A strategy for tumor-selective chemotherapy by enzymatic liberation of seco-duocarmycin SA-derivatives from nontoxic prodrugs

Article abstract of DOI:10.1016/S0968-0896(01)00098-0

Immuno-conjugates obtained by linking enzymes with appropriate monoclonal antibodies, which bind to tumor-associated antigens, can be employed in a tumor-selective antibody directed enzyme prodrug therapy (ADEPT). For this strategy the glycosides 17a-c we

Full text of DOI:10.1016/S0968-0896(01)00098-0

Bioorganic & Medicinal Chemistry 9 (2001) 1929–1939
A Strategy for Tumor-Selective Chemotherapy
by Enzymatic Liberation of seco-duocarmycin
SA-derivatives from Nontoxic Prodrugs
Lutz F. Tietze,* Monika Lieb, Tobias Herzig, Frank Haunert and Ingrid Schuberth
Institut fur Organische Chemie der Georg-August-Universitat Gottingen, Tammannstraße 2, D-37077 Gottingen, Germany
¨
¨ ¨ ¨
Received 19 February 2001; accepted 5 April 2001
Abstract—Immuno-conjugates obtained by linking enzymes with appropriate monoclonal antibodies, which bind to tumor-asso-
ciated antigens, can be employed in a tumor-selective antibody directed enzyme prodrug therapy (ADEPT). For this strategy the
glycosides 17a–c were prepared as prodrugs of CI-TMI 14 which is a structurally simplified analogue of the highly potent antitumor
agent duocarmycin SA 2. Exposure of 17a–c to cultured carcinoma cells of line A549 displayed a very low toxicity; however, after
addition of the corresponding enzymes and exposure for 24 h at prodrug concentrations of <0.1 mM the proliferation of the car-
cinoma cells was inhibited almost completely with ED50prodrug/ED50drug of up to 270 in the presence and in the absence of the
enzyme. The synthesis of 17a–c was achieved by transformation of nitroanisidine 6 into 12 which was glycosidated to give 16a–c.
Removal of the silyl groups, introduction of a chlorine atom and solvolysis of the acetal groups led to 17a–c, of which 17a and 17c
are promising candidates for further elaboration. # 2001 Elsevier Science Ltd. All rights reserved.
Introduction
have been identified which express a similar pharmaco-
phoric group.
Tumor-selective chemotherapy must be based on the
exploitation of phenotypic or genetic differences of
malignant cells from normal tissue. Several rather spe-
cific antigens of tumor cells have been identified in
recent years1 and monoclonal antibodies were raised
against these domains. For therapeutic purposes, these
antibodies have been linked to radioactive species,2
natural toxines or cytotoxic agents.3 Very promising is
the ADEPT concept (antibody directed enzyme prodrug
therapy)3a,4 in which a little toxic prodrug is enzymati-
cally converted into a toxic species selectively at the
surface of malignant cells employing enzyme–immuno-
conjugates (Fig. 1). The success of this concept clearly
depends on the difference of toxicity between the pro-
drugs and the corresponding drugs as well as on a high
biological activity of the drug itself.
The cytotoxicity of CC-1065 and the duocarmycins is
caused by an alkylation of N-3 of adenine in AT-rich
parts of the minor groove of the DNA by reaction with
the spirocyclopropylcyclohexadienone moiety in 1 or 2.
In various studies it was shown that the natural anti-
biotic CC-1065 1, which was first isolated from Strepto-
myces zelensis, is a very potent cytostatic agent (Scheme
1).5 Recently, the duocarmycins as duocarmycin SA 2
*Corresponding author. Tel.: +49-551-393271; fax: +49-551-399476;
e-mail: ltietze@gwdg.de
Figure 1. Model of the antibody directed enzyme prodrug therapy
(ADEPT) concept.
0968-0896/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00098-0

1930
L. F. Tietze et al. / Bioorg. Med. Chem. 9 (2001) 1929–1939
However, seco-compounds of 1 or 2 as 3 which cannot
form the spirocyclopropyl moiety as in 4 have a drasti-
cally reduced cytotoxicity (Scheme 2).
the seco-CI-unit without the TMI moiety; however,
these compounds did not reveal a sufficient cytotoxicity
after enzymatic cleavage.
Thus, we6 and others7 have shown that the formation of
this structural element is halted by blocking the phe-
nolic hydroxy group as an ether. However, ethers are
very stable functionalities which can usually not be
cleaved under physical conditions. We therefore intro-
duced the glycosides which can be hydrolyzed by a gly-
cohydrolase with liberation of the corresponding toxin.
These compounds are now suitable for ADEPT as pro-
drugs. CC-1065 cannot be used in cancer therapy due to
a delayed deadly liver toxicity. But structurally related
simplified compounds as CI-TMI 5 (CI=1,2,7,7a-tetra-
hydrocyclopropa[c]indol-4-one, TMI=5,6,7-trimethoxy-
indole-2-carboxylate) are appropriate as anticancer
agents.
Results
Synthesis of seco-CI-TMI 14 and the glycosides 17a–c
The synthesis of seco-CI-TMI 14 and the glycosides
17a–c was accomplished by transformation of nitroani-
sidine 6 into the known seco-CI-methanesulfonamide 7
in eight steps with an overall yield of 27% (Scheme 4).8a
Removal of the methanesulfonyl moiety was accom-
plished by reduction with an excess of bismethoxy-
ethoxy sodium dihydroaluminate (RedAl1) in refluxing
toluene and the resulting secondary amine 8 was imme-
diately coupled without purification with 5,6,7-tri-
methoxy-indole-2-carboxylic acid 98b in DMF using 1-
(3-dimethylaminopropyl)-3-ethylcarbodiimide hydro-
chloride (EDC) as coupling reagent to give the amide 10
in 57% yield.
Here, we present our results using different glycosides of
seco-CI-TMI 57a,8 (Scheme 3) containing the seco-CI-
unit as a simplified pharmacophoric group of duo-
carmycin SA 2 with the trimethoxyindole moiety for
binding to the minor groove of DNA. In a former short
communication6 we had already described glycosides of
Compound 10 was employed as the key intermediate for
the synthesis of the CI-TMI glycosides 17a–c. On the
other hand, it was also used to prepare the free seco-CI-
TMI 14 as well as the benzyl-protected compound 13
for comparison in the cell tests. Substitution of the
hydroxy group in 10 by chloride using the Appel proce-
dure with triphenylphosphane in tetrachloromethane
and dichloromethane for 5 h at 40 ꢀC gave 13. Hydro-
genolytic removal of the benzyl group using palladium
on carbon yielded seco-CI-TMI 14.8b
For the synthesis of the seco-CI-TMI glycosides 17a–c,
the hydroxy group in 10 was protected as a tert-butyl-
diphenylsilyl ether to give 11. Which was then depro-
tected at the phenolic hydroxy group by hydrogenation
to yield 12. Peracetylated trichloroacetimidates of
galactose 15a, glucose 15b and mannose 15c were used
.
for the glycosidation of 12 in the presence of BF3 OEt2
in dichloromethane at room temperature (Scheme 5).9
In all cases the 1,2-trans-glycosides were obtained in
acceptable yields of 53–75%.
The configuration at the anomeric center was deter-
1
mined by H NMR spectroscopy showing a coupling
00 00
constant of J1 ,2 =8.0 Hz for the b-galactoside 16a and
Scheme 1. Structure of (+)-CC-1065 (1) and (+)-duocarmycin SA
(2).
