Synthesis and biological evaluation of novel analogues and prodrugs of the cytotoxic antibiotic CC-1065 for selective cancer therapy

Article abstract of DOI:10.1002/1099-0690(200205)2002:10<1634::AID-EJOC1634>3.0.CO;2-Y

This paper describes novel seco-analogues 25-27 of the cytotoxic antibiotic CC-1065 (1) and their prodrugs 5, 6a, and 6b, for antibody-directed enzyme prodrug therapy (ADEPT). The partially hydrogenated seco-CCI-analogue 7 and the corresponding methyl-CCI

Full text of DOI:10.1002/1099-0690(200205)2002:10<1634::AID-EJOC1634>3.0.CO;2-Y

FULL PAPER
Synthesis and Biological Evaluation of Novel Analogues and Prodrugs of the
Cytotoxic Antibiotic CC-1065 for Selective Cancer Therapy
Lutz F. Tietze,*[a] Tobias Herzig,[a] Tim Feuerstein,[a] and Ingrid Schuberth[a]
Keywords: Antitumor agents / Cancer therapy / Enzymes / Glycosides / Prodrugs
This paper describes novel seco-analogues 25−27 of the
cytotoxic antibiotic CC-1065 (1) and their prodrugs 5, 6a, and
6b, for antibody-directed enzyme prodrug therapy (ADEPT).
The partially hydrogenated seco-CCI-analogue 7 and the
respectively. Compounds 25−27 were prepared by treatment
of 7, 8a, and 8b with 20 after deprotection. In vitro tests
showed a strong cytotoxicity for 25 and a fairly low toxicities
for 26 and 27. However, the selectivity of the prodrugs 5, 6a,
corresponding methyl-CCI analogues 8a and 8b were syn- and 6b was not sufficient for ADEPT. Interestingly, 8a and 8b
thesized by alkylation of 9 with 15 and 16, respectively, fol-
lowed by radical cyclization and deprotection. Treatment of
7, 8a, and 8b with the galactose trichloroacetimidate 21 and
the bisindolyl-carboxylic acid 20 in the presence of EDC fol-
lowed by solvolysis gave the desired prodrugs 5, 6a, and 6b,
did not undergo Winstein cyclization to produce the spirocy-
clopropylcyclohexadienone moiety.
( Wiley-VCH Verlag GmbH, 69451 Weinheim, Germany,
2002)
Introduction
lethal hepatotoxicity. Synthetic analogues of 1, such as CBI
(2), are highly potent cytostatic agents but do not display
this hepatotoxicity.[9] The antiproliferative effect of 1 and
its analogues was shown to derive from a selective al-
kylation of N-3 of adenine in DNA by a nucleophilic attack
at the spirocyclopropylcyclohexadienone moiety as the
pharmacophoric group.[10]
One of the main problems of anticancer therapy is insuf-
ficient differentiation between normal and malignant cells
by the known antiproliferant agents, resulting in severe side
effects. Approaches for more selective therapy might be
based on the use of phenotypic differences[1Ϫ4] such as the
increased rate of glycolysis and the expression of specific
antigens, as well as on genetic[5] differences. This may result
in a specific liberation of a toxin from a suitable nontoxic
or little toxic prodrug.
A promising concept is antibody-directed enzyme prodrug
therapy (ADEPT).[5Ϫ7] In this approach, the toxic effector
is liberated from a prodrug by enzymatic cleavage by a con-
jugate of an appropriate enzyme and a monoclonal anti-
body that binds to tumor-associated antigens. A crucial re-
quirement for this approach is high cytotoxicity in the cor-
responding drug, in the range of an ED50 value Ͻ 10 n
(ED50, drug concentration required for 50% biological ef-
fect on target cells) and a high Q ED50 value (ED50 of pro-
drug/ED50 of prodrug ϩ enzyme).
Synthetically, the pharmacophoric group in 1 and 2
might be formed by a Winstein cyclization[11] of a corres-
ponding phenol, such as 3a, with a leaving group at the β-
position (Scheme 2).[9] The cyclization, which also proceeds
under physiological conditions, can be blocked by glycosyl-
ation of the phenolic hydroxyl group.[12Ϫ14] In order to
employ this effect in ADEPT, we prepared the seco-CBI-
galactoside 3b. However, this compound surprisingly
showed a decrease in cytotoxicity relative to 3a by a factor
of only 32, which is clearly not sufficient for selective ther-
apy. Speculating that the comparably high cytoxicity of 3a
may derive from a direct attack of N-3 in adenine at the
chloromethyl group in 3a without formation of the spirocy-
clopropylcyclohexadiene moiety, we prepared the branched
compound anti-4b, containing a secondary rather than a
primary chloride as in 3b. Enzymatic cleavage resulted in
anti-4a, which cyclized to give the spirocyclopropylcy-
clohexadienone moiety. This indeed showed a much higher
selectivity, with a Q ED50 ϭ 770 with human bronchial car-
cinoma cells of line A549.[14] To obtain further information
about the structure activity relationship of seco-CC-1065
analogues, we prepared a new class of antiproliferating
agents containing a partially hydrogenated ring A. Here we
describe the synthesis and the evaluation of the biological
activity of compounds 5, 6a, and 6b, and 25Ϫ27.
One class of compounds with extraordinarily high cyto-
toxicities, with ED50 values of about 0.03 nM (cell line
L1210), are the natural occurring antibiotics CC-1065 (1)
and the duocarmycins (Scheme 1).[8] However, CC-1065 (1)
cannot be used for clinical treatment because of its delayed
[a]
Georg-August-Universität Göttingen, Institut für Organische
Chemie
Tammannstraße 2, 37077 Göttingen, Germany
Fax: (internat.) ϩ 49-(0)551/399476
E-mail: ltietze@gwdg.de
1634
 WILEY-VCH Verlag GmbH, 69451 Weinheim, Germany, 2002 1434Ϫ193X/02/0510Ϫ1634 $ 20.00ϩ.50/0 Eur. J. Org. Chem. 2002, 1634Ϫ1645

Novel Analogues and Prodrugs of the Cytotoxic Antibiotic CC-1065
FULL PAPER
(Scheme 3). There are three possible approaches for the syn-
thesis of 9: selective hydrogenation of an aromatic ring in a
naphthalene derivative, construction of a second alicyclic
ring starting from a benzene derivative, or preparation from
tetralene 10 by aromatic substitution reactions. The first
way would involve the danger of incomplete reduction and
hence potential contamination of the prodrugs 5 and 6 with
traces of the CBI analogue, which could seriously falsify the
results of biological tests. While exploring the second way,
we observed that the final closure of the alicyclic ring re-
quired harsh conditions that were not compatible with the
substituents already present in the aromatic ring.
We therefore employed the commercially available tetra-
lene 10 as starting material. Compound 10 was transformed
into 12 via the dinitro derivative 11, by use of a mixture of
concentrated nitric and sulfuric acid with careful control
over the reaction conditions, followed by reduction of the
nitro group at position 1 with SnCl2 to give the amine 12
(Scheme 4).[15] The yields of both reactions were poor and
the workup procedure lengthy, but it was possible to work
on mole scales and thus obtain sufficient amounts of mat-
erial for the following conversions. Transformation of the
amine 12 to give phenol 13 was performed by formation
of a diazonium salt followed by addition of water. After
protection of the phenolic hydroxyl group in 13 as its benzyl
ether 14, the remaining nitro group was reduced, and the
amino group formed was protected as its BOC-carbamate.
Regioselective iodination in the position para to the benzyl
ether moiety was achieved by use of the Königstein proced-
ure to give 9 in 75% yield based on 13.[16]
Ring C in 5 was introduced by alkylation of the amide 9
with 1,3-dichloride 15 followed by radical cyclization with
nBu3SnH, which provided chloride 17 in a good overall
yield of 77% based on 9 (Scheme 5).[17,18] Debenzylation of
17 to give 7 was achieved under mild conditions without
dehalogenation in a transfer hydrogenation with Pd/C in
the presence of ammonium formate.[19] The introduction of
the sugar moiety and the side chain, which is important for
binding to the minor groove of the DNA, was performed
in one process, since we were able to remove the protecting
group at the nitrogen simultaneously during the glycosid-
ation. Thus, treatment of 7 with the trichloroacetimidate
21[20] in the presence of BF3·OEt2 gave the corresponding
β--galactoside, containing a free secondary amino moiety.
This was transformed into the desired prodrug 5 by addi-
tion of the bisindolyl-carboxylic acid 20 and N-(3-dimethyl-
aminopropyl)-NЈ-ethylcarbodiimide hydrochloride (EDC),
followed by solvolysis of the acetate moieties to give a 25%
yield over three steps.
Scheme 1. Structure of (ϩ)-CC-1065 (1) and the analogues CBI
(2), seco-CBI 3a, and methyl-seco-CBI 4a, and their prodrugs 3b
and 4b
Scheme 2. Formation of the spirocyclopropylcyclohexadienone
moiety of CC-1065 and its analogues by Winstein cyclization from
the seco-compounds
Compound 5 contains a stereogenic center in the seco-
CCI unit.[21] This was formed nonselectively, and so the
compound, since it also contains a sugar moiety, was ob-
tained as a 1:1 mixture of two diastereomers. However, no
attempts were made to separate the stereoisomers, since (as
shown by us for related compounds) both diastereomers
could be cleaved by galactosidase at similar rates. In addi-
Results
Synthesis
Retrosynthesis of the glycosidic prodrugs 5 and 6 sug- tion, we prepared the benzyl ether 22 and the phenol 25 as
gested the tetralene derivative 9, via 7 and 8, respectively model substances for the cytotoxicity tests, by coupling of
Eur. J. Org. Chem. 2002, 1634Ϫ1645
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L. F. Tietze, T. Herzig, T. Feuerstein, I. Schuberth
FULL PAPER
Scheme 3. Retrosynthesis of the novel partially hydrogenated prodrugs 5 and 6
Scheme 4. Synthesis of the key intermediate 9
N-deprotected 7 and 17, respectively, with acid 20 in the the same procedure from 19, via 8b, gave nearly identical
presence of EDC. yields of 6b. We also prepared the benzyl ethers 23 and 24
In a fashion similar to that described for the synthesis in this series, as well as the unprotected phenols 26 and 27,
of 5, we also synthesized the methyl-substituted prodrug 6. all containing the bisindolyl-carboxylic acid for binding to
Compound 6 contains two stereogenic centers in the seco- the minor groove of the DNA.
