (t, J ) 9.6
Hz, 1 H), 4.37 (dd, J ) 11.0, 3.5 Hz, 1 H), 4.15-4.08 (m, 2 H),
3.96 (dd, J ) 11.8, 8.2 Hz, 1 H), 3.92 (s, 3 H), 3.87 (s, 3 H),
By the same procedure (+)-(S)-25 gave (+)-(S)-26 (74%):
mp 208 °C dec; [R]D +16.8° (c 0.298, CHCl3).
(+)- a n d (-)-11. (-)-(R)-26 (72 mg, 0.18 mmol) was stirred
in HCl-saturated dioxane (10 mL) for 2 h (until TLC indicated
complete reaction), and the suspension was evaporated. EDCI
(77 mg, 0.4 mmol) and 5,6,7-trimethoxyindole-2-carboxylic acid
(44.5 mg, 0.18 mmol) in DMA (2.5 mL) were added, and the
mixture was stirred at 20 °C for 16 h. Aqueous NaCl was added
and the mixture was extracted with CH2Cl2 (×4). The extracts
were dried (Na2SO4), evaporated onto silica, and purified by
chromatography (50% EtOAc/petroleum ether) to give (-)-(R)-
11 as an orange powder (60 mg, 63%): mp 236-237 °C; [R]D
-46.5° (c 0.342, CHCl3).
3.82 (s, 3 H), 3.79 (s, 3 H), 2.78 (s, 6 H). Anal. Calcd for C27H29
-
ClN4O6: C, 59.94; H, 5.40; N, 10.36. Found: C, 60.25; H, 5.61;
N, 10.25.
Meth yl 1-(Ch lor om eth yl)-5-[(4-n itr oben zyloxyca r bon -
yl)a m in o]-3-[(5,6,7-t r im et h oxyin d ol-2-yl)ca r b on yl]-1,2-
d ih yd r o-3H-p yr r olo[3,2-e]in d ole-7-ca r boxyla te (29). 4-Ni-
trobenzyl chloroformate (0.10 g, 0.46 mmol) was added to a
solution of 8 (214 mg, 0.42 mmol) in pyridine (7 mL) and the
mixture stirred at 20 °C. More 4-nitrobenzyl chloroformate (2
× 0.10 g) was added after 30 and 90 min, and after a further
1 h the mixture was diluted with H2O and stirred for 30 min.
The solid was filtered off, dried, and triturated with hot EtOAc
to give 29 as a cream solid (209 mg, 72%): mp 257-258.5 °C;
1H NMR (d6-DMSO) δ 11.86 (s, 1 H), 11.37 (s, 1 H), 9.82 (s, 1
H), 8.89 (s, 1 H), 8.29 (d, J ) 8.6 Hz, 2 H), 7.75 (d, J ) 8.6 Hz,
2 H), 7.38 (s, 1 H), 7.01 (s, 1 H), 6.96 (s, 1 H), 5.37 (s, 2 H),
4.71 (t, J ) 10.2 Hz, 1 H), 4.38 (dd, J ) 11.0, 4.2 Hz, 1 H),
4.19-4.12 (m, 1 H), 4.11 (dd, J ) 10.9, 3.3 Hz, 1 H), 4.01 (dd,
J ) 10.8, 6.6 Hz, 1 H), 3.93 (s, 3 H), 3.91 (s, 3 H), 3.82 (s, 3 H),
By the same procedure (+)-(S)-26 gave (+)-(S)-11 (63%):
mp 237-238 °C; [R]D +45.9° (c 0.331, CHCl3).
(R)- a n d (S)-8. By the same procedure as described above
(-)-(R)-11 and (+)-(S)-11 were converted to (R)-8 and (S)-8,
each mp 210-220 °C dec. Neither enantiomer possessed
measurable optical activity, but HPLC on a Daicel Chiralcel
OD column (30% EtOH/hexane, RT 58 and 66 min, respec-
tively), confirmed that epimerisation had not occurred and the
ee values were no lower than those of the corresponding

Synthesis and Cytotoxicity of Amino-seco-DSA
J . Org. Chem., Vol. 64, No. 16, 1999 5953
enantiomers (-)- and (+)-25 from which they were derived.
In Vitr o Cytotoxicity Assa y. Growth inhibitory potency
under aerobic conditions was determined using log-phase
cultures in 96-well plates, as described previously.8a Com-
pounds were freshly dissolved in DMSO or acetone. IC50 values
were calculated as the drug concentration providing 50%
inhibition of growth relative to the controls.
Ack n ow led gm en t. The authors thank Karin Tan
and Alison Coleman for technical assistance. This work
was supported by the Auckland Division of the Cancer
Society of New Zealand.
J O990464J