00
00
J1 , 2 =3.5 Hz for the a-mannoside 16c. In the spec-
trum of glucoside 16b the signal for 100-H is hidden in a
multiplet.
Scheme 2. Formation of the spirocyclopropylcyclohexadienone moi-
ety (4) from a seco-compound (3).
Scheme 3. Prodrugs of the seco-CI-TMI unit (5).

L. F. Tietze et al. / Bioorg. Med. Chem. 9 (2001) 1929–1939
1931
Scheme 4. Synthesis of seco-CI-TMI derivatives 13 and 14: (a) RedAl, 3 h 110 ꢀC, 94%; (b) tBuPh2SiCl, 4-dimethylamino-pyridine, NEt3, 16 h
ꢀ
40 ꢀC, 92%; (c)Pd/C, 3 bar H2, 16 h, 64%; (d) PPh3, CCl4, 2 0 h, 60 C, 55%; (e) Pd/C, 3 bar H2, 24 h, 55%.
The tert-butyldiphenylsilyl moiety was removed with
silica gel coated with tetrabutylammonium fluoride
(TBAF) in tetrahydrofuran, and the deprotected primary
hydroxy group transformed into the chloride in an Appel
reaction with triphenylphosphane in tetrachloromethane
and dichloromethane. Solvolysis of the acetyl groups
using sodium methoxide in methanol completed the
synthesis to give 17a–c in 38–70% yield over three steps.
Determination of the cytotoxicity of seco-CI-TMI
glycosides and seco-CI-TMI derivatives 13 and 14
The cytotoxic effect of the seco-CI-TMI glycosides and
other seco-CI-TMI derivatives was evaluated in vitro by
exposure of human bronchial carcinoma cells of line
A549 to the substances for 24 h. As expected, the seco-
CI-TMI 14 with a free phenolic hydroxy group exhib-
ited a relatively high cytotoxicity with an ED50=2.2 nM
for the relative clone formation, while the benzylated
seco-CI-TMI 13 showed a much lower toxicity with an
ED50=650 nM (Fig. 2A–C).
For the determination of the cytotoxicity of the seco-CI-
TMI glycosides we used a serum free culture medium in
order to avoid an enzymatic hydrolysis of the glycosides
since we have shown that fetal calf serum and even the
basal medium supplement contain some b-d-galacto-
sidase and b-d-glucosidase.6 The testing revealed that
the cytotoxicity of the galactoside 17a (Fig. 2A) with an
ED50=1050 nM and of the mannoside 17c (Fig. 2C)
with an ED50=2200 nM is as low as found for the
stable benzyl protected seco-CI-TMI 13, whereas the
glucoside 17b (Fig. 2B) proved to be unexpectedly toxic
with an ED50=100 nM.
In the presence of the corresponding enzyme, which
Scheme 5. Synthesis of the glycosidic prodrugs 17a–c of seco-CI-TMI
(14).
removes the sugar moiety from the phenolic hydroxy
group and thus releases the cytotoxic seco-CI-TMI, all
glycosides displayed an ED50 in the range of the cyto-
toxicity of seco-CI-TMI 14.
some cases it was possible to separate the diastereomers
and prepare enantiopure 12 by enzymatic removal of
the sugar moiety. However, in the described work the
mixtures of diastereomers were used for the biological
tests since a difference in the rate of enzymatic cleavage
of the diastereomers was not observed and in addition, it
was known that the enantiomers of 1 show comparable
cytotoxicity.
For the glycosidations racemic 12 was used and thus
16a–c were obtained as mixtures of diastereomers. Since
an enantiomer-differentiating reaction does not occur
due to a large distance between the stereogenic centers
in 12 and the sugar moieties, a 1:1 mixture is formed. In

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L. F. Tietze et al. / Bioorg. Med. Chem. 9 (2001) 1929–1939
The toxicity of the galactoside 17a was also examined at
a lower pH of 6.2(Fig. 2D). These investigations were
stimulated by our work on the design of new selective
antitumor agents based on the difference of pH in nor-
mal and tumor cells under hyperglycemic conditions.10
Thus, we developed compounds showing a 100 times
higher cytotoxicity at pH 6.2, the mean pH value in the
tissue of transplanted solid tumors of hygerglycemic
hosts, compared to the cytotoxicity at pH 7.4, the pH
value in normal cells of that host. However, a difference
of cytotoxicity of 17a at pH 6.2was not observed since
the galactoside is stable at these pH values.
blocking the phenolic hydroxy group thus preventing
the formation of the spirocyclopropyl moiety which
causes alkylation at N-3 of adenine units in the DNA.
The use of seco-CI-TMI in the ADEPT concept requires
a blocking of the phenolic hydroxy group in a way that
allows its release under physiological conditions by use
of an enzyme. This is possible for the glycosides 17a–c
which can be cleaved by glycohydrolases in order to
release the seco-CI-TMI unit. The difference of cyto-
toxicity of seco-CI-TMI galactoside 17a and seco-CI-
TMI mannoside 17c is very high in comparison with
seco-CI-TMI 14 and sufficient for a selective approach
(Table 1).
Surprisingly, the difference of cytotoxicity between seco-
CI-TMI glucoside 17b and seco-CI-TMI 14 is rather low
with an ED50prodrug/ED50drug of 40. So far we do not
have an explanation for the relatively high cytotoxicity
of the seco-CI-TMI glucoside 17b. However, the pro-
drug might penetrate the cell membrane by an active
Discussion
In spite of its simplified structure the seco-CI-TMI 14
shows a remarkable high cytotoxicity which is only
diminished by a factor of less than 100 compared to CC-
1065. It is easily accessible and can be detoxified by
Figure 2. In vitro cytotoxicity of 13, 14 and 17a–c against human bronchial carcinoma cells of line A549. (A) in vitro cytotoxicity of compound 13
(~), 14 (&) and 17a without (*) and with (*) addition of b-d-galactosidase (0.4 U/mL); (B) in vitro cytotoxicity of compound 13 (~), 14 (&) and
17b without (*) and with (*) addition of b-d-glucosidase (0.4 U/mL); (C) in vitro cytotoxicity of compound 13 (~), 14 (&) and 17c without (*)
and with (*) addition of a-d-mannosidase (0.4 U/mL); (D) in vitro cytotoxicity of compound 17a at pH 6.2( *) and pH 7.4 (*) and compound 14
(&). Cells were exposed to various concentrations of the test substances for 24 h at 37 ꢀC; after 12days of incubation clone formation was compared
to untreated control assay and the relative clone forming rate was determined.

L. F. Tietze et al. / Bioorg. Med. Chem. 9 (2001) 1929–1939
1933
Table 1. In vitro cytotoxicity of compounds 13, 14 and 17a–c against
human bronchial carcinoma cells of line A549
standard methods. All reagents obtained from commer-
cial sources were used without further purification.
Thin-layer chromatography was performed on pre-
coated silica gel plates (SIL G/UV254, Macherey-Nagel
GmbH & Co. KG). Silica gel 32–63 (0.032–0.064 mm)
(Macherey-Nagel GmbH & Co. KG) was used for col-
umn chromatography.