CCI unit, and thus can exist as four diastereomers. Here a
Structure Determination
pronounced difference in the biological activities had to be
expected; we therefore prepared the diastereomers 6a and
The structures of the newly prepared compounds were
6b (with a syn and an anti orientation of the hydrogens at mainly determined by NMR spectroscopy. As examples, the
C-1 and C-10) separately. Alkylation of 9 with 16 followed 1H NMR spectroscopic data of compounds 5؊8 are discus-
by radical cyclization gave a 1:1 mixture of the diastereo- sed. It should be noted that the compounds were measured
mers 18 and 19, which could be separated by column chro- in different solvents.
matography (18: 37%; 19: 42%). For further transforma-
The hydrogens 7-CH2 and 8-CH2 of the alicylic moiety
tions, the single diastereomers were used separately. Thus, of these compounds resonated as multiplets around δ ϭ
debenzylation of 18 to give 8a followed by glycosidation 1.65 and 1.80, and the corresponding benzylic hydrogens
with 21 and introduction of the bisindolyl-carboxylic acid around δ ϭ 2.60. For the aromatic hydrogens in 7, 8a, and
moiety 20 afforded 6a in 42% yield after solvolysis, whereas 8b, singlets were found at δ ϭ 7.28Ϫ7.33, whereas for the
1636
Eur. J. Org. Chem. 2002, 1634Ϫ1645

Novel Analogues and Prodrugs of the Cytotoxic Antibiotic CC-1065
FULL PAPER
Scheme 5. Preparation of the seco-CCI derivatives for the biological tests; AIBN ϭ α,αЈ-Azoisobutyronitrile, EDC ϭ N-ethyl-NЈ-(3-
dimethylaminopropyl)carbodiimide hydrochloride
glycosidic compounds 5, 6a, and 6b, singlets were observed, 4.10Ϫ4.18. The resonances for 10-H in 8a and 8b, as quad-
as expected, further downfield at δ ϭ 8.00Ϫ8.06. For deter- ruplets of doublets at δ ϭ 4.40 and 4.44 with J ϭ 7.2/7.0
mination of the configurations of the glycosidic bonds in 5, and 3.5/3.6 Hz, respectively, also did not allow differenti-
6a, and 6b, the resonances of the hydrogens at the anomeric ation.
centers are of importance; the corresponding signals were
found at δ ϭ 4.90Ϫ4.92 as doublets with J ϭ 7.5Ϫ8.0 Hz,
In vitro Cytotoxicity Assays
clearly indicating a β configuration. The assignment of the
The in vitro cytotoxicity assays were carried out with co-
relative configurations of the two pairs of diastereomers 6a herent cells of the bronchial carcinoma cell line A549 in
and 6b and 8a and 8b was hampered by the fact that 8a triplicate in 6 multiwell plates of concentrations of 102, 103,
and 8b could not be transformed into the spirocyclopropyl- 104, and 105 cells per cavity. Incubation with the phenolic
cyclohexadienone moiety, as shown for the corresponding compounds 25, 26, and 27, the benzyl ethers 22, 23, and
diastereomeric methyl-seco-CBI analogues syn- and anti- 24, and the galactosides 5, 6a, and 6b, both in the absence
4a.[14] However, comparison of the chemical shift values for and in the presence of galactosidase, was performed in ul-
1-H and the methyl groups at C-10 of syn- and anti-4a, and traculture medium of various concentrations for 24 h. An
also their derivatives, with those of 8a and 8b allowed the important criterion is the comparison of the ED50 value of
structures to be assigned. Thus, 1-H of the syn compounds the phenolic compound and the ED50 value of a mixture of
always resonated at lower field than 1-H of the anti com- the glycosidic compound and β--galactosidase. These
pounds, while the opposite was true of the methyl groups values should be similar, otherwise a suicide mechanism
at C-10. We therefore assumed that 8a, with signals for 1- must be assumed. In such a case the system is not suitable
H at δ ϭ 3.66 and for 11-CH3 at δ ϭ 1.18, had a syn ori- for ADEPT.
entation of the hydrogens at C-1 and C-10 (1SR, 10SR).
The seco-CCI derivative 25 showed very high cytotox-
Correspondingly, 1-H of 8b resonated at δ ϭ 3.40 and 11- icity, with an ED50 of 0.14 nM (Figure 1 and Table 1) in our
CH3 at δ ϭ 1.54, and 8b should therefore have an anti ori- HTCFA (Human Tumor Colony Forming Ability) assay on
entation. In accordance with this assignment, 6a must have A549 cells. Its biological activity was only less than that of
a syn and 6b an anti orientation. In contrast to 1-H and 11- CC-1065 (1) by a factor of five, and by a factor of fifteen
CH3, the diastereotopic protons at C-2 in 7, 8a, and 8b relative to that of its CBI analogue 2, according to the pub-
resonated at very similar fields, with δ ϭ 3.85Ϫ3.90 and lished data[22] for these compounds on L1210 cells. This
Eur. J. Org. Chem. 2002, 1634Ϫ1645
1637

L. F. Tietze, T. Herzig, T. Feuerstein, I. Schuberth
FULL PAPER
shows that the insertion of 25 into the minor groove of the that the enzyme was not deactivated in the hydrolytic pro-
DNA still takes place, even though it does not have the cess. However the cytotoxicity of prodrug 5 was only
ideal flat aromatic ring system. Thus, partially hydrogen- slightly less than that of 25, with a Q ED50 ϭ 62.
ated derivatives of CC-1065 (1) are a novel, highly potent
class of CC-1065 analogues.
In accordance with the results found for anti-4b, a much
higher selectivity was expected for the methyl-CCI com-
pounds 6a and 6b, since the direct attack of N-3 of adenine
should be slower due to steric hindrance. We first investig-
ated the cytotoxicity of the diastereomeric compounds 26
and 27, containing free phenolic hydroxy groups. The syn
isomer 26 displayed a modest cytotoxicity of ED50
ϭ
600 nM, whereas the anti isomer 27 showed a cytotoxicity
fifteen times higher, with an ED50 ϭ 40 nM. The syn-galac-
toside 6a was nontoxic in the investigated concentration
range, even in the presence of β--galactosidase (ED50
Ͼ
35000 nM). The anti-galactoside 6b showed a cytotoxicity
of ED50 ϭ 73000 nM, whereas the antiproliferative effect in
the presence of β--galactosidase was increased to an
ED50 ϭ 800 nM, corresponding to a selectivity factor (Q
ED50) of 91.
Astoundingly, the decrease in the cytotoxicity of the
benzylated compounds 23 and 24 compared to the free
phenols 26 and 27, respectively, was rather low, with a fac-
tor of 20Ϫ55.
Discussion
The reason for the comparatively high cytotoxicity of the
galactoside 5 still remains unknown. Since serum-free in-
cubation medium was employed, any enzyme activity of the
medium (as observed in previous studies[12] with other me-
dia) could be ruled out. We must therefore assume either
that nonenzymatic cleavage of the glycosidic bond in 5 oc-
curs, or that direct alkylation of the DNA with 5 takes place
without formation of the spirocyclopropane moiety. The
latter effect must be caused by the galactose moiety, since
the benzylated compound 22 shows a rather low cytotox-
icity. Similar observations were made with the correspond-
ing CBI prodrug, showing a selectivity factor of 32;[14] com-
pared to this result, the partially hydrogenated prodrug 5
displays a slightly better selectivity, but this is not sufficient
in our opinion for selective cancer therapy.
The lack of any cytotoxicity of the syn-galactoside 6a in
the presence of β--galactosidase Ϫ the corresponding
phenolic compound 26 has an ED50 ϭ 600 nM Ϫ might be
due to the fact that 6a is a poor substrate for galactosidase.
The same might also be true for the anti-galactoside 6b,
since 6b in the presence of galactosidase is 20 times less
toxic than the phenolic compound 27. We do not assume a
suicide mechanism in these cases, since the activity of the
enzyme is nearly unaltered for suitable substrates in the
mixture. An important aspect is the low difference in the
cytotoxicities of the benzyl ethers 23 and 24 relative to
those of the corresponding phenols 26 and 27, respectively,
clearly indicating that direct alkylation of adenine or some
Figure 1. In vitro cytotoxicity of 5, 6a, 6b, and 22؊27 against hu-
man bronchial carcinoma cells of line A549; A, in vitro cytotoxicity
of compound 22 (black square), 25 (black triangle), and 5 without
(black circle) and with (white circle) addition of β--galactosidase
(0.4 U/mL); B, in vitro cytotoxicity of syn-compounds 23 (black
square), 26 (black triangle), and 6a without (black circle) and with
(white circle) addition of β--galactosidase (0.4 U/ml); C, in vitro
cytotoxicity of anti-compounds 24 (black square), 27 (black tri-
angle), and 6b without (black circle) and with (white circle) addi-
tion of β--galactosidase (0.4 U/ml); cells were exposed to various
concentrations of the test substances for 24 h at 37 °C; after 12
days of incubation clone formation was compared to untreated
control assay and the relative clone forming rate was determined
A cytotoxicity similar to that found for the phenol 25 was other base in DNA takes place with the chloromethyl group
observed for the prodrug 5 in the presence of the enzyme in 23 and 24. We therefore postulate that the formation of
β--galactosidase, with an ED50 ϭ 1.3 nM, which indicated the spirocyclopropyl moiety is somehow hampered by the
1638
Eur. J. Org. Chem. 2002, 1634Ϫ1645

Novel Analogues and Prodrugs of the Cytotoxic Antibiotic CC-1065
Table 1. In vitro cytotoxicity of compounds 5, 6a, 6b, and 22Ϫ27 against human bronchial carcinoma cells of line A549
FULL PAPER
Compound[a]
Substituent at the phenolic oxygen
ED50 [nM][b]
Selectivity factor[c] (Q ED50)
22
25
5
Benzyl
H
β--Galactose
β--Galactose[d]
Benzyl
290
0.14
80
1.3
12400
600
Ͼ35000
Ͼ35000
2200
40
73000
800
62
5
23
26
6a
6a
24
27
6b
6b
H
β--Galactose
β--Galactose[d]
Benzyl
Ϫ
H
β--Galactose
91
β--Galactose[d]
[a]
[b]
Compounds 5, 6a, and 6b were used as mixtures of diastereomers and 22؊27 as racemic mixtures of the respective diastereomer.
Cells were exposed to the compounds for 24 h at 37 °C; after 12 days of incubation clone formation was compared to untreated control
[c]
assay. ED50 ϭ drug concentration required for 50% biological effect on target cells. Selectivity factor (Q ED50) ϭ ED50 of compound/
ED50 of compound in the presence of β--galactosidase. 0.4 U β--galactosidase/mL were added for 24 h.
[d]
cyclohexane moiety. To verify this assumption we investig-
ated the Winstein cyclization of the phenols 7, 8a, and 8b
Conclusion
under basic conditions (Scheme 6). Treatment of 7 with so-
dium hydride in THF afforded the desired spirocyclopropyl
compound 28 in 80% yield. In contrast, the phenols 8a and
8b did not react at all to give 29 under these conditions.
Since the seco-methyl-CBI analogue anti-4a reacted
smoothly, the observed behavior of 8a and 8b must arise
from the combined steric bulk of the methyl group and the
cyclohexene moiety. On the basis of these results, it is un-
derstandable that the introduction of a methyl group at the
chloromethyl moiety in 7 does not improve the selectivity
(Q ED50), as found for methyl-seco-CBI. Further work will
thus concentrate on this aspect, and so we intend to prepare
the corresponding cyclobutano, cyclopentano, and cyclo-
heptano compounds and compare their cytotoxicities and
selectivities.
A new type of a seco-CC-1065 analogue with an annu-
lated cyclohexane ring has been prepared. The antiprolifer-
ative activity was similar to that of CC-1065 (1) and CBI
(2). The quotient of the ED50 of the corresponding glycosi-
dated prodrug and the drug was slightly higher than that
found for the glycosylated seco-CBI compound, but still not
sufficient for use in ADEPT. Of interest is the observation
that the methylated seco-CCI 8, with a free phenolic hy-
droxy group, did not undergo cyclization under basic condi-
tions to give the corresponding spirocyclopropylcyclohex-
adienone moiety. A possible explanation for this finding is
the steric bulk of methyl seco-CCI 8 relative to methyl seco-
CBI anti-4a and seco-CCI 7.
Experimental Section
General: Reactions were performed under an inert gas atmosphere
in flame-dried flasks. Solvents were dried by standard methods.
Reagents obtained from commercial sources were used without fur-
ther purification. Thin-layer chromatography was performed on
precoated silica gel plates (SIL G/UV254, MachereyϪNagel & Co.).
Silica gel 32-64 (0.032Ϫ0.064 mm) (MachereyϪNagel & Co.) was
used for column chromatography (PE: petroleum ether).
Elemental Analysis: Because of the high value of several prepared
compounds, elemental analyses were not performed in these cases.