Substituent at the
phenolic oxygena
Addition of
glycohydrolase
ED50 (nM)b
Specifity
factorc
H (13)
Bn (14)
ꢁ
ꢁ
2.20
650
1050
6.10
1000
100
14.2
2200
8.20
b-d-Galactose (17a)
b-d-Galactose (17a)
b-d-Galactose (17a)
b-d-Glucose (17b)
b-d-Glucose (17b)
a-d-Mannose (17c)
a-d-Mannose (17c)
ꢁ
180
+
d
UV–vis spectra were recorded in CH3CN on a Mettler
Lambda 2spectrometer. IR spectra were recorded as
KBr pellets or as films on a Bruker IFS 25 or Vector 22
ꢁ
ꢁ
+
ꢁ
7
270
1
spectrometer. H and 13C NMR spectra were recorded
+
on a Varian XL 200, VXR 200 and VXR 500 or a Bru-
ker AM 300 with tetramethylsilane (TMS) as the inter-
nal standard in [D]chloroform or [D4]methanol.
Multiplicities of 13C NMR peaks were determined with
the APT pulse sequence. Mass spectra were measured at
70 eV on a Varian MAT311A, high-resolution mass
spectra on a Varian MAT731 instrument. Melting
points were determined on a Mettler FR 61 and are
uncorrected.
aCompounds 13 and 14 were used as racemic mixtures; compounds
17a–c were used as mixtures of diastereomers.
bCells were exposed to the compounds for 24 h at 37 ꢀC; after 12days
of incubation the clone formation was compared to an untreated con-
trol assay.
cSpecificity factor=ED50 in the absence of the enzyme/ED50 in the
presence of the enzyme.
dMeasured at pH 6.2; all other measurements were carried out at pH
7.4.
transport mechanism and then be cleaved in the lyso-
some which contains glycohydrolases. This effect may
be of interest in designing novel selective anticancer
agents which are not based on the ADEPT concept,
since the glucose intake of cancer cells is much higher
compared to normal cells.
(3R/S) - 6 - Benzyloxy - 3 - hydroxymethyl - 1 - (50,60,70 - tri-
methoxyindol-20-yl-carbonyl)-2,3-dihydro-1H-indole (10).
6-Benzyloxy-3-hydroxymethyl-N-methanesulfonyl-2,3-
dihydro-1H-indole (10.35 g, 31.04 mmol) 78a was dis-
solved in oxygen free toluene (140 mL), and sodium(bis-
methoxyethoxy)-dihydroaluminate (RedAl1) (46 mL,
0.155 mol, 5.0 equiv) was slowly added while stirring.
After refluxing for 3 h the solution was cooled to 0 ꢀC,
poured into ice cold brine (200 mL), extracted with die-
thyl ether (3Â200 mL) and dried over Na2SO4. Con-
In the presence of the corresponding glycohydrolases,
all three seco-CI-TMI glycosides 17a–c displayed nearly
the same cytotoxicity as found for seco-CI-TMI 14. This
shows that the glycosides are completely cleaved by the
enzymes meaning that the enzyme is not destroyed by
the formed toxin; thus, a suicide mechanism with regard
to the enzymes does not occur. ED50prodrug/ED50drug
values of 270 and 170 were reached for the mannoside
and the galactoside by comparing the cytotoxicity in the
presence and in the absence of the enzyme.
centration
in
vacuo
yielded
6-benzyloxy-3-
hydroxymethyl-2,3-dihydro-1H-indole 8 (7.44 g, 29.14
mmol, 94%) as a light yellow oil. This was dissolved in
DMF (200 mL) and 5,6,7-trimethoxyindole-2-car-
boxylic acid 98b (5.98 g, 23.78 mmol, 1.0 equiv) and 1-
(3-dimethylaminopropyl)-3-ethylcarbodiimide hydro-
chloride (EDC) (9.12g, 47.56 mmol, 3.0 equiv) were
added. The solution was stirred for 20 h at 50 ꢀC, cooled
to rt and poured into water (200 mL). The organic layer
was extracted with ethyl acetate (3Â200 mL), washed
with brine (200 mL) and dried over Na2SO4. Evapora-
tion and purification by column chromatography (PE/
EtOAc=2:1) afforded 10 (6.62g, 13.55 mmol, 57%) as
a white solid. Rf=0.28 (PE/EtOAc=1:1); mp 149 ꢀC;
Conclusion
The seco-CI-TMI galactoside 17a and the seco-CI-TMI
mannoside 17c are both accessible by efficient synthetic
routes. The compounds seem to be very suitable for
ADEPT since they display a high difference of cyto-
toxicity in the presence and in the absence of b-d-
galactosidase and a-d-mannosidase, respectively, and a
high biological activity of the drug itself. In contrast,
the seco-CI-TMI glucoside shows a relatively high
cytotoxicity. Further investigations with different cell
lines and other types of assays will now be undertaken.
Appropriate immuno-conjugates of antibodies and
enzymes will be employed for the in vivo cleavage of the
prodrugs according to the concept of the ADEPT. The
results of the these tests will be reported in due course.
~
IR (pellet): ꢀ=3442 (OH), 2928, 2854 (aliphat. C–H),
1626 (C¼O, amide), 1592, 1494 (C¼C), 1390 (CH3),
1310 (OH), 1236, 1108 (C–O), 948, 836 cmꢁ1 (1,3,4-tri-
substitution); UV/vis (CH3CN): lmax (lg e)=207.0
1
(4.733), 324.0 nm (4.495); H NMR (300 MHz, CDCl3):
d 0.93 (sb; 1H, OH), 3.62–3.67 (m; 1H, 3-H), 3.76–3.82
(m; 1H, 8-Ha), 3.85–3.89 (m; 1H, 8-Hb), 3.91 (s; 3H,
OCH3), 3.94 (s; 3H, OCH3), 4.08 (s; 3H, OCH3), 4.48
(dd, J=4,5, 10.5 Hz; 1H, 2-Ha), 4.61 (dd, J=9.5, 10.5
Hz; 1H, 2-Hb), 5.10 (s; 2H, OCH2Ph), 6.73 (dd, J=2.5,
8.0 Hz; 1H, 5-H), 6.83 (s; 1H, 40-H), 6.96 (d, J=2.5 Hz;
1H, 30-H), 7.16 (dd, J=1.0, 8.0 Hz; 1H, 4-H), 7.32(t,
J=7.0 Hz; 1H, Ph–H), 7.39 (t, J=7.0 Hz; 2H, Ph–H),
7.46 (d, J=7.0 Hz; 2H, Ph–H), 8.13 (d, J=2.5 Hz; 1H,
7-H), 9.40 (sb; 1H, indole NH); 13C NMR (50 MHz,
CDCl3): d 42.79 (C-3), 53.85 (C-2), 56.26 (OCH3), 61.09
(OCH3), 61.45 (OCH3), 65.25 (C-8), 70.20 (OCH2Ph),
Experimental
All reactions were performed under an inert gas atmo-
sphere in flame-dried flasks. All solvents were dried by

1934
L. F. Tietze et al. / Bioorg. Med. Chem. 9 (2001) 1929–1939
97.67 (C-30), 104.7 (C-7), 106.5 (C-40), 111.6 (C-5), 123.6
(C-60), 123.7 (C-3a), 124.4 (C-4), 125.4 (C-7a0), 127.5
(Ph–C), 127.9 (Ph–C), 128.5 (Ph–C), 130.0 (C-3a0),
137.0 (C-100), 138.8 (C-70),140.6 (C-20), 145.2(C-7a),
150.1 (C-50), 159.2(C-6), 160.3 (NCO); MS (70 eV, EI):
m/z (%) 488 (90) [M]+, 457 (2) [M–CH2OH]+, 397 (5)
[M–Bn]+, 255 (15) [Mꢁ(TMI–OH–H)]+, 234 (100)
[TMI–OH]+, 224 (30) [Mꢁ(TMI–OH–H)–CH2OH]+,
91 (55) [Bn]+; C28H28N2O6 (488.54) calcd (%) C 68.84,
H 5.78; found C 68.79, H 5.79; HRMS calcd 488.1947;
found 488.1947.