1-Hydroxy-3-nitro-5,6,7,8-tetrahydronaphthalene (13): Pulverized
amine 12[15] (6.48 g, 33.7 mmol), obtained from 10 by nitration and
reduction, was suspended in 75% H2SO4 (100 mL) and treated at
Ϫ5 °C with a solution of NaNO2 (2.44 g, 35.4 mmol, 1.05 equiv.)
in conc. H2SO4 (15 mL). After the mixture had been stirred for 1 h
at this temperature, water (100 mL) was added and the mixture was
warmed to 60 °C until the evolution of gas ceased. The dark brown
solution was extracted twice with EtOAc, and the extracts were
washed with water and brine and dried with Na2SO4. Evaporation
Scheme 6. Winstein cyclization of partially hydrogenated seco-
CBI analogues
Eur. J. Org. Chem. 2002, 1634Ϫ1645
1639

L. F. Tietze, T. Herzig, T. Feuerstein, I. Schuberth
FULL PAPER
of the solvent and column chromatography (PE/EtOAc, 5:1)
yielded phenol 13 (3.49 g, 18.1 mmol, 54%) as a brown solid. Rf ϭ
0.45 (PE/EtOAc, 5:1), m.p. 100Ϫ101 °C. 1H NMR (200 MHz,
CDCl3): δ ϭ 1.84 (mc, 4 H, 6-CH2, 7-CH2), 2.71, 2.84 (each t, J ϭ
5.2 Hz, each 2 H, 5-CH2, 8-CH2), 5.59 (s, 1 H, OH), 7.46 (d, J ϭ
2.0 Hz, 1 H, 2-H), 7.58 (s, 1 H, 4-H). 13C NMR (50 MHz, CDCl3):
δ ϭ 22.0, 22.3 (C-6, C-7), 23.4 (C-8), 29.8 (C-5), 106.6 (C-2), 116.5
(C-4), 132.0 (C-8a), 140.0 (C-4a), 145.9 (C-3), 153.9 (C-1). MS (EI,
70 eV): m/z (%) ϭ 193 (100) [M]ϩ, 176 (22) [M Ϫ OH]ϩ, 147 (28)
[M Ϫ NO2]ϩ. C10H11NO3 (193.205).
111.1 (C-4), 121.1 (C-8a), 127.1 (o-C), 127.6, (p-C), 128.4 (m-C),
136.2 (C-3), 137.6 (i-C), 138.8 (C-4a), 152.8 (CϭO), 156.8 (C-1).
MS (EI, 70 eV): m/z (%) ϭ 353 (4) [M]ϩ, 297 (15) [M Ϫ C4H9 ϩ
H]ϩ, 91 (100) [C7H7]ϩ. C22H27NO3 (353.465).
4-Benzyloxy-2-(tert-butyloxycarbonylamino)-1-iodo-5,6,7,8-tetra-
hydronaphthalene (9): A solution of amide 31 (4.65 g, 13.3 mmol)
in methanol/water (4:1, 350 mL) was treated with HIO3 (492 mg,
2.79 mmol, 0.21 equiv.) and iodine (1.38 g, 5.45 mmol, 0.41 equiv.)
and heated under reflux with vigorous stirring for 1.5 h. Cooling
of the mixture in an ice bath gave a white, voluminous precipitate,
which was filtered off and washed with water. Thorough drying
1-Benzyloxy-3-nitro-5,6,7,8-tetrahydronaphthalene (14): Phenol 13
(3.49 g, 18.1 mmol) was dissolved in an acetone/DMF mixture (2:1, provided iodide 9 (6.00 g, 12.5 mmol, 94%) as white flakes. Rf ϭ
27 mL), and K2CO3 (3.48 g, 25.2 mmol, 1.5 equiv.), benzyl bromide 0.64 (PE/EtOAc, 10:1), m.p. 135Ϫ137 °C. UV (CH3CN): λmax (lg
(2.43 mL, 21.7 mmol, 1.2 equiv.), and tetrabutylammonium iodide ε) ϭ 219.5 nm (4.664), 242.0 (4.194), 286.0 (3.289). IR (KBr): ν˜
(333 mg, 905 µmol, 0.05 equiv.) were added. After stirring for 14 h
at room temperature, the solution was concentrated and taken up
in EtOAc. The organic extract was washed five times with water,
(cmϪ1) ϭ 3388, 2937, 1730, 1595Ϫ1H NMR (200 MHz, CDCl3):
δ ϭ 1.56 (s, 9 H, Boc-CH3), 1.75 (mc, 4 H, 6-CH2, 7-CH2), 2.67
(mc, 4 H, 5-CH2, 8-CH2), 5.09 (s, 2 H, OCH2), 7.04 (br. s, 1 H,
then with brine, dried with Na2SO4, and concentrated in vacuo. NH), 7.30Ϫ7.46 (m, 5 H, o-H, m-H, p-H), 7.72 (s, 1 H, 3-H). 13C
Benzyl ether 14 (4.96 g, 17.5 mmol, 97%) was isolated as a brown,
amorphous solid. Rf ϭ 0.56 (PE/EtOAc, 4:1), m.p. 96Ϫ98 °C. UV
(CH3CN): λmax (lg ε) ϭ 294.5 nm (3.852). IR (KBr): ν˜ (cmϪ1) ϭ
NMR (50 MHz, CDCl3): δ ϭ 22.3, 23.7, 24.0 (C-5, C-6, C-7), 28.4
(Boc-CH3), 37.4 (C-8), 69.8 (OCH2), 80.7 (Boc-C), 87.3 (C-1),
101.5 (C-3), 123.7 (C-4a), 127.3 (o-C), 127.7, (p-C), 128.4 (m-C),
3425, 2927, 1523, 1340. 1H NMR (200 MHz, CDCl3): δ ϭ 1.82 136.7 (C-2), 137.1 (i-C), 139.9 (C-8a), 152.8 (CϭO), 156.9 (C-1).
(mc, 4 H, 6-CH2, 7-CH2), 2.82 (mc, 4 H, 5-CH2, 8-CH2), 5.14 (s, 2 MS (EI, 70 eV): m/z (%) ϭ 479 (20) [M]ϩ, 423 (40) [M Ϫ
H, OCH2), 7.35Ϫ7.46 (m, 5 H, o-H, m-H, p-H), 7.56 (d, J ϭ C4H9 ϩ H]ϩ, 296 (21) [M Ϫ I Ϫ C4H9 ϩ H]ϩ, 91 (100) [C7H7]ϩ.
2.1 Hz, 1 H, 2-H), 7.63 (s, 1 H, 4-H). 13C NMR (50 MHz, CDCl3): C22H26INO3 (479.357): calcd. (%) C 55.12, H 5.47; found C 55.18,
δ ϭ 22.1, 22.3 (C-6, C-7), 23.9 (C-8), 29.8 (C-5), 70.2 (OCH2), H 5.32.
102.8 (C-2), 116.9 (C-4), 127.2 (o-C), 128.1, (p-C), 128.7 (m-C),
5-Benzyloxy-3-(tert-butyloxycarbonyl)-1-chloromethyl-1,2,6,7,8,9-
134.5 (C-8a), 136.2 (i-C), 139.4 (C-4a), 146.1 (C-3), 156.6 (C-1).
hexahydro-3H-benz[e]indole (17). Alkylation: Amide 9 (500 mg,
MS (EI, 70 eV): m/z (%) ϭ 283 (18) [M]ϩ, 91 (100) [C7H7]ϩ.
1.04 mmol) was dissolved in DMF (12 mL) and treated with NaH
C17H17NO3 (283.330).
(60% in oil, 100 mg, 2.50 mmol, 2.5 equiv.). After the mixture had
3-Amino-1-benzyloxy-5,6,7,8-tetrahydronaphthalene (30): A mixture
of benzyl ether 14 (4.61 g, 16.3 mmol) in 1,4-dioxane (22 mL) and
PtO2·H2O (20 mg, 0.86 mmol, 0.05 equiv.) was placed under an at-
mosphere of hydrogen (3 bar) and stirred for 24 h. After the cata-
lyst had been removed by filtration through celite, the solution was
been stirred at room temperature for 30 min, chloride 15 (231 mg,
2.08 mmol, 2.0 equiv.) was added, and after 14 h the reaction was
quenched with saturated aq. NH4Cl. After extraction with EtOAc,
the extracts were washed with water and brine and dried with
Na2SO4. The solution was concentrated under reduced pressure
evaporated to dryness. Amine 30 (4.12 g, 16.3 mmol, quantitative and the residue was purified by flash chromatography (PE/EtOAc,
yield) was obtained as a brown solid and was used in the next step
20:1) to give 564 mg (11.02 mmol, 98%) of a yellow oil as a mixture
without further purification. Rf ϭ 0.29 (PE/EtOAc, 5:1). 1H NMR of stable rotamers. Rf ϭ 0.49, 0.59 (PE/EtOAc, 10:1). 1H NMR
(200 MHz, CDCl3): δ ϭ 1.74 (mc, 4 H, 6-CH2, 7-CH2), 2.64 (mc, (200 MHz, CDCl3): δ ϭ 1.34/1.58 (each s, together 9 H, Boc-CH3),
4 H, 5-CH2, 8-CH2), 3.32 (br. s, 2 H, NH2), 5.01 (s, 2 H, OCH2), 1.76 (mc, 4 H, 6-CH2, 7-CH2), 2.70 (mc, 4 H, 5-CH2, 8-CH2),
6.08, 6.14 (each s, each 1 H, 2-H, 4-H), 7.30Ϫ7.44 (m, 5 H, o-H,
3.55Ϫ4.10 (m, 1 H, 1Ј-Ha), 4.43Ϫ4.51 (m, 1 H, 1Ј-Hb), 4.96Ϫ5.12
(m, 2 H, OCH2), 5.93Ϫ6.05 (m, 2 H, 2Ј-H, 3Ј-H), 6.52Ϫ6.66 (m, 1
m-H, p-H). 13C NMR (50 MHz, CDCl3): δ ϭ 22.7, 23.0, 23.1 (C-
6, C-7, C-8), 29.8 (C-5), 69.6 (OCH2), 97.3 (C-2), 107.8 (C-4), 116.7 H, 3-H), 7.30Ϫ7.49 (m, 5 H, o-H, m-H, p-H). 13C NMR (50 MHz,
(C-8a), 126.9 (o-C), 127.6, (p-C), 128.4 (m-C), 137.6 (i-C), 139.2 CDCl3): δ ϭ 22.0, 23.7, 23.9 (C-5, C-6, C-7), 28.3/28.4 (Boc-CH3),
(C-4a), 144.4 (C-3), 157.3 (C-1). MS (EI, 70 eV): m/z (%) ϭ 253 37.1 (C-8), 46.0/49.0 (C-1Ј), 69.7 (OCH2), 80.3/80.8 (Boc-C), 98.5/
(71) [M]ϩ, 162 (38) [M Ϫ C7H7]ϩ, 91 (100) [C7H7]ϩ. C17H19NO
(253.346).
98.6 (C-1), 109.9/110.5 (C-3), 119.9/120.0 (C-3Ј), 121.1/121.4 (C-
4a), 126.9/127.0 (o-C), 127.8 (2 signals) (p-C), 128.5 (2 signals) (m-
C), 128.8/129.1 (C-2Ј), 136.7/136.8 (i-C), 141.0, 141.2, 141.4, 141.8
(C-2, C-8a), 153.8/154.0 (CϭO), 156.2/156.3 (C-4). MS (EI, 70 eV):
m/z (%) ϭ 553 (2) [M]ϩ, 497 (9) [M Ϫ C4H9 ϩ H]ϩ, 426 (8) [M Ϫ
I]ϩ, 370 (98) [M Ϫ I Ϫ C4H9 ϩ H]ϩ, 91 (100) [C7H7]ϩ. HRMS
calcd. for C25H29ClINO3 (553.167): 553.0840; found 553.0880.