filtration through Celite and the solution was con-
centrated in vacuo. Purification by column chromato-
graphy (PE/EtOAc=3:1) yielded 12 (2.24 g, 3.52 mmol,
64%) as a white solid. Rf=0.35 (PE/EtOAc=3:1); IR
~
(pellet): ꢀ=3458 (NH), 2934, 2858 (aliphat. C–H), 1624
(C¼O, Amid), 1586, 1494 (C¼C), 1528 (NCO), 1462,
1428, 1386 (CH3), 1312 (OH), 1258, 1236, 1198, 1156
(C–O), 944, 860 cmꢁ1 (1,3,4-trisubst.); UV–vis
(CH3CN): lmax (lg e)=192.5 (4.992), 324.5 nm (4.457);
1H NMR (300 MHz, CDCl3): d 1.05 (s; 9H, (CH3)3C),
3.62(m ; 1H, 3-H), 3.65 (dd, J=7.5, 9.5 Hz; 1H, 8-Ha),
c
3.86 (dd, J=5.0, 9.5 Hz; 1H, 8-Hb), 3.91 (s; 3H, OCH3),
3.96 (s; 3H, OCH3), 4.14 (s; 3H, OCH3), 4.41 (dd,
J=4.5, 10.5 Hz; 1H, 2-Ha), 4.52(dd, J=9.5, 10.5 Hz;
1H, 2-Hb), 6.61 (dd, J=2.5, 8.0 Hz; 1H, 5–H), 6.82 (s;
1H, 40-H), 6.89 (d, J=2.5 Hz; 1H, 30-H), 7.03 (d, J=8.0
Hz; 1H, 4-H), 7.26–7.43 (m; 6H, Ph–H), 7.52–7.60 (m;
4H, Ph–H), 7.95 (sb; 1H, phenol OH), 8.21 (d, J=2.5
Hz; 1H, 7-H), 9.51 (sb; 1H, indole NH); 13C NMR
(125 MHz, CDCl3): d 19.23 ((CH3)3C), 26.80 ((CH3)3C),
43.00 (C-3), 54.33 (OCH3), 61.26 (OCH3), 61.57
(OCH3), 66.86 (C-2), 97.71 (C-40) 106.0 (C-7), 107.2(C-
30), 111.7 (C-5), 123.3 (C-3a), 123.5 (C-3a0), 125.0 (C-4),
125.8 (C-7a0), 127.7 (Ph–C), 129.6 (C-20), 129.7 (Ph–C),
133.2(C-1 00), 135.5 (Ph–C), 138.1 (C-70), 140.7 (C-60),
144.3 (C-7a), 150.0 (C-50), 157.1 (C-6), 160.7 (NCO);
MS (70 eV, EI): m/z (%) 636.2(25) [M] +, 579.1 (30)
[M–(CH3)C]+, 234.0 (35) [TMI]+, 199.0 (100)
[Ph2SiOH]+; C37H40N2O6Si (636.82) calcd C 69.79, H
6.33; found C 69.62, H 6.36.
(3R/S)-6-Benzyloxy-3-(tert-butyl-diphenyl-silyl)-oxy-
methyl-1-(50,60,70-trimethoxyindol-20-yl-carbonyl)-2,3-di-
hydro-1H-indole (11). To a solution of alcohol 10 (2.12
g, 4.34 mmol), triethylamine (10 mL) and 4-dimethyla-
minopyridine (DMAP) (50 mg, 0.87 mmol, 0.2equiv) in
dichloromethane (55 mL) was added dropwise tert-
butyldiphenylsilyl chloride (2.26 mL, 2.39 g, 8.65 mmol,
2.0 equiv) at rt. The reaction mixture was stirred for 16
h at 60 ꢀC, washed with brine (100 mL) and extracted
with dichloromethane (3Â100 mL). After drying of the
extracts over Na2SO4 the solvents were removed in
vacuo and the residue purified by column chromato-
graphy (PE/EtOAc=3:1) to afford 11 (2.90 g, 3.99
mmol, 92%) as
a colorless oil. Rf=0.68 (PE/
EtOAc=3:1); IR (pellet): ꢀ~=3458 (NH), 2934, 2858
(aliphat. C-H), 1628 (C¼O, amide), 1598 (C¼C), 1526
(NCO), 1428, 1412, 1388 (CH3), 1230, 1194, 1160 (C–
O), 950, 836 (1,3,4-trisubst.), 744, 706 cmꢁ1 (Si–Ph);
UV–vis (CH3CN): lmax (lg e)=324.5 nm (4.478); 1H
NMR (300 MHz, CDCl3): d 1.03 (s; 9H, (CH3)3C), 3.64
(mc; 1H, 3-H), 3.69 (dd, J=7.5, 9.5 Hz; 1H, 8-Ha), 3.99
(dd, J=5.0, 9.5 Hz; 1H, 8-Hb), 3.94 (s; 3H, OCH3), 3.96
(s; 3H, OCH3), 4.09 (s; 3H, OCH3), 4.43 (dd, J=4.5,
10.5 Hz; 1H, 2-Ha), 4.55 (dd, J=9.5, 10.5 Hz; 1H, 2-
Hb), 5.11 (s; 2H, OCH2Ph), 6.69 (dd, J=2.5, 8.0 Hz;
1H, 5-H), 6.84 (s; 1H, 40-H), 6.87 (d, J=2.5 Hz; 1H, 30-
H), 7.07 (d, J=8.0 Hz; 1H, 4-H), 7.28–7.50 (m; 11H,
Ph–H), 7.54–7.63 (m; 4H, Ph–H), 8.12(d, J=2.5 Hz;
1H, 7-H), 9.40 (sb; 1H, indole NH); 13C NMR (50 MHz,
CDCl3): d 19.20 ((CH3)3C), 26.77 ((CH3)3C), 42.96 (C-
3), 53.93 (OCH3), 61.06 (OCH3), 61.43 (OCH3), 66.78
(3R/S)-6-Benzyloxy-3-chloromethyl-1-(50,60,70-trimethoxy-
indol-20 -yl-carbonyl)-2,3-dihydro-1H-indole (13). To a
solution of amide 7 (100 mg, 0.20 mmol) in dichloro-
methane (5 mL) and tetrachloromethane (5 mL) was
added triphenylphosphane (161 mg, 0.61 mmol, 3.0
equiv), and the mixture was stirred for 5 h at 40 ꢀC.