1-Benzyloxy-3-(tert-butyloxycarbonylamino)-5,6,7,8-tetrahydro-
naphthalene (31): Di-tert-butyl dicarbonate (4.44 g, 20.3 mmol, 1.25
equiv.) was added to a solution of amine 30 (4.10 g, 16.2 mmol) in
1,4-dioxane (90 mL). After the mixture had been stirred for 24 h at
room temperature, the solvent was evaporated and the residue was
recrystallized from ethanol/water (1:1) to give amide 31 (4.70 g,
13.3 mmol, 82%) as light brown needles. Rf ϭ 0.74 (PE/EtOAc,
Cyclization:
A solution of the alkylated product (564 mg,
1.02 mmol) in toluene (17.0 mL) was degassed thoroughly and
5:1), m.p. 123 °C. UV (CH3CN): λmax (lg ε) ϭ 215.5 nm (4.686), treated with nBu3SnH (0.36 mL, 1.28 mmol, 1.25 equiv.) and AIBN
244.5 (4.151), 281.5 (3.324), 287.5 (3.321). IR (KBr): ν˜ (cmϪ1) ϭ
(18 mg, 101 µmol, 0.1 equiv.) After the mixture had been stirred
3295, 2928, 1686, 1612, 1536. 1H NMR (200 MHz, CDCl3): δ ϭ for 2.5 h at 80 °C, the volatiles were removed in vacuo and the
1.52 (s, 9 H, Boc-CH3), 1.76 (mc, 4 H, 6-CH2, 7-CH2), 2.68 (mc, 4 residue was dissolved in Et2O. The solution was washed with 10%
H, 5-CH2, 8-CH2), 5.05 (s, 2 H, OCH2), 6.36 (br. s, 1 H, NH), 6.60,
aq. KF and dried with Na2SO4. The solution was concentrated
6.95 (each s, each 1 H, 2-H, 4-H), 7.30Ϫ7.46 (m, 5 H, o-H, m-H, under reduced pressure and the residue was purified by flash chro-
p-H). 13C NMR (50 MHz, CDCl3): δ ϭ 22.8, 22.9 (C-6, C-7, C-8), matography (PE/EtOAc, 20:1). Benzyl ether 17 (336 mg, 786 µmol,
28.3 (Boc-CH3), 29.8 (C-5), 69.6 (OCH2), 80.2 (Boc-C), 99.9 (C-2),
1640
77%) was obtained as a white solid. Rf ϭ 0.38 (PE/EtOAc, 20:1),
Eur. J. Org. Chem. 2002, 1634Ϫ1645

Novel Analogues and Prodrugs of the Cytotoxic Antibiotic CC-1065
FULL PAPER
m.p. 157 °C. UV (CH3CN): λmax (lg ε) ϭ 220.5 nm (4.596), 254.5 Compound 19: Rf ϭ 0.42 (PE/EtOAc, 10:1), m.p. 186Ϫ188 °C, dec.
(4.101), 297.5 (3.638). IR (KBr): ν˜ (cmϪ1) ϭ 3445, 2939, 1693,
UV (CH3CN): λmax (lg ε) ϭ 219.0 nm (4.651), 252.0 (4.172), 298.0
1
1595, 1480. H NMR (200 MHz, CDCl3): δ ϭ 1.57 (s, 9 H, Boc- (3.718). IR (KBr): ν˜ (cmϪ1) ϭ 3443, 2934, 1688, 1593, 1483. 1H
CH3), 1.68, 1.88 (each mc, each 2 H, 7-CH2, 8-CH2), 2.55Ϫ2.83 NMR (200 MHz, CDCl3): δ ϭ 1.56 (s, 9 H, Boc-CH3), 1.57 (d,
(m, 4 H, 6-CH2, 9-CH2), 3.34 (dd, J ϭ 11.1, 10.5 Hz, 1 H, 10-Ha),
3.50 (dddd, J ϭ 9.1, 10.5, 2.0, 2.1 Hz, 1 H, 1-H), 3.70 (dd, J ϭ 8-CH2), 2.49Ϫ2.88 (m, 4 H, 6-CH2, 9-CH2), 3.44 (ddd, J ϭ 10.1,
11.1, 2.1 Hz, 1 H, 10-Hb), 3.93 (dd, J ϭ 11.8, 9.1 Hz, 1 H, 2-Ha), 2 ϫ 3.5 Hz, 1 H, 1-H), 3.90 (dd, J ϭ 10.1, 10.5 Hz, 1 H, 2-Ha),
J ϭ 7.0 Hz, 3 H, 11-CH3), 1.70, 1.92 (each mc, each 2 H, 7-CH2,
4.14 (br. d, J ϭ 11.8 Hz, 2-Hb), 5.07 (s, 2 H, OCH2), 7.31Ϫ7.47 (m, 4.13 (br. d, J ϭ 10.5 Hz, 2-Hb), 4.47 (dq, J ϭ 3.5, 7.0 Hz, 1 H, 10-
6 H, 4-H, o-H, m-H, p-H). 13C NMR (50 MHz, CDCl3): δ ϭ 22.5, H), 5.07 (s, 2 H, OCH2), 7.30Ϫ7.54 (m, 6 H, 4-H, o-H, m-H, p-H).
22.6, 23.2 (C-6, C-7, C-8), 26.7 (C-9), 28.4 (Boc-CH3), 41.5 (C-1),
45.8 (C-10), 52.5 (C-2), 69.7 (OCH2), 80.7 (Boc-C), 97.1 (C-4),
13C NMR (50 MHz, CDCl3): δ ϭ 22.5, 22.6, 23.3 (C-6, C-7, C-8),
23.4 (C-11), 27.3 (C-9), 28.5 (Boc-CH3), 45.9 (C-1), 49.5 (C-2), 59.4
119.6 (C-9b), 120.3 (C-5a), 127.1 (o-C), 127.6 (p-C), 128.4 (m-C), (C-10), 69.6 (OCH2), 80.4 (Boc-C), 96.7 (C-4), 120.0 (C-9b), 121.2
134.4 (C-9a), 137.4 (i-C), 141.2 (C-3a), 152.5 (CϭO), 157.1 (C-5). (C-5a), 127.1 (o-C), 127.5 (p-C), 128.4 (m-C), 134.2 (C-9a), 137.5
MS (DCI, 200 eV): m/z (%) ϭ 445 (100) [M ϩ NH4]ϩ, 389 (9) [M (i-C), 141.5 (C-3a), 152.2 (CϭO), 156.8 (C-5). MS (EI, 70 eV):
Ϫ C4H9 ϩ NH4]ϩ. C25H30ClNO3 (427.975).
m/z (%) ϭ 441 (20) [M]ϩ, 385 (35) [M Ϫ C4H9 ϩ H]ϩ, 322 (100)
[M Ϫ C4H9 Ϫ C2H4Cl ϩ H]ϩ, 91 (65) [C7H7]ϩ Ϫ C26H32ClNO3
(442.002).
syn-Benzyl Ether 18 and anti-Benzyl Ether 19. Alkylation: In ac-
cordance with the procedure given above, amide 9 (500 mg,
1.04 mmol) was treated with NaH (60% in oil, 100 mg, 2.50 mmol,
2.5 equiv.) and chloride 16 (261 mg, 2.09 mmol, 2.0 equiv.) to give
General Procedure 1. Cleavage of the Benzyl Ether 17؊19: Pd/C
(10%, 1.1 weight-equiv.) and ammonium formate (0.9 weight-
590 mg (1.04 mmol, 99%) of a yellow oil as a mixture of stable equiv.) were added to a solution of the benzyl ether in acetone
1
rotamers. Rf ϭ 0.40, 0.48 (PE/EtOAc, 10:1). H NMR (200 MHz,
CDCl3): δ ϭ 1.34/1.55 (each s, together 9 H, Boc-CH3), 1.76 (mc, (1Ϫ24 h) at the specified temperature (25Ϫ56 °C). The solid was
4 H, 6-CH2, 7-CH2), 1.88/2.05 (each s, together 3 H, 4Ј-CH3), 2.70 removed by filtration through Celite, the Celite was washed thor-
(m, 4 H, 5-CH2, 8-CH2), 3.64Ϫ4.05 (m, 1 H, 1Ј-Ha), 4.43Ϫ4.49 (m, oughly with acetone, and the filtrate was concentrated. Flash chro-
(25 mL/mmol). The mixture was stirred for the specified time
1 H, 1Ј-Hb), 4.95Ϫ5.12 (m, 2 H, OCH2), 5.68Ϫ5.75 (m, 1 H, 2Ј-
H), 6.54Ϫ6.66 (m, 1 H, 3-H), 7.27Ϫ7.44 (m, 5 H, o-H, m-H, p-H).
13C NMR (50 MHz, CDCl3): δ ϭ 22.0, 23.7, 23.8 (C-5, C-6, C-7),
20.9/26.1 (C-4Ј), 28.2/28.4 (Boc-CH3), 37.1 (C-8), 46.4/47.9//48.8
(C-1Ј), 69.7 (OCH2), 80.0/80.5 (Boc-C), 98.6 (C-1), 109.8/110.4 (C-
3), 121.9/122.1 (C-4a), 123.0 (C-2Ј), 126.8 (o-C), 127.7 (2 signals)
(p-C), 128.4 (2 signals) (m-C), 132.0 (C-3Ј), 136.7 (2 signals) (i-C),
140.9, 141.0, 141.3, 142.1 (C-2, C-8a), 153.7/153.9 (CϭO), 156.2/
156.7 (C-4). MS (DCI, 200 eV): m/z (%) ϭ 585 (100) [M ϩ NH4]ϩ,
529 (22) [M Ϫ C4H9 ϩ H]ϩ. C26H31ClINO3 (567.894).
matography (PE/EtOAc, 10:1) provided the product.
3-(tert-Butyloxycarbonyl)-1-chloromethyl-5-hydroxy-1,2,6,7,8,9-
hexahydro-3H-benz[e]indole (7): According to General Procedure 1,
benzyl ether 17 (135 mg, 315 µmol) was treated with 10% Pd/C
(150 mg, 141 µmol) and NH4HCO2 (122 mg, 1.93 mmol) for 2.5 h
at room temperature. Phenol 7 (104 mg, 308 µmol, 97%) was isol-
ated as a brownish solid. Rf ϭ 0.16 (PE/EtOAc, 10:1), m.p.
137Ϫ140 °C. UV (CH3CN): λmax (lg ε) ϭ 219.5 nm (4.585), 252.0
(4.137), 298.0 (3.670). IR (KBr): ν˜ (cmϪ1) ϭ 3394, 2936, 1675,
1
1601, 1416. H NMR (200 MHz, CDCl3): δ ϭ 1.55 (s, 9 H, Boc-
Cyclization:
A
solution of the alkylated product (590 mg,
CH3), 1.67, 1.88 (each mc, each 2 H, 7-CH2, 8-CH2), 2.48Ϫ2.72
(m, 4 H, 6-CH2, 9-CH2), 3.31 (dd, J ϭ 11.1, 10.5 Hz, 1 H, 10-Ha),
3.47 (dddd, J ϭ 9.0, 10.5, 2 ϫ 2.0 Hz, 1 H, 1-H), 3.68 (dd, J ϭ
11.1, 2.0 Hz, 1 H, 10-Hb), 3.90 (dd, J ϭ 11.8, 9.0 Hz, 1 H, 2-Ha),
4.10 (br. d, J ϭ 11.8 Hz, 2-Hb), 6.11 (s, 1 H, OH), 7.33 (br. s, 1 H,
4-H). 13C NMR (50 MHz, CDCl3): δ ϭ 22.5, 22.7 (C-6, C-7, C-8),
1.04 mmol) in toluene (17 mL) was degassed thoroughly and
treated with nBu3SnH (0.40 mL, 1.44 mmol, 1.39 equiv.) and AIBN
(45 mg, 276 µmol, 0.25 equiv.). After the mixture had been stirred
for 3.5 h at 80 °C, all volatile components were removed in vacuo
and the residue was dissolved in Et2O. The solution was washed
with 10% equiv. KF and dried with Na2SO4. The solution was con- 26.7 (C-9), 28.4 (Boc-CH3), 41.5 (C-1), 45.8 (C-10), 52.5 (C-2), 81.2
centrated under reduced pressure and the residue was purified by
flash chromatography (PE/EtOAc, 20:1). The syn-benzyl ether 18
(171 mg, 387 µmol, 37%) and anti-benzyl ether 19 (193 mg, 437
(Boc-C), 100.2 (C-4), 118.0 (C-9b), 119.5 (C-5a), 134.6 (C-9a),
140.7 (C-3a), 152.7 (CϭO), 154.4 (C-5). MS (EI, 70 eV): m/z (%) ϭ
337 (26) [M]ϩ, 281 (77) [M Ϫ C4H9 ϩ H]ϩ, 232 (100) [M Ϫ C4H9
µmol, 42%) were both obtained as white solids containing traces Ϫ CH2Cl ϩ H]ϩ, 188 (42) [M Ϫ Boc Ϫ CH2Cl]ϩ. C18H24ClNO3
of nBu3Sn-compounds.
(337.850): calcd. (%)C 63.99, H 7.16; found C 63.76, H 6.97.
Compound 18: Rf ϭ 0.55 (PE/EtOAc, 10:1), m.p. 179Ϫ180 °C, dec. syn-Phenol 8a: According to General Procedure 1, benzyl ether 18
UV (CH3CN): λmax (lg ε) ϭ 221.0 nm (4.641), 255.5 (4.143), 298.0 (144 mg, 326 µmol) was treated with 10% Pd/C (158 mg, 148 µmol)
(3.685). IR (KBr): ν˜ (cmϪ1) ϭ 3442, 2935, 1692, 1593, 1452. 1H and NH4HCO2 (130 mg, 2.06 mmol) for 1.5 h at 50 °C. Phenol 8a
NMR (200 MHz, CDCl3): δ ϭ 1.20 (d, J ϭ 7.4 Hz, 3 H, 11-CH3), (85 mg, 242 µmol, 74%) was isolated as a white solid. Rf ϭ 0.43
1.57 (s, 9 H, Boc-CH3), 1.58Ϫ1.95 (m, 4 H, 7-CH2, 8-CH2), (PE/EtOAc, 5:1), m.p. 193Ϫ196 °C, dec. UV (CH3CN): λmax (lg
2.50Ϫ2.86 (m, 4 H, 6-CH2, 9-CH2), 3.69 (br. d, J ϭ 9.5 Hz, 1 H,
ε) ϭ 220.0 nm (4.553), 252.5 (4.084), 298.5 (3.622). IR (KBr): ν˜
1-H), 3.89 (dd, J ϭ 11.8, 9.9 Hz, 1 H, 2-Ha), 4.22 (br. d, J ϭ (cmϪ1) ϭ 3383, 2970, 1683, 1603, 1487. 1H NMR (200 MHz,
11.8 Hz, 2-Hb), 4.42 (dq, J ϭ 4.0, 7.0 Hz, 1 H, 10-H), 5.07 (s, 2 H,
CDCl3): δ ϭ 1.18 (d, J ϭ 7.2 Hz, 3 H, 11-CH3), 1.57 (s, 9 H, Boc-
OCH2), 7.31Ϫ7.48 (m, 6 H, 4-H, o-H, m-H, p-H). 13C NMR CH3), 1.66, 1.91 (each mc, each 2 H, 7-CH2, 8-CH2), 2.43Ϫ2.74
(50 MHz, CDCl3): δ ϭ 17.9 (C-11), 22.6, 23.2 (C-6, C-7, C-8), 27.0 (m, 4 H, 6-CH2, 9-CH2), 3.66 (ddd, J ϭ 9.8, 2 ϫ 3.5 Hz, 1 H, 1-
(C-9), 28.5 (Boc-CH3), 45.9 (C-1), 49.4 (C-2), 57.8 (C-10), 69.7
(OCH2), 80.5 (Boc-C), 97.0 (C-4), 119.8, 120.2 (C-5a, C-9b), 127.1 1 H, 2-Hb), 4.40 (dq, J ϭ 3.5, 7.2 Hz, 1 H, 10-H), 5.51 (br. s, 1 H,
(o-C), 127.6 (p-C), 128.4 (m-C), 134.2 (C-9a), 137.4 (i-C), 141.5 (C-
OH), 7.28 (br. s, 1 H, 4-H). 13C NMR (50 MHz, CDCl3): δ ϭ 17.8
H), 3.85 (dd, J ϭ 11.9, 9.8 Hz, 1 H, 2-Ha), 4.18 (br. d, J ϭ 11.9 Hz,
3a), 152.2 (CϭO), 157.0 (C-5). MS (EI, 70 eV): m/z (%) ϭ 441 (C-11), 22.6, 22.8 (C-6, C-7, C-8), 26.9 (C-9), 28.5 (Boc-CH3), 45.9
(18) [M]ϩ, 385 (36) [M Ϫ C4H9 ϩ H]ϩ, 322 (38) [M Ϫ C4H9
C2H4Cl ϩ H]ϩ, 91 (100) [C7H7]ϩ.
Ϫ
(C-1), 49.4 (C-2), 57.8 (C-10), 81.1 (Boc-C), 100.3 (C-4), 117.9 (C-
5a), 119.8 (C-9b), 134.5 (C-9a), 141.1 (C-3a), 152.4 (CϭO), 154.3
Eur. J. Org. Chem. 2002, 1634Ϫ1645
1641

L. F. Tietze, T. Herzig, T. Feuerstein, I. Schuberth
(C-5). MS (DCI, 200 eV): m/z (%) ϭ 369 (100) [M ϩ NH4]ϩ. (p-C), 128.5 (m-C), 131.4 (C-2ЈЈ), 131.7, 131.9 (C-2Ј, C-5Ј), 133.4
FULL PAPER
C19H26ClNO3 (351.877): calcd. C 64.85, H 7.45; found C 64.64,
H 7.15.
(C-7aЈ), 134.4 (C-9a), 136.8 (C-7aЈЈ), 137.4 (i-C), 141.9 (C-3a),
155.9 (C-5), 159.7 (CONHЈЈ), 159.9 (CONHЈ). MS (DCI, 200 eV):
m/z (%) ϭ 646 (100) [M ϩ NH4]ϩ, 629 (16) [M ϩ H]ϩ, 610 (19)
[M Ϫ HCl ϩ NH4]ϩ. C38H33ClN4O3 (629.163).
anti-Phenol 8b: According to General Procedure 1, benzyl ether 19
(183 mg, 414 µmol) was treated with 10% Pd/C (201 mg, 189 µmol)
and NH4HCO2 (165 mg, 2.62 mmol) for 2 h at 50 °C. Phenol 8b syn-Benzyl Ether 23: According to General Procedure 2, syn-benzyl
(117 mg, 333 µmol, 80%) was isolated as a brownish solid. Rf ϭ
0.35 (PE/EtOAc, 5:1), m.p. 168Ϫ171 °C, dec. UV (CH3CN): λmax
(lg ε) ϭ 217.5 nm (4.537), 250.0 (4.113), 298.5 (3.647). IR (KBr):
ether 18 (70 mg, 158 µmol) was stirred in 4  HCl/1,4-dioxane for
1 h. The residue was treated with EDC (93 mg, 480 µmol) and acid
20 (50 mg, 160 µmol). Indole derivative 23 (46 mg, 72 µmol, 45%)
ν˜ (cmϪ1) ϭ 3379, 2974, 1674, 1602, 1487. 1H NMR (200 MHz, was obtained as a white powder. Rf ϭ 0.93 (PE/acetone/MeOH,
CDCl3): δ ϭ 1.54Ϫ1.76 (m, 14 H, 7-CH2/8-CH2, 11-CH3, Boc- 10:6:1). UV (CH3CN): λmax (lg ε) ϭ 207.5 nm (4.852), 319.0
CH3), 1.98 (mc, 2 H, 7-CH2/8-CH2), 2.43Ϫ2.76 (m, 4 H, 6-CH2, 9- (4.705). IR (KBr): ν˜ (cmϪ1) ϭ 3419, 3282, 2930, 1615, 1523. 1H
CH2), 3.40 (mc, 1 H, 1-H), 3.86 (dd, J ϭ 11.3, 9.5 Hz, 1 H, 2-Ha),
NMR (500 MHz, [D6]DMSO): δ ϭ 1.09 (d, J ϭ 7.0 Hz, 3 H, 11-
4.11 (br. d, J ϭ 11.3 Hz, 1 H, 2-Hb), 4.44 (dq, J ϭ 3.6, 7.0 Hz, 1 CH3), 1.61, 1.83 (each mc, each 2 H, 7-CH2, 8-CH2), 2.50Ϫ2.57
H, 10-H), 5.05 (br. s, 1 H, OH), 7.32 (br. s, 1 H, 4-H). 13C NMR
(m, 1 H, 9-Ha), 2.64Ϫ2.74 (m, 3 H, 6-CH2, 9-Hb), 3.86 (mc, 1 H,
(50 MHz, CDCl3): δ ϭ 22.5, 22.6, 22.8 (C-6, C-7, C-8), 23.4 (C-
1-H), 4.57 (mc, 3 H, 2-CH2, 10-H), 5.08 (s, 2 H, OCH2), 7.07 (t,
11), 27.2 (C-9), 28.5 (Boc-CH3), 45.9 (C-1), 49.5 (C-2), 59.4 (C-10), J ϭ 8.0 Hz, 1 H, 5ЈЈ-H), 7.17 (s, 1 H, 3Ј-H), 7.21 (t, J ϭ 8.0 Hz, 1
80.7 (Boc-C), 100.1 (C-4), 117.8 (C-5a), 120.3 (C-9b), 134.4 (C-9a), H, 6ЈЈ-H), 7.32 (t, J ϭ 7.5 Hz, 1 H, p-H), 7.38 (t, J ϭ 8.0 Hz, 2 H,
141.1 (C-3a), 152.4 (CϭO), 154.1 (C-5). MS (DCI, 200 eV): m/z
m-H), 7.42 (s, 1 H, 3ЈЈ-H), 7.45 (d, J ϭ 7.5 Hz, 2 H, o-H), 7.49 (d,
(%) ϭ 369 (100) [M ϩ NH4]ϩ, 313 (92) [M Ϫ C4H9 ϩ NH4]ϩ. J ϭ 8.1 Hz, 2 H, 6Ј-H, 7ЈЈ-H), 7.59 (d, J ϭ 8.1 Hz, 1 H, 7Ј-H), 7.67
C19H26ClNO3 (351.877): calcd. (%) C 64.85, H 7.45; found C 64.98,
H 7.29.
(d, J ϭ 8.0 Hz, 1 H, 4ЈЈ-H), 7.78 (br. s, 1 H, 4-H), 8.24 (s, 1 H, 4Ј-
H), 10.15 (s, 1 H, CONH), 11.69 (s, 2 H, indole-NH). 13C NMR
(125 MHz, [D6]DMSO): δ ϭ 17.9 (C-11), 22.0 (2 signals), 23.0 (C-
6, C-7, C-8), 26.0 (C-9), 46.1 (C-1), 51.7 (C-2), 58.1 (C-10), 69.2
(OCH2), 99.4 (C-4), 103.4 (C-3ЈЈ), 105.4 (C-3Ј), 112.1, 112.3 (C-7Ј,
C-7ЈЈ), 113.0 (C-6ЈЈ), 119.3, 119.8 (C-4Ј, C-6Ј), 121.1, 121.4, 121.5
(C-5a, C-9b, C-4ЈЈ), 123.5 (C-5ЈЈ), 127.0, 127.1 (C-3aЈ, C-3aЈЈ),
127.1 (o-C), 127.6 (p-C), 128.4 (m-C), 131.2 (C-2ЈЈ), 131.7, 131.9
(C-2Ј, C-5Ј), 133.3 (C-7aЈ), 134.3 (C-9a), 136.7 (C-7aЈЈ), 137.2 (i-
C), 141.7 (C-3a), 155.9 (C-5), 159.5 (CONHЈЈ), 159.7 (CONHЈ).
MS (DCI, 200 eV): m/z (%) ϭ 660 (100) [M ϩ NH4]ϩ, 624 (9) [M
Ϫ HCl ϩ NH4]ϩ. C39H35ClN4O3 (643.190).