Removal of the solvent and purification by column
chromatography (PE/EtOAc=3:1) afforded 13 (55 mg,
0.11 mmol, 55%) as a colorless solid. Rf=0.50 (PE/
~
EtOAc=3:1); IR (pellet): ꢀ=936 (aliphat. C–H), 1628
(C¼O, amide), 1600, 1494 cmꢁ1 (C¼C); UV–vis
(CH3CN): lmax (lg e)=206.0 (4.743), 324.5 nm (4.449);
1H NMR (300 MHz, C6D6): d 3.10–3.22 (m; 2H,
CH2Cl), 3.55 (s; 3H, OCH3), 3.70 (s; 3H, OCH3), 3.73–
3.78 (mc; 1H, 3-H), 3.80 (s; 3H, OCH3), 3.98–4.07 (m;
2H, 2-H2), 4.92s; 2H, OCH 2Ph), 6.69 (s; 1H, 40-H), 6.72
(dd, J=2.5, 8.0 Hz; 1H, 5-H), 7.00–7.44 (m; 7H, 5-H,
30-H, Ph–H), 8.70 (d, J=2.5 Hz; 1H, 7-H), 9.75 (sb; 1H,
indole NH); 13C NMR (50 MHz, CDCl3): d 43.12(C-3),
47.16 (C-2), 54.84 (C-8), 56.25 (OCH3), 61.13 (OCH3),
61.47 (OCH3), 70.20 (OCH2Ph), 97.61 (C-30), 104.6 (C-
7), 106.4 (C-40), 111.7 (C-5), 123.3 (C-60), 123.6 (C-3a),
124.5 (C-4), 125.5 (C-7a0), 127.5 (Ph–C), 128.0 (Ph–C),
128.6 (Ph–C), 129.8 (C-3a0), 136.8 (C-100), 138.9 (C-70),
140.5 (C-20), 144.9 (C-7a), 150.1 (C-50), 159.6 (C-6), 160.2
(NCO). MS (70 eV, EI): m/z (%) 506.5 (3) [M]+, 2 34.2 (5)
[TMI]+, 2 2 3.2 (5) [MꢁTMI–CH2Cl]+, 91.1 (100) [Bn]+.
0
(C-2), 70.12 (OCH2Ph), 97.62(C–2), 104.4 (C–4 ), 106.1
(C–7), 111.4 (C–30), 123.6 (C–3a), 124.4 (C–3a0), 124.7
(C–4), 125.3 (C–7a0), 127.4 (Ph–C, Bn), 127.7 (Ph–C,
TBDPS), 127.8 (Ph–C, Bn), 128.4 (Ph–C, Bn), 129.7
(Ph–C, TBDPS), 130.1 (C-20), 133.1 (C-1000, TBDPS),
135.3 (Ph–C, TBDPS), 137.0 (C-100, Bn), 138.8 (C-70),
140.3 (C-60), 144.9 (C-7a), 150.0 (C-50), 159.0 (C-6),
160.0 (NCO); MS (70 eV, EI): m/z (%) 726.7 (30) [M]+,
669.5 (55) [M–(CH3)3C]+, 234 (100) [TMI]+;
C44H46N2O6Si (726.95): calcd (%) C 72.70, H 6.38;
found C 72.65, H 6.39; HR-MS calcd 726.3125; found
726.3125.
(3R/S)-3-(tert-Butyl-diphenyl-silyl)-oxymethyl-6-hydroxy-1
-(50,60,70-trimethoxyindol-20-yl-carbonyl)-2,3-dihydro-1H-
indole (12). Benzyl ether 11 (4.2g, 5.53 mmol) was dis-
solved in ethyl acetate (30 mL) and palladium (10% on
charcoal, 1.18 g, 1.11 mmol, 0.2equiv) was added. The
suspension was stirred in a hydrogen pressure apparatus
(3 bar) at rt for 16 h. The solid was removed by
(3R/S)-3-Chloromethyl-6-hydroxy-1-(50,60,70-trimethoxy-
indol-20-yl-carbonyl)-2,3-dihydro-1H-indole (14). Benzyl
ether 13 (55 mg, 0.11 mmol) was dissolved in ethyl acetate
(10 mL) and palladium (10% on charcoal, 17 mg, 0.02

L. F. Tietze et al. / Bioorg. Med. Chem. 9 (2001) 1929–1939
1935
mmol, 0.2equiv) was added. The suspension was stirred
in a hydrogen pressure apparatus (3 bar) at rt for 24 h.
The solid was removed by filtration through Celite and
washed with dichloromethane (50 mL) and methanol
(50 mL). The filtrate was concentrated in vacuo to yield
14 (25 mg, 0.06 mmol, 55%). Rf=0.52(PE/
EtOAc=1:1); IR (pellet): ꢀ~=3432(OH), 2934 (aliphat.
C–H), 1622 (C¼O, amide), 1492(C ¼C), 1304 (OH),
7.36–7.44 (mc; 2H, Ph–H), 7.52–7.61 (m; 4H, Ph–H),
8.06 (sb; 1H, 7-H), 9.35 (sb; 1H, indole NH); 13C
NMR (75 MHz, CDCl3):11 d 19.13 ((CH3)3C), 20.50,
20.58, 20.67 (Ac–CH3), 26.69 ((CH3)3C), 42.91 (C-
3), 53.82 (C–2), 56.20 (OCH3), 61.04 (OCH3), 61.25
(C-600), 61.31 (OCH3), 66.61 (C-8), 66.84 (C-400),
68.57 (C-300), 70.81 (C-200), 70.97 (C-500), 97.57 (C-
40), 99.72(C-1 00), 106.3 (C-30), 106.6/106.8 (C-7),
113.0/113.1 (C-5), 123.5 (C-3a0), 124.8 (C-4), 125.4
(C-7a0), 127.0 (C-3a), 127.7 (Ph–C), 129.7 (Ph–C),
129.9 (C-20), 133.0 (C-1000), 135.4 (Ph–C), 138.8 (C-70),
140.4 (C-60), 144.9 (C-7a), 150.0 (C-50), 157.0 (C-6),
160.0 (NCO), 169.4, 170.0, 170.2, 170.4 (Ac–CO); MS
(70 eV, DCI): m/z (%) 984.5 (100) [M+NH4]+, 967.5
(50) [M+H]+.
ꢁ1
1264 (CH3), 1112cm
(C–O); UV–vis (CH3CN): lmax
(lg e) 206.0 (4.554), 324.5 nm (4.328); 1H NMR
(300 MHz, 80% CDCl3, 20% DMSO-d6): d 3.59 (dd,
J=8.0, 10.0 Hz; 1H, 8–Ha), 3.70–3.87 (m; 2H, 8-Hb, 3-
H), 3.89 (s; 3H, OCH3), 3.91 (s; 3H, OCH3), 4.07 (s; 3H,
OCH3), 4.40 (dd, J=4.5, 10.5 Hz; 1H, 2-Ha), 4.62(dd,
J=6.0, 10.5 Hz; 1H, 2–Hb), 6.57 (dd, J=2.5, 8.0 Hz;
1H, 5-H), 6.88 (s; 1H, 40-H), 6.95 (d, J=2.0 Hz; 30-H),
7.10 (d, J=8.0 Hz; 1H, 4 H), 7.78 (sb; 1H, 7-H), 9.18 (sb;
1H, indole NH), 10.28 (sb; 1H, OH); 13C NMR
(50 MHz, 80% CDCl3, 20% DMSO-d6): d 42.86 (C-3),
47.32(C-8), 54.77 (C–2), 56.17 (OCH 3), 61.11 (OCH3),
[(3R/S)-3-(tert-Butyl-diphenyl-silyl)-oxymethyl-1-(5,6,7-
trimethoxyindol-2-yl-carbonyl)-2,3-dihydro-1H-indol-6-
yl]-2,3,4,6-tetra-O-acetyl-ꢀ-D-glucopyranoside (16b). To
a solution of phenol 12 (1.14 g, 1.78 mmol), molecular
sieves (4 A, 12g) and trichloroacetimidate 15b (0.91 g,
1.78 mmol, 1.07 equiv) in dichloromethane (80 mL) was
added dropwise a solution of boron trifluoride diethyl
ether complex (0.253 g, 1.78 mmol, 1.0 equiv) in di-
chloromethane (1.78 mL) at 0 ꢀC and the mixture was
stirred for 16 h at rt. The molecular sieves were filtered
off, and the filtrate was washed with saturated NaHCO3
solution. The aqueous layer was extracted with di-
chloromethane (3Â100 mL) and the combined organic
phases were washed with brine and dried over Na2SO4.
Evaporation and purification by column chromato-
graphy (PE/EtOAc=3:1) afforded 16b (1.30 g, 1.34
mmol, 75%) as a colorless foam. Rf=0.09 (PE/
0
61.37 (OCH3), 97.65 (C-30), 105.6 (C-7), 106.2(C–4 ),
111.5 (C-5), 121.7 (C-60), 123.5 (C-3a), 124.5 (C-4),
0
125.3 (C-7a0), 130.1 (C-3a0), 138.8 (C-70), 140.2(C-2 ),
144.6 (C-7a), 149.9 (C-50), 158.1 (C-6), 160.0 (NCO);
MS (70 eV, EI): m/z 416.3 (69) [M]+, 234.2 (100)
[TMI]+.
[(3R/S)-3-(tert-Butyl-diphenyl-silyl)-oxymethyl-1-(5,6,7-
trimethoxyindol-2-yl-carbonyl)-2,3-dihydro-1H-indol-6-
yl]-2,3,4,6-tetra-O-acetyl-ꢀ-D-galactopyranoside (16a).
To a mixture of phenol 12 (2.19 g, 3.44 mmol), mole-
cular sieves (4 A, 10 g) and trichloroacetimidate 15a
(1.80 g, 3.78 mmol, 1.1 equiv) in dichloromethane (140
mL) was added dropwise a solution of boron trifluoride
diethyl ether complex (0.49 g, 3.44 mmol, 1.0 equiv) in
dichloromethane (25 mL) at 0 ꢀC and the mixture was
stirred for 16 h at rt. The molecular sieves were filtered
off, and the filtrate was washed with saturated NaHCO3
solution. The aqueous layer was extracted with di-
chloromethane (3Â150 mL) and the combined organic
phases were washed with brine and dried over Na2SO4.
Evaporation and purification by column chromato-
graphy (PE/EtOAc=3:1) afforded 16a (2.062 g, 2.13
mmol, 62%) as a colorless foam. Rf=0.10 (PE/
~
EtOAc=2:1); IR (pellet): ꢀ=3464 (NH), 3076, 3048
(aromat. C–H), 2934, 2858 (aliphat. C–H), 1758 (C¼O,
ester), 1630 (C¼O, amide), 1598, 1490 (C¼C), 1526
(NCO), 1440, 1430, 1376 (CH3), 1228, 1162, 1112, 1070
(C–O), 942, 852 (1,3,4-trisubst.), 744, 706 cmꢁ1 (Si–Ph);
UV–vis (CH3CN): lmax (lg e)=266.0 (3.854), 321.0 nm
1
(4.463); H NMR (200 MHz, CDCl3):11 d 1.04 (s; 9H,
(CH3)3C), 2.04, 2.05, 2.07, 2.08 (s; each 3H, Ac–CH3),
3.63 (mc; 1H, 3-H), 3.69 (m; 1H, 8-Ha), 3.82(m;
1H, 500-H), 3.88 (m; 1H, 8-Hb), 3.92(s; 3H, OCH 3),
3.96 (s; 3H, OCH3), 4.08 (s; 3H, OCH3), 4.18 (mc;
1H, 600-Ha), 4.30 (mc; 1H, 600-Hb), 4.42(m c; 1H, 2-
Ha), 4.54 (mc; 1H, 2-Hb), 5.16 (m; 2H, 100-H, 200-
H), 5.29 (m; 2H, 300–H, 400-H), 6.70 (dt, J=2.5, 8.0
Hz; 1H, 5–H), 6.83 (s; 1H, 40-H), 6.85 (d, J=2.0
Hz; 1H, 30-H), 7.08 (d, J=8.0 Hz; 1H, 4-H), 7.30–
7.42(m c; 6H, Ph–H), 7.53–7.61 (mc; 4H, Ph–H),
8.05 (sb; 1H, 7-H), 9.29 (sb; 1H, indole NH); 13C
NMR (75 MHz, CDCl3)11: d ((CH3)3C), 20.54, 20.55,
20.60, 20.64 (Ac–CH3), 26.81 ((CH3)3C), 43.01 (C-3),
53.93 (C-2), 56.31 (OCH3), 61.07 (OCH3), 61.47
(OCH3), 61.74 (C600), 66.71 (C-8), 68.16 (C-4000), 71.13
(C-300), 72.01 (C-200), 72.90 (C-500), 97.72(C-4 ), 99.17
(C-100), 106.3 (C-30), 106.6 (C-7), 113.2(C-5), 123.6 (C-
3a0), 124.9 (C-4), 125.5 (C-7a0), 127.0 (C-3a), 127.8 (Ph–
C), 129.8 (Ph–C), 133.1 (C-20), 134.8 (C-1000), 135.5 (Ph–
C), 138.9 (C-70), 140.5 (C-60), 145.0 (C-7a), 150.1 (C-50),
157.0 (C-6), 160.2(NCO), 169.3, 169.4, 170.,2 170.7
(Ac–CO); MS (70 eV, DCI): m/z (%) 985.0 (95)
[M+NH4]+, 967.9 (100) [M+H]+.
~
EtOAc=2:1); IR (pellet): ꢀ=3328 (aromat. C–H), 2972,
2936, 2862 (aliphat. C–H), 1754 (C¼O, ester), 1626
(C¼O, amide), 1596, 1492(C ¼C), 1526 (NCO), 1466,
1440, 1374 (CH3), 1228, 1156, 1114, 1076 (C–O), 954,
824 (1,3,4-trisubst.), 744, 706 cmꢁ1 (Si–Ph); UV–vis
(CH3CN): lmax (lg e)=319.5 (4.086), 266.0 nm (3.797);
1H NMR (300 MHz, CDCl3)11: d 1.02(s; 9H, (CH 3)3C),
2.01, 2.03, 2.09, 2.19 (s; each 3H, Ac–CH3), 3.63 (mc;
1H, 3-H), 3.70 (ddd, J=7.5, 9.0, 9.5 Hz; 1H, 8-Ha), 3.86
(ddd, J=5.0, 9.5, 11.5 Hz; 1H, 8-Hb), 3.92(s; 3H,
OCH3), 3.95 (s; 3H, OCH3), 4.08 (s; 3H, OCH3), 4.09
(mc; 1H, 500-H), 4.20 (d, J=6.5 Hz; 2H, 600-H2), 4.42
(ddd, J=4.5, 10.5, 13.5 Hz; 1H, 2–Ha), 4.54 (ddd,
J=7.5, 9.5, 10.5 Hz; 1H, 2-Hb), 5.10 (t, J=8.0 Hz; 1H,
200-H), 5.11 (ddd, J=0.5, 3.5, 10.5 Hz; 1H, 100-H), 5.46
(dt, J=1.0, 3.5 Hz; 1H, 400-H), 5.50 (ddd, J=1.0, 8.0,
10.5 Hz; 1H, 300-H), 6.72(dt, J=2.5, 8.0 Hz; 1H, 5–H),
0
6.82(s; 1H, 4 -H), 6.85 (dd, J=1.0, 2.5 Hz; 1H, 30-H),
7.08 (d, J=8.0 Hz; 1H, 4-H), 7.28–7.36 (mc; 4H, Ph–H),

1936
L. F. Tietze et al. / Bioorg. Med. Chem. 9 (2001) 1929–1939
~
[(3R/S)-3-(tert-Butyl-diphenyl-silyl)-oxymethyl-1-(5,6,7-