General Procedure 2: Coupling of 7, 8, 17؊19 with 20: The sub-
strates were dissolved in 4  HCl/1,4-dioxane (25 mL/mmol) and
stirred at room temperature for the specified time. The solution was
concentrated in vacuo, and the residue was thoroughly dried in
vacuo. It was then dissolved in dry degassed DMF (17 mL/mmol)
and treated with 3.0 equiv. of EDC and 1.0 equiv. of bisindolyl-
carboxylic acid 20.[9] After the mixture had been stirred for 18 h at
room temperature, a small amount of silica gel was added, and the
solvents were evaporated to dryness. Flash chromatography (solv-
ent gradient PE/acetone, 5:1 // PE/acetone, 5:3 // PE/acetone/
MeOH, 10:6:1) provided the title compound, which was isolated
by concentration of the eluate to a small volume and addition of
petroleum ether. The precipitate was collected by a glass frit,
washed with Et2O and a small amount of acetone, and thor-
oughly dried.
anti-Benzyl Ether 24: According to General Procedure 2, anti-
benzyl ether 19 (70 mg, 158 µmol) was stirred in 4  HCl/1,4-diox-
ane for 1 h. The residue was treated with EDC (93 mg, 480 µmol)
and acid 20 (50 mg, 160 µmol). Indole derivative 24 (55 mg,
86 µmol, 54%) was obtained as a beige powder. Rf ϭ 0.94 (PE/
acetone/MeOH, 10:6:1). UV (CH3CN): λmax (lg ε) ϭ 205.5 nm
(4.796), 317.5 (4.624). IR (KBr): ν˜ (cmϪ1) ϭ 3410, 3286, 2930,
1619, 1543. 1H NMR (500 MHz, [D6]DMSO): δ ϭ 1.58 (d, J ϭ
6.9 Hz, 3 H, 11-CH3), 1.59, 1.87 (each mc, each 2 H, 7-CH2, 8-
CH2), 2.54 (mc, 1 H, 9-Ha), 2.66Ϫ2.77 (m, 3 H, 6-CH2, 9-Hb), 3.77
(d, J ϭ 9.2 Hz, 1 H, 1-H), 4.49Ϫ4.56 (m, 2 H, 2-CH2), 4.61 (dq,
J ϭ 2.3, 6.8 Hz, 1 H, 10-H), 5.08 (s, 2 H, OCH2), 7.07 (t, J ϭ
8.1 Hz, 1 H, 5ЈЈ-H), 7.22 (t, J ϭ 8.1 Hz, 1 H, 6ЈЈ-H), 7.23 (s, 1 H,
3Ј-H), 7.31 (t, J ϭ 7.8 Hz, 1 H, p-H), 7.39 (t, J ϭ 7.8 Hz, 2 H, m-
H), 7.42 (s, 1 H, 3ЈЈ-H), 7.44Ϫ7.48 (m, 4 H, 6Ј-H, 7ЈЈ-H, o-H), 7.57
(d, J ϭ 7.9 Hz, 1 H, 7Ј-H), 7.67 (d, J ϭ 8.0 Hz, 1 H, 4ЈЈ-H), 7.85
(br. s, 1 H, 4-H), 8.25 (s, 1 H, 4Ј-H), 10.14 (s, 1 H, CONH), 11.66 (s,
1 H, indole-NH), 11.68 (s, 1 H, indole-NH). 13C NMR (125 MHz,
[D6]DMSO): δ ϭ 22.0, 22.1, 23.1 (C-6, C-7, C-8), 23.4 (C-11), 26.1
(C-9), 45.7 (C-1), 51.2 (C-2), 60.7 (C-10), 69.2 (OCH2), 99.2 (C-4),
103.4 (C-3ЈЈ), 105.5 (C-3Ј), 112.1, 112.3 (C-7Ј, C-7ЈЈ), 113.0 (C-6ЈЈ),
119.2, 119.8 (C-4Ј, C-6Ј), 121.1, 121.5, 122.2 (C-5a, C-9b, C-4ЈЈ),
123.5 (C-5ЈЈ), 127.1, 127.2 (C-3aЈ, C-3aЈЈ), 127.1 (o-C), 127.6 (p-C),
128.4 (m-C), 131.3 (C-2ЈЈ), 131.6, 131.9 (C-2Ј, C-5Ј), 133.3 (C-7aЈ),
134.0 (C-9a), 136.7 (C-7aЈЈ), 137.3 (i-C), 141.9 (C-3a), 155.6 (C-5),
159.5, 159.6 (CONHЈ, CONHЈЈ). MS (DCI, 200 eV): m/z (%) ϭ
660 (59) [M ϩ NH4]ϩ, 643 (18) [M ϩ H]ϩ, 624 (100) [M Ϫ HCl ϩ
NH4]ϩ, 607 (24) [M Ϫ HCl ϩ H]ϩ. C39H35ClN4O3 (643.190).
5-Benzyloxy-1-chloromethyl-3-[(5Ј-{[(1H-indol-2ЈЈ-yl)carbonyl]-
amino}-1H-indol-2Ј-yl)carbonyl]-1,2,6,7,8,9-hexahydro-3H-benz-
[e]indole (22): According to General Procedure 2, benzyl ether 17
(70 mg, 160 µmol) was stirred in 4  HCl/1,4-dioxane for 1 h. The
residue was treated with EDC (93 mg, 480 µmol) and acid 20
(51 mg, 160 µmol). Indole derivative 22 (45 mg, 72 µmol, 45%) was
obtained as a yellowish powder. Rf ϭ 0.68 (PE/acetone/MeOH,
10:6:1). UV (CH3CN): λmax (lg ε) ϭ 207.0 nm (4.810), 319.0
(4.666). IR (KBr): ν˜ (cmϪ1) ϭ 3417, 3282, 2930, 1618, 1523. 1H
NMR (500 MHz, [D6]DMSO): δ ϭ 1.60, 1.83 (each mc, each 2 H,
7-CH2, 8-CH2), 2.51 (mc, 1 H, 9-Ha), 2.63Ϫ2.72 (m, 3 H, 6-CH2,
9-Hb), 3.67 (dd, J ϭ 11.2, 9.5 Hz, 1 H, 10-Ha), 3.76 (mc, 1 H, 1-
H), 3.87 (dd, J ϭ 11.2, 2.6 Hz, 1 H, 10-Hb), 4.45 (d, J ϭ 11.5 Hz,
1 H, 2-Ha), 4.58 (dd, 2 ϫ J ϭ 11.5 Hz, 2-Hb), 5.06 (s, 2 H, OCH2),
7.06 (t, J ϭ 7.9 Hz, 1 H, 5ЈЈ-H), 7.13 (s, 1 H, 3Ј-H), 7.22 (t, J ϭ
7.9 Hz, 1 H, 6ЈЈ-H), 7.31 (t, J ϭ 8.1 Hz, 1 H, p-H), 7.38 (t, J ϭ
8.0 Hz, 2 H, m-H), 7.41 (s, 1 H, 3ЈЈ-H), 7.44 (d, J ϭ 7.8 Hz, 2 H,
o-H), 7.49 (d, J ϭ 7.8 Hz, 2 H, 6Ј-H, 7ЈЈ-H), 7.57 (d, J ϭ 7.8 Hz,
1 H, 7Ј-H), 7.66 (d, J ϭ 7.9 Hz, 1 H, 4ЈЈ-H), 7.77 (br. s, 1 H, 4-H),
8.19 (s, 1 H, 4Ј-H), 10.14 (s, 1 H, CONH), 11.65 (s, 2 H, indole-
NH). 13C NMR (125 MHz, [D6]DMSO): δ ϭ 22.1, 23.2 (C-6, C-7,
C-8), 25.9 (C-9), 41.1 (C-1), 46.8 (C-10), 54.5 (C-2), 69.3 (OCH2),
99.4 (C-4), 103.5 (C-3ЈЈ), 105.5 (C-3Ј), 112.3, 112.4 (C-7Ј, C-7ЈЈ),
113.1 (C-6ЈЈ), 119.4, 119.9 (C-4Ј, C-6Ј), 121.3, 121.4, 121.7 (C-5a,
C-9b, C-4ЈЈ), 123.7 (C-5ЈЈ), 127.2 (C-3aЈ, C-3aЈЈ), 127.2 (o-C), 127.8
Phenol 25: According to General Procedure 2, amide 7 (65 mg, 192
µmol) was stirred in 4  HCl/1,4-dioxane for 1 h. The residue was
1642
Eur. J. Org. Chem. 2002, 1634Ϫ1645

Novel Analogues and Prodrugs of the Cytotoxic Antibiotic CC-1065
FULL PAPER
treated with EDC (111 mg, 577 µmol) and acid 20 (61 mg, 192
µmol). Phenol 25 (16 mg, 30 µmol, 15%) was obtained as a grayish
1 H, 3ЈЈ-H), 7.57 (d, J ϭ 8.1 Hz, 1 H, 7Ј-H), 7.60 (d, J ϭ 8.1 Hz,
1 H, 7ЈЈ-H), 7.67 (d, J ϭ 8.0 Hz, 2 H, 6Ј-H, 4ЈЈ-H), 7.78 (br. s, 1
powder. Rf ϭ 0.69 (PE/acetone/MeOH, 10:6:1). UV (CH3CN): λmax H, 4-H), 8.38 (s, 1 H, 4Ј-H), 10.22 (s, 1 H, CONH), 11.52 (s, 1
(lg ε) ϭ 205.0 nm (4.534), 316.5 (4.390). IR (KBr): ν˜ (cmϪ1) ϭ
3428, 3275, 2932, 1658, 1521. 1H NMR (500 MHz, [D7]DMF): δ ϭ
1.66, 1.88 (each mc, each 2 H, 7-CH2, 8-CH2), 2.55 (mc, 1 H, 9-
H, indole-NH), 11.68 (s, 1 H, indole-NH). 13C NMR (125 MHz,
[D7]DMF): δ ϭ 23.2, 23.3, 23.8, 23.9 (C-6, C-7, C-8, C-11), 27.2
(C-9), 47.0 (C-1), 52.2 (C-2), 61.5 (C-10), 102.8 (C-4), 103.9 (C-
Ha), 2.68Ϫ2.78 (m, 3 H, 6-CH2, 9-Hb), 3.70 (dd, J ϭ 11.2, 9.7 Hz, 3ЈЈ), 106.2 (C-3Ј), 112.7, 113.0 (C-7Ј, C-7ЈЈ), 113.5 (C-4Ј), 119.6 (C-
1 H, 10-Ha), 3.79 (mc, 1 H, 1-H), 3.94 (dd, J ϭ 11.2, 2.6 Hz, 1 H, 6Ј), 120.1 (C-5a), 120.5 (C-5ЈЈ), 121.4 (C-9b), 122.3 (C-4ЈЈ), 124.2
10-Hb), 4.59 (d, J ϭ 11.5 Hz, 1 H, 2-Ha), 4.65 (dd, J ϭ 11.5, (C-6ЈЈ), 128.5 (2 signals) (C-3aЈ, C-3aЈЈ), 132.7 (C-2Ј), 132.9, 133.1
10.3 Hz, 2-Hb), 7.10 (t, J ϭ 8.0 Hz, 1 H, 5ЈЈ-H), 7.20 (s, 1 H, 3Ј- (C-5Ј, C-2ЈЈ), 134.3, 134.7 (C-9a, C-7aЈ), 137.8 (C-7aЈЈ), 142.7 (C-
H), 7.25 (t, J ϭ 8.0 Hz, 1 H, 6ЈЈ-H), 7.51 (s, 1 H, 3ЈЈ-H), 7.58 (d, 3a), 155.9 (C-5), 160.3, 160.5 (CONHЈ, CONHЈЈ). MS (ESI): m/z
J ϭ 8.2 Hz, 1 H, 7Ј-H), 7.61 (d, J ϭ 8.2 Hz, 1 H, 7ЈЈ-H), 7.69 (d, (%) ϭ 552 (100) [M Ϫ H]Ϫ, 516 (41) [M Ϫ HCl Ϫ H]ϩ.
J ϭ 8.0 Hz, 1 H, 6Ј-H), 7.71 (d, J ϭ 8.0 Hz, 1 H, 4ЈЈ-H), 7.72 (br. C32H29ClN4O3 (553.065).
s, 1 H, 4-H), 8.36 (s, 1 H, 4Ј-H), 10.24 (s, 1 H, CONH), 11.55 (s, 1
H, indole-NH), 11.70 (s, 1 H, indole-NH). 13C NMR (125 MHz,
[D7]DMF): δ ϭ 23.2 (2 signals), 23.4 (C-6, C-7, C-8), 26.7 (C-9),
42.6 (C-1), 47.2 (C-10), 55.4 (C-2), 102.8 (C-4), 103.9 (C-3ЈЈ), 106.1
(C-3Ј), 112.8, 113.0 (C-7Ј, C-7ЈЈ), 113.4 (C-4Ј), 119.6 (C-6Ј), 120.3,
120.4 (C-5a, C-9b), 120.6 (C-5ЈЈ), 122.4 (C-4ЈЈ), 124.3 (C-6ЈЈ),
128.4, 128.5 (C-3aЈ, C-3aЈЈ), 132.7 (C-2Ј), 133.0, 133.1 (C-5Ј, C-
2ЈЈ), 134.3, 134.9 (C-9a, C-7aЈ), 137.9 (C-7aЈЈ), 142.5 (C-3a), 156.2
(C-5), 160.5, 160.6 (CONHЈ, CONHЈЈ). MS (ESI): m/z (%) ϭ 537
(100) [M Ϫ H]Ϫ, 501 (71) [M Ϫ HCl Ϫ H]ϩ. C31H27ClN4O3
(539.038).