trimethoxyindol-2-yl-carbonyl)-2,3-dihydro-1H-indol-6-
ꢀ=3464 (OH), 2938 (aliphat. C-H), 1752 (C¼O, ester),
1628 (C¼O, amide), 1604, 1490 (C¼C), 1526 (NCO),
1440, 1384 (CH3), 1308 (OH), 1232, 1160, 1108, 1076
(C–O), 954, 874 cmꢁ1 (1,3,4-trisubst.); UV–vis
(CH3CN): lmax (lg e)=206.0 (4.641), 320.5 nm (4.443);
1H NMR (300 MHz, CDCl3):11 d 0.79 (sb; 1H, OH),
1.92, 1.95, 2.00, 2.10 (s; each 3H, Ac–CH3), 3.56 (mc;
1H, 3-H), 3.72(m ; 2H, 8-H2), 3.82(s; 3H, OCH ), 3.87
(s; 3H, OCH3), 3.95 (s; 2H, 600-H2), 4.00 (s; 3H, OCH3),
4.08 (t, J=6.5 Hz; 1H, 500-H), 4.49 (m; 2H, 2-H2), 5.03
(m; 2H, 100-H, 200-H), 5.38 (m; 2H, 300-H, 400-H), 6.67
(dd, J=2.5, 8.0 Hz; 1H, 5-H), 6.74 (s; 1H, 40-H),
6.87 (s; 1H, 30-H), 7.08 (d, J=8.0 Hz; 1H, 4-H),
8.02/8.04 (d, J=2.5 Hz; 1H, 7-H), 9.46/9.82 (sb; 1H,
indole NH); 13C NMR (75 MHz, CDCl3):11 d 20.47,
20.54, 20.63, 20.78 (Ac–CH3), 42.82/43.01 (C-3),
53.84/53.98 (C-2), 56.20 (OCH3), 61.02(OCH 3),
61.19/61.23 (C-600), 61.36 (OCH3), 64.89 (C-8), 66.84/
66.92(C-4 00), 68.60 (C-300), 70.74/70.79 (C-200), 70.81/
70.93 (C-500), 97.68/97.72(C-4 0), 99.55/99.59 (C-100),
106.5 (C-7), 106.6 (C-30), 113.3 (C-5), 123.5 (C-3a0),
124.5 (C-4), 125.5/125.6 (C-7a0), 126.6 (C-3a), 129.8/
129.9 (C-20), 138.7 (C-70), 140.4/140.6 (C-60), 144.9/
145.0 (C-7a), 150.0 (C-50), 157.0/157.1 (C-6), 160.3/
160.5 (NCO), 169.4, 170.0, 170.2, 170.4 (Ac–CO); MS
(70 eV, DCI): m/z (%) 746.6 (100) [M+NH4]+, 729.6
(65) [M+H]+.
yl]-2,3,4,6-tetra-O-acetyl-ꢁ-D-mannopyranoside
(16c).
To a solution of phenol 12 (200 mg, 0.31 mmol), mole-
cular sieves (4 A, 2g) and trichloroacetimidate 15c (106
mg, 0.38 mmol, 1.2equiv) in dichloromethane (20 mL)
was added dropwise a solution of boron trifluoride die-
thyl ether complex (26 mg, 0.31 mmol, 1.0 equiv) in di-
chloromethane (2.0 mL) at 0 ꢀC and the mixture was
stirred for 24 h at rt. The molecular sieves were filtered
off, and the filtrate was washed with saturated NaHCO3
solution. The aqueous layer was extracted with di-
chloromethane (3Â100 mL) and the combined organic
phases were washed with brine and dried over Na2SO4.
Evaporation and purification by column chromato-
graphy (PE/EtOAc=3:1) afforded 16c (161 mg, 0.166
mmol, 53%) as a colorless foam. Rf=0.13 (PE/
c
3
~
EtOAc=2:1); IR (pellet): ꢀ=3462(NH), 2936 (aliphat.
C-H), 1754 (C¼O, ester), 1630 (C¼O, amide), 1602,
1490 (C¼C), 1526 (NCO), 1468, 1440, 1430, 1412, 1386
(CH3), 1228, 1162, 1112, 1074 (C–O), 956, 856 (1,3,4-
trisubst.), 744, 706 cmꢁ1 (Si–O); UV–vis (CH3CN): lmax
(lg e)=321.0 nm (4.379); 1H NMR (500 MHz,
CDCl3):11 d 1.00/1.01 (s; 9H, (CH3)3C), 1.97/1.98, 2.01,
2.02, 2.03 (s; each 3H, Ac–CH3), 3.64 (mc; 1H, 3-H),
3.67/3.68 (dd, J=8.0, 10.0 Hz; 1H, 8–Ha), 3.85/3.86
(dd, J=5.5, 10.0 Hz; 1H, 8–Hb), 3.90 (s; 3H, OCH3),
3.93 (s; 3H, OCH3), 4.03/4.05 (dd, J=2.0, 12.5 Hz; 1H,
600-Ha), 4.05 (s; 3H, OCH3), 4.11 (m; 1H, 500-H), 4.32–
4.34 (dd, J=3.5, 12.5 Hz; 1H, 600-Hb), 4.39/4.41 (dd,
J=4.5, 10.5 Hz; 1H, 2-Ha), 4.51/4.53 (dd, J=4.5, 9.5
Hz; 1H, 2-Hb), 5.36 (t, J=10.0 Hz; 1H, 400-H), 5.43
(dd, J=2.0, 3.5 Hz; 1H, 200-H), 5.54 (m; 2H, 100-H, 300-
H), 6.74 (2dd, J=2.5, 8.0 Hz; 1H, 5-H), 6.81 (s; 1H,
40-H), 6.83 (2d, J=2.0 Hz; 1H, 30-H), 7.07 (d, J=8.0
Hz; 1H, 4-H), 7.25–7.35 (mc; 4H, Ph–H), 6.35–7.42
(mc; 2H, Ph–H), 7.53–7.60 (mc; 4H, Ph–H), 8.13 (2d,
J=2.5 Hz; 1H, 7-H), 9.37 (sb; 1H, indole NH); 13C
NMR (75 MHz, CDCl3):11 d 19.21 ((CH3)3C), 20.63,
20.69, 20.85, 21.00 (Ac–CH3), 26.78 ((CH3)3C), 43.00
(C-3), 53.78 (C-2), 56.26 (OCH3), 61.08 (OCH3), 61.44
(OCH3), 62.16 (C-6‘‘), 66.02 (C-400), 66.65 (C-8), 68.92
(C-300), 69.08 (C-200), 69.42(C-5 00), 95.96/96.05 (C-40),
97.67 (C-100), 106.2(C-3 0), 107.0/107.1 (C-7), 112.0/
112.2 (C-5), 123.7 (C-3a0), 124.8/124.9 (C-4), 125.4 (C-
3a), 126.8 (C-7a0), 127.8 (Ph–C), 129.8/130.0 (Ph–C),
133.0 (C-20), 133.1 (C-1000), 133.5 (Ph–C), 138.9 (C-70),
140.5 (C-60), 145.1 (C-7a), 150.0 (C-50), 155.7 (C-6),
160.0 (NCO), 169.7, 169.8, 170.0, 170.1 (Ac–CO); MS
(70 eV, DCI): m/z 984.5 (100) [M+NH3]+, 967.5 (35)
[M]+.