General Procedure 3: Glycosidation and coupling of phenols 7, 8a,
and 8b with 20. The phenol was dissolved in dry CH2Cl2 (50 mL/
mmol) and treated with molecular sieves (4A, 6.90 g/mmol) and
˚
tetraacetyl trichloroacetimidate 21 (1.05 equiv.).[20] The mixture
was stirred for 30 min at room temperature, and BF3·OEt2
(7.7 equiv.) was added dropwise at Ϫ10 °C. Stirring was continued
for 1 h at this temperature, and the mixture was then allowed to
reach room temperature and stirred for further 4.5 h. The solution
was separated from the molecular sieves by a transfer cannula, and
the molecular sieves were washed three times with CH2Cl2. The
combined solutions were concentrated in vacuo, and the residue
was thoroughly dried in vacuo. The residue was dissolved in dry
degassed DMF (17 mL/mmol) and treated with EDC (2.2 equiv.)
and acid 20 (0.9 equiv.).[9] After the mixture had been stirred for
18 h, a small amount of silica gel was added, and the solvents were
evaporated to dryness. Flash chromatography (PE/EtOAc, 2:1 //
PE/EtOAc, 1:2 // PE/acetone/MeOH, 10:6:1) gave the peracetylated
syn-Phenol 26: According to General Procedure 2, syn-amide 8a
(64 mg, 182 µmol) was stirred in 4  HCl/1,4-dioxane for 1 h. The
residue was treated with EDC (111 mg, 577 µmol) and acid 20
(58 mg, 182 µmol). syn-Phenol 26 (10 mg, 18 µmol, 10%) was ob-
tained as a beige powder. Rf ϭ 0.76 (PE/acetone/MeOH, 10:6:1).
UV (CH3CN): λmax (lg ε) ϭ 206.0 nm (4.453), 318.5 (4.363). IR
(KBr): ν˜ (cmϪ1) ϭ 3426, 3291, 2932, 1621. 1H NMR (500 MHz, galactosides, which were dissolved in dry MeOH (40 mL/mmol)
[D7]DMF): δ ϭ 1.16 (d, J ϭ 7.2 Hz, 3 H, 11-CH3), 1.65, 1.88 (each and treated with a solution of NaOMe in MeOH (5.4 , 2.0 equiv.).
mc, each 2 H, 7-CH2, 8-CH2), 2.51Ϫ2.57 (m, 1 H, 9-Ha), 2.69Ϫ2.76
Water (20 mL/mmol) was added after 4 h of stirring, and the pre-
(m, 3 H, 6-CH2, 9-Hb), 3.88 (d, J ϭ 10.1 Hz, 1 H, 1-H), 4.60Ϫ4.64 cipitate was collected with a glass frit, washed with water, and thor-
(m, 2 H, 2-Ha, 10-H), 4.70 (d, J ϭ 10.8 Hz, 1 H, 2-Hb), 7.10 (t,
J ϭ 8.0 Hz, 1 H, 5ЈЈ-H), 7.23 (s, 1 H, 3Ј-H), 7.25 (t, J ϭ 8.0 Hz, 1
H, 6ЈЈ-H), 7.51 (s, 1 H, 3ЈЈ-H), 7.56 (d, J ϭ 8.0 Hz, 1 H, 7Ј-H),
7.59 (d, J ϭ 8.0 Hz, 1 H, 7ЈЈ-H), 7.68 (d, J ϭ 8.1 Hz, 1 H, 4ЈЈ-H),
7.71 (d, J ϭ 8.0 Hz, 1 H, 6Ј-H), 7.72 (br. s, 1 H, 4-H), 8.39 (s, 1 H,
4Ј-H), 10.22 (s, 1 H, CONH), 11.57 (s, 1 H, indole-NH), 11.67 (s,
1 H, indole-NH). 13C NMR (125 MHz, [D7]DMF): δ ϭ 18.2 (C-
11), 23.2 (2 signals), 23.9 (C-6, C-7, C-8), 27.0 (C-9), 47.4 (C-1),
52.5 (C-2), 58.9 (C-10), 103.0 (C-4), 103.9 (C-3ЈЈ), 106.1 (C-3Ј),
112.8, 113.0 (C-7Ј, C-7ЈЈ), 113.5 (C-4Ј), 119.8 (C-6Ј), 120.4, 120.5
(2 signals) (C-5a, C-9b, C-5ЈЈ), 122.3 (C-4ЈЈ), 124.2 (C-6ЈЈ), 128.4,
128.5 (C-3aЈ, C-3aЈЈ), 132.5 (C-2Ј), 133.0, 133.1 (C-5Ј, C-2ЈЈ), 134.3,
134.9 (C-9a, C-7aЈ), 137.9 (C-7aЈЈ), 142.6 (C-3a), 156.2 (C-5),
160.4, 160.5 (CONHЈ, CONHЈЈ). MS (ESI): m/z (%) ϭ 1104 (33)
[2 M Ϫ 2H]2Ϫ, 552 (100) [M Ϫ H]Ϫ, 516 (54) [M Ϫ HCl Ϫ H]ϩ.
C32H29ClN4O3 (553.065).
oughly dried to provide the desired prodrugs.
Galactoside 5: According to General Procedure 3, phenol 7
(100 mg, 296 µmol) was treated with tetraacetyl trichloroacetimid-
ate 21 (152 mg, 304 µmol), BF3·OEt2 (0.30 mL, 2.28 mmol), and
molecular sieves (2.06 g), followed by EDC (128 mg, 658 µmol) and
acid 20 (83 mg, 263 µmol) to give 116 mg of the peracetylated gal-
actoside (133 µmol), which was treated with NaOMe/MeOH (5.4
, 49 µL, 266 µmol). Galactoside 5 (51 mg, 73 µmol, 25%) was
obtained as a beige powder, as a mixture of diastereomers. Rf (in-
termediate product) ϭ 0.65 (PE/acetone/MeOH, 10:6:1). UV
(CH3CN): λmax (lg ε) ϭ 206.5 nm (4.694), 317.0 (4.619). IR (KBr):
ν˜ (cmϪ1) ϭ 3405, 3289, 2929, 1616, 1519. 1H NMR (500 MHz,
[D7]DMF): δ ϭ 1.64, 1.86 (each mc, each 2 H, 7-CH2, 8-CH2),
2.53Ϫ2.58 (m, 1 H, 9-Ha), 2.71Ϫ2.79 (m, 3 H, 6-CH2, 9-Hb),
3.58Ϫ3.63 (m, 1 H, 6*-Ha), 3.69 (mc, 1 H, 6*-Hb), 3.75Ϫ3.79 (m,
2 H, 1-H, 10-Ha), 3.82 (m, 2 H, 3*-H, 5*-H), 3.95Ϫ4.00 (m, 2 H,
10-Hb, 2*-H), 4.60Ϫ4.75 (m, 3 H, 2-CH2, 4*-H), 4.90 (d, J ϭ
8.0 Hz, 1 H, 1*-H), 7.10 (t, J ϭ 8.1 Hz, 1 H, 5ЈЈ-H), 7.20/7.22 (each
s, together 1 H, 3Ј-H), 7.26 (t, J ϭ 8.1 Hz, 1 H, 6ЈЈ-H), 7.52 (s, 1
anti-Phenol 27: According to General Procedure 2, anti-amide 8b
(64 mg, 182 µmol) was stirred in 4  HCl/1,4-dioxane for 1 h. The
residue was treated with EDC (111 mg, 577 µmol) and acid 20
(58 mg, 182 µmol). anti-Phenol 27 (23 mg, 42 µmol, 23%) was ob- H, 3ЈЈ-H), 7.59Ϫ7.63 (m, 2 H, 7Ј-H, 7ЈЈ-H), 7.71 (mc, 2 H, 6Ј-H,
tained as a beige powder. Rf ϭ 0.77 (PE/acetone/MeOH, 10:6:1).
UV (CH3CN): λmax (lg ε) ϭ 205.0 nm (4.741), 318.0 (4.646). IR
4ЈЈ-H), 8.00 (br. s, 1 H, 4-H), 8.38 (s, 1 H, 4Ј-H), 10.22 (s, 1 H,
CONH), 11.63 (s, 1 H, indole-NH), 11.67 (s, 1 H, indole-NH). 13C
(KBr): ν˜ (cmϪ1) ϭ 3428, 3279, 2930, 1655, 1521. 1H NMR NMR (125 MHz, [D7]DMF): δ ϭ 23.0 (2 signals), 23.8, 23.9 (C-6,
(500 MHz, [D7]DMF): δ ϭ 1.63, 1.90 (each mc, each 2 H, 7-CH2,
C-7, C-8), 26.8 (C-9), 42.4/42.6 (C-1), 47.0 (C-10), 55.3/55.5 (C-2),
8-CH2), 1.66 (d, J ϭ 6.9 Hz, 3 H, 11-CH3), 2.51Ϫ2.57 (m, 1 H, 9- 61.4 (C-6*), 69.2/69.3 (C-4*), 71.9/72.0 (C-2*), 74.8/74.9 (C-3*),
Ha), 2.71Ϫ2.75 (m, 3 H, 6-CH2, 9-Hb), 3.80 (d, J ϭ 10.0 Hz, 1 H, 76.3 (C-5*), 102.9/103.1 (C-1*), 103.3 (C-4), 103.9 (C-3ЈЈ), 106.1
1-H), 4.59Ϫ4.68 (m, 3 H, 2-CH2, 10-H), 7.10 (t, J ϭ 7.9 Hz, 1 H, (C-3Ј), 112.8, 113.0 (C-7Ј, C-7ЈЈ), 113.5 (2 signals) (C-4Ј), 119.7 (C-
5ЈЈ-H), 7.25 (t, J ϭ 8.0 Hz, 1 H, 6ЈЈ-H), 7.28 (s, 1 H, 3Ј-H), 7.52 (s,
6Ј), 120.5 (C-5ЈЈ), 122.3 (C-4ЈЈ), 123.2 (2 signals) (C-5a, C-9b),
Eur. J. Org. Chem. 2002, 1634Ϫ1645
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L. F. Tietze, T. Herzig, T. Feuerstein, I. Schuberth
FULL PAPER
124.2 (C-6ЈЈ), 128.3, 128.5 (C-3aЈ, C-3aЈЈ), 132.6 (2 signals) (C-2Ј), (C-6, C-7, C-8), 24.3 (C-11), 27.2 (C-9), 46.2 (C-1), 51.4/52.5 (C-
133.0, 133.1 (C-5Ј, C-2ЈЈ), 134.3, 134.9 (C-9a, C-7aЈ), 137.9 (C- 2), 60.4 (C-10), 61.4 (C-6*), 69.8 (2 signals) (C-4*), 71.8/72.0 (C-
7aЈЈ), 142.7 (C-3a), 156.4/156.5 (C-5), 160.5, 160.8 (CONHЈ, 2*), 74.1 (2 signals) (C-3*), 75.4 (C-5*), 102.9/103.2 (C-1*), 104.5
CONHЈЈ). MS (ESI): m/z (%) ϭ 724 (100) [M ϩ Na]ϩ, 700 (100) (C-4), 104.6 (C-3ЈЈ), 105.6 (C-3Ј), 112.9, 113.0 (C-7Ј, C-7ЈЈ), 113.5/
[M Ϫ H]Ϫ. C37H37ClN4O8 (701.184).
113.6 (C-4Ј), 119.9 (2 signals) (C-6Ј), 120.3 (C-5ЈЈ), 121.2 (C-4ЈЈ),
121.6, 121.7, 122.9 (C-5a, C-9b), 124.3 (C-6ЈЈ), 128.5 (C-3aЈ, C-
3aЈЈ), 132.5, 132.6 (C-2Ј), 133.0 (2 signals), 133.1 (C-5Ј, C-2ЈЈ),
134.3, 134.6 (C-9a, C-7aЈ), 137.8 (C-7aЈЈ), 142.9 (C-3a), 156.2 (2
signals) (C-5), 160.5 (2 signals) (CONHЈ, CONHЈЈ). MS (ESI):
m/z (%) ϭ 737 (100) [M ϩ Na]ϩ. C38H39ClN4O8 (715.211).
syn-Galactoside 6a: According to General Procedure 3, phenol 8a
(105 mg, 299 µmol) was treated with tetraacetyl trichloroacetimid-
ate 21 (154 mg, 306 µmol), BF3·OEt2 (304 µL, 2.30 mmol), and mo-
lecular sieves (2.07 g), followed by EDC (129 mg, 663 µmol) and
acid 20 (83 mg, 264 µmol) to give 190 mg of the peracetylated gal-
actoside (215 µmol), which was treated with NaOMe/MeOH (5.4
, 79 µL, 430 µmol). syn-Galactoside 6a (65 mg, 91 µmol, 42%)
was obtained as a gray powder, as a mixture of diastereomers. Rf
(intermediate product) ϭ 0.41 (PE/EtOAc, 1:1). UV (CH3CN):
λmax (lg ε) ϭ 207.0 nm (4.643), 316.5 (4.569). IR (KBr): ν˜ (cmϪ1) ϭ
3406, 2931, 1619, 1525, 1464. 1H NMR (500 MHz, [D7]DMF): δ ϭ
1.16/1.19 (each d, J ϭ 7.3 Hz, together 3 H, 11-CH3), 1.64, 1.86
(each mc, each 2 H, 7-CH2, 8-CH2), 2.53Ϫ2.59 (m, 1 H, 9-Ha),
2.70Ϫ2.80 (m, 3 H, 6-CH2, 9-Hb), 3.63 (mc, 1 H, 6*-Ha), 3.68Ϫ3.72
(m, 1 H, 6*-Hb), 3.77 (mc, 1 H, 1-H), 3.82Ϫ3.86 (m, 2 H, 3*-H,
5*-H), 3.96Ϫ4.00 (m, 1 H, 2*-H), 4.63Ϫ4.75 (m, 4 H, 2-CH2, 10-
H, 4*-H), 4.92 (d, J ϭ 7.5 Hz, 1 H, 1*-H), 7.11 (t, J ϭ 8.1 Hz, 1
H, 5ЈЈ-H), 7.25Ϫ7.28 (m, 2 H, 3Ј-H, 6ЈЈ-H), 7.54 (s, 1 H, 3ЈЈ-H),
7.61Ϫ7.64 (m, 2 H, 7Ј-H, 7ЈЈ-H), 7.69 (d, J ϭ 8.0 Hz, 1 H, 4ЈЈ-H),
7.72 (d, J ϭ 8.1 Hz, 1 H, 6Ј-H), 8.02 (br. s, 1 H, 4-H), 8.39 (d, J ϭ
2.0 Hz, 1 H, 4Ј-H), 10.24 (s, 1 H, CONH), 11.66Ϫ11.70 (m, 2 H,
indole-NH). 13C NMR (125 MHz, [D7]DMF): δ ϭ 18.2/18.3 (C-
11), 23.0 (2 signals), 23.8, 23.9 (C-6, C-7, C-8), 27.0 (C-9), 47.4 (C-
1), 52.7 (C-2), 58.7 (2 signals) (C-10), 61.4 (C-6*), 69.2 (2 signals)
(C-4*), 71.8/72.0 (C-2*), 74.8 (2 signals) (C-3*), 76.3 (C-5*), 102.9/
103.0 (C-1*), 103.4 (C-4), 103.9 (C-3ЈЈ), 106.3 (C-3Ј), 112.9, 113.0
(C-7Ј, C-7ЈЈ), 113.5/113.6 (C-4Ј), 119.8 (C-6Ј), 120.5 (C-5ЈЈ), 122.3
(C-4ЈЈ), 123.2 (2 signals), 123.4 (C-5a, C-9b), 124.2 (C-6ЈЈ), 128.3,
128.4 (C-3aЈ, C-3aЈЈ), 132.4 (2 signals) (C-2Ј), 133.0 (2 signals) (C-
5Ј, C-2ЈЈ), 134.4, 134.5, 134.9 (C-9a, C-7aЈ), 137.8 (C-7aЈЈ), 142.7
(C-3a), 156.5 (2 signals) (C-5), 160.5, 160.6, 160.7 (CONHЈ,
CONHЈЈ). MS (ESI): m/z (%) ϭ 1451 (10) [2 M ϩ Na]ϩ, 737 (100)
[M ϩNa]ϩ. C38H39ClN4O8 (715.211).
2-(tert-Butyloxycarbonyl)-1,2,5,6,7,8,9,10-octahydrocyclopropa-
[c]benz[e]indol-4-one (28): Phenol 7 (10 mg, 30 µmol) was dissolved
in THF (0.75 mL) and treated with NaH (60% in oil, 3.0 mg, 75
µmol, 2.5 equiv.). After 24 h at room temperature the mixture was
concentrated, and the residue was purified by column chromato-
graphy. Compound 28 (7.2 mg, 24 µmol, 80%) was obtained as a
yellow oil. Rf ϭ 0.33 (PE/EtOAc, 1:1). 1H NMR (300 MHz,
CDCl3): δ ϭ 1.06 (dd, 2 ϫ J ϭ 5.5 Hz, 1 H, 9-Ha), 1.46 (s, 9 H,
Boc-CH3), 1.61 (dd, J ϭ 8.0, 5.4 Hz, 1 H, 9-Hb), 1.55Ϫ1.66 (m, 4
H, 6-CH2, 7-CH2), 1.78Ϫ1.86, 2.20Ϫ2.41 (each m, each 2 H, 5-
CH2, 8-CH2), 2.37 (ddd, J ϭ 8.0, 2 ϫ 5.5 Hz, 1 H, 10-H), 3.75 (dd,
J ϭ 5.5, 12.0 Hz, 1 H, 1-Ha), 3.86 (d, J ϭ 12.0 Hz, 1 H, 1-Hb),
6.45 (br. s, 1 H, 3-H). 13C NMR (75 MHz, CDCl3): δ ϭ 21.7, 22.0,
22.1 (C-5, C-6, C-7), 22.7, 23.2, 24.3 (C-8, C-9, C-10), 28.2 (Boc-
CH3), 36.6 (C-8b), 52.3 (C-1), 83.0 (Boc-C), 108.8 (C-3), 134.4 (C-
4a), 145.4 (C-8a), 151.8 (CϭO), 158.8 (C-2a), 187.8 (C-4) Ϫ
C18H23NO3 (301.389).
Cell Culture: Human bronchial carcinoma cells of line A549
(ATCC CCL 185) were kindly provided by the Institut für Zellbiol-
ogie, Universität Essen, and were maintained as exponentially
growing cultures at 37 °C and 7.5% CO2 in air in Dulbecco’s modi-
fied Eagle’s medium (DMEM) (Biochrom, Berlin, Germany) sup-
plemented with 10% fetal calf serum (heat-inactivated for 30 min
at 56 °C, GibcoBRL, Karlsruhe, Germany), 44 m NaHCO3 (Bi-
ochrom, Berlin, Germany), and 4 m -glutamine (GibcoBRL,
Karlsruhe, Germany).
In Vitro Cytotoxicity Assays: Adherent cells of line A549 were sown
in triplicate in 6 multiwell plates at concentrations of 102, 103, 104,
and 105 cells per cavity. Culture medium was removed (suction)
after 24 h, and cells were washed in the incubation medium Ultra-
culture (UC, serum-free special medium, purchased from BioWhit-
taker Europe, Verviers, Belgium). Incubation with compounds
10؊12 was then performed in Ultraculture medium at various con-
centrations for 24 h. All substances were used as freshly prepared
solutions in DMSO (Merck, Darmstadt, Germany) diluted with
incubation medium to a final concentration of DMSO of 1% in the
wells. After 24 h of exposure, the test substance was removed and
the cells were washed with fresh medium. Cultivation was per-
formed at 37 °C and 7.5% CO2 in air for 12 days. The medium
was removed and the clones were dried and stained with Löffler’s
methylene blue (Merck, Darmstadt, Germany). They were then
counted macroscopically.
anti-Galactoside 6b: According to General Procedure 3, phenol 8b
(58 mg, 165 µmol) was treated with tetraacetyl trichloroacetimidate
21 (85 mg, 169 µmol), BF3·OEt2 (168 µL, 1.27 mmol), and molecu-
lar sieves (1.14 g), followed by EDC (71 mg, 366 µmol) and acid 20
(46 mg, 146 µmol) to give 132 mg of the peracetylated galactoside
(149 µmol), which was treated with NaOMe/MeOH (5.4 , 55 µL,
299 µmol). anti-Galactoside 29 (48 mg, 67 µmol, 41%) was ob-
tained as a gray powder, as a mixture of diastereomers. Rf (per-
acetylated galactoside) ϭ 0.77 (PE/EtOAc, 1:2). UV (CH3CN):
λmax (lg ε) ϭ 206.0 nm (4.698), 311.5 (4.630). IR (KBr): ν˜ (cmϪ1) ϭ
3406, 3330, 2930, 1620, 1531. 1H NMR (500 MHz, [D7]DMF): δ ϭ
1.60, 1.89 (each mc, each 2 H, 7-CH2, 8-CH2), 1.65/1.67 (each d,
J ϭ 7.0 Hz, 3 H, 11-CH3), 2.51Ϫ2.56 (m, 1 H, 9-Ha), 2.68Ϫ2.80
(m, 3 H, 6-CH2, 9-Hb), 3.62 (mc, 1 H, 6*-Ha), 3.68Ϫ3.72 (m, 1
H, 6*-Hb), 3.76 (mc, 1 H, 1-H), 3.81Ϫ3.85 (m, 2 H, 3*-H, 5*-H),
3.98Ϫ4.00 (m, 1 H, 2*-H), 4.63Ϫ4.71 (m, 4 H, 2-CH2, 10-H, 4*-
H), 4.91 (d, J ϭ 7.5 Hz, 1 H, 1*-H), 7.10 (t, J ϭ 8.0 Hz, 1 H, 5ЈЈ-
H), 7.21/7.30 (each s, together 1 H, 3Ј-H), 7.26 (t, J ϭ 8.0, 1 H,
6ЈЈ-H), 7.53 (s, 1 H, 3ЈЈ-H), 7.61 (d, J ϭ 8.2 Hz, 1 H, 7ЈЈ-H), 7.56/
7.62 (each d, J ϭ 8.0 Hz, together 1 H, 7Ј-H), 7.70 (d, J ϭ 8.1 Hz,
The relative clone forming rate was determined according to the
following formula:
relative clone-forming rate [%] ϭ 100 ϫ (number of clones counted
after exposure)/(number of clones counted in the control)
1 H, 4ЈЈ-H), 7.70/7.75 (each d, J ϭ 8.0 Hz, together 1 H, 6Ј-H), Liberation of the drugs from their glycosidic prodrugs was achieved
8.06 (br. s, 1 H, 4-H), 8.33/8.40 (each s, together 1 H, 4Ј-H), 10.23
by addition of 0.4 U/ml β--galactosidase (EC 3.2.1.23, Grade X,
(s, 1 H, CONH), 11.62/11.65/11.68/11.82 (each s, together 2 H, in-
purchased from Sigma Germany, Deisenhofen, Germany) to the
dole-NH). 13C NMR (125 MHz, [D7]DMF): δ ϭ 23.0, 23.2, 24.0 cells during incubation with the substances.
1644
Eur. J. Org. Chem. 2002, 1634Ϫ1645

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This work was supported by the Deutsche Forschungsgemeinschaft
(SFB 416) and the Fonds der Chemischen Industrie. The authors
would like to thank Stefanie Heidrich for her excellent work in the
cytotoxicity tests. We are also indebted to BASF AG, Bayer AG,
Degussa AG, Dragoco Gerberding & Co. AG, and Wacker-Chemie
GmbH for generous gifts of chemicals.
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