[(3R/S)-3-Hydroxymethyl-1-(5,6,7-trimethoxyindol-2-yl-
carbonyl)-2,3-dihydro-1H-indol-6-yl]-2,3,4,6-tetra-O-ace-
tyl-ꢀ-D-glucopyranoside (18b). To a solution of gluco-
side 16b (1.30 g, 1.34 mmol) in tetrahydrofuran (100
mL) was added tetrabutylammonium fluoride on silica
gel (1.1 mmol/1 g, 2.45 g, 2.69 mmol, 2.0 equiv) at 0 ꢀC.
The reaction mixture was stirred at 0 ꢀC for 4 h. After
removal of the solvents in vacuo the residue was pur-
ified by column chromatography (PE/EtOAc=1:1) to
yield 18b (596 mg, 0.82mmol, 61%) as a colorless foam.
Rf=0.13 (PE/EtOAc=1:2); IR (pellet): ꢀ~=3468 (NH),
2940 (aliphat. C-H), 1756 (C¼O, ester), 1626 (C¼O,
amide), 1600, 1492(C ¼C), 1526 (NCO), 1468, 1440,
1414, 1378 (CH3), 1308 (OH), 1232, 1162, 1110, 1044
(C–O), 958, 878 cmꢁ1 (1,3,4-trisubst.); UV–vis
(CH3CN): lmax (lg e)=207.5 (4.614), 320.5 nm (4.458);
1H NMR (500 MHz, CDCl3):11 d 2.01/2.02, 2.04/2.05,
2.06, 2.07/2.08 (s; each 3H, Ac–CH3), 3.64 (mc; 1H, 3-
H), 3.78 (dd, J=7.0, 10.5 Hz; 1H, 8-Ha), 3.80 (m; 2H,
500-H), 3.86 (dd, J=5.0, 10.5 Hz; 1H, 8-Hb), 3.89/3.90 (s;
3H, OCH3), 3.94/3.96 (s; 3H, OCH3), 3.99/4.07 (s; 3H,
OCH3), 4.18 (dd, J=1.5, 12.5 Hz; 1H, 600-Ha), 4.26 (dd,
J=4.5, 12.5 Hz; 1H, 600-Hb), 4,48 (dd, J=4.5, 10.5 Hz;
1H, 2-Ha), 4.61 (dd, J=2.5, 10.5 Hz; 1H 2-Hb), 5.17 (m;
2H, 100-H, 200-H), 5.28 (m; 2H, 300-H, 400-H), 6.74/6.75
(dd, J=2.5, 8.0 Hz; 1H, 5–H), 6.84 (s; 1H, 40-H), 6.96/
6.99 (s; 1H, 30-H), 7.17/7.18 (d, J=8.0 Hz; 1H, 4-H),
8.09/8.14 (sb; 1H, 7-H), 9.43/10.23 (sb; 1H, indole NH);
13C NMR (125 MHz, CDCl3):11 d 20.21, 20.47, 20.54/
20,59, 20.61/20.63, (Ac–CH3), 42.89/43.85 (C-3), 53.19/
54.22 (C-2), 56.29/56.32 (OCH3), 60.96/61.57 (C-600),
61.04/61.08 (OCH3), 61.52/61.69 (OCH3), 64.75/64.92
(C-8), 67.40/67.97 (C-400), 70.85/71.06 (C-300), 71.29/
71.81 (C-200), 72.86 (C-500), 97.82/98.14 (C-40), 98.49/
98.82(C-1 00), 105.3/106.3 (C-7), 106.7/106.9 (C-30),
[(3R/S)-3-Hydroxymethyl-1-(5,6,7-trimethoxyindol-2-yl-
carbonyl)-2,3-dihydro-1H-indol-6-yl]-2,3,4,6-tetra-O-ace-
tyl-ꢀ-D-galactopyranoside (18a). To a solution of galac-
toside 16a (1.50 g, 1.55 mmol) in tetrahydrofuran (100
mL) was added tetrabutylammonium fluoride on silica
gel (1.1 mmol/1 g, 2.82 g, 3.10 mmol, 2.0 equiv) at 0 ꢀC.
The reaction mixture was warmed to 40 ꢀC and stirred
for 5 h. After removal of the solvents in vacuo the resi-
due was purified by column chromatography (PE/
EtOAc=1:1) to yield 18a (1.05 g, 1.44 mmol, 93%) as a
colorless foam. Rf=0.15 (PE/EtOAc=1:2); IR (pellet):

L. F. Tietze et al. / Bioorg. Med. Chem. 9 (2001) 1929–1939
1937
113.4/113.6 (C-5), 123.4/123.6 (C-3a0), 124.5/124.7 (C-
4), 125.7 (C-7a0), 126.3/126.5 (C-3a), 130.0/130.4 (C-20),
138.6/138.8 (C-70), 140.7/141.3 (C-60), 144.7/145.1 (C-
7a), 150.1 (C-50), 157.0 (C-6), 160.6/161.2(NCO), 169.0/
169.3, 169.4, 170.1/170.2, 171.1/172.0 (Ac–CO); MS (70
eV, DCI): m/z (%) 746.4 (100) [M+NH4]+, 729.4 (25)
[M+H]+, 704.4 (20) [MꢁOAc]+.
321.5 nm (4.649); 1H NMR (300 MHz, CDCl3):11 d 1.94,
1.96/1.99, 2.01/2.02, 2.11 (s; each 3H, Ac–CH3), 3.50
(mc; 1H, 3-H), 3.74 (m; 2H, 8-H2), 3.83 (s; 3H, OCH3),
3.87 (s; 3H, OCH3), 4.01 (s; 3H, OCH3), 4.04 (mc; 1H,
500-H), 4.12(dd, J=1.0, 6.0 Hz; 2H, 600-H2), 4.41 (dt,
J=5.0, 6.0 Hz; 1H, 2-Ha), 4.54 (dt, J=5.0, 10.0 Hz;
1H, 2-Hb), 5.05 (m; 2H, 100-H, 200-H), 5.40 (d,
J=3.5 Hz; 1H, 400-H), 5.43 (dd, J=8.0, 10.5 Hz;
1H, 300-H), 6.70 (dd, J=2.5, 8.0 Hz; 1H, 5-H), 6.80
(s; 1H, 40-H), 6.83 (d, J=1.5, Hz; 1H, 30-H), 7.10
(d, J=8.0 Hz; 1H, 4-H), 8.00 (d, J=2.5 Hz; 1H, 7-H),
9.31 (sb; 1H, indole NH); 13C NMR (75 MHz,
CDCl3):11 d 20.58, 20.65, 20.77 (Ac–CH3), 43.03 (C-3),
[(3R/S)-3-Hydroxymethyl-1-(5,6,7-trimethoxyindol-2-yl-
carbonyl)-2,3-dihydro-1H-indol-6-yl]-2,3,4,6-tetra-O-ace-
tyl-ꢁ-D-mannopyranosid (18c). To a solution of manno-
side 16c (572mg, 0.59 mmol) in tetrahydrofuran (20
mL) was added tetrabutylammonium fluoride on silica
gel (1.1 mmol/1 g, 1.14 g, 1.20 mmol, 2.0 equiv) at 0 ꢀC.
The reaction mixture was stirred at rt for 2h. After
removal of the solvents in vacuo the residue was pur-
ified by column chromatography (PE/EtOAc=1:1) to
yield 18c (268 mg, 0.37 mmol, 62%) as a colorless foam.
46.95 (C-2), 54.77 (C-600), 56.28 (OCH3), 61.12(OCH ),
3
61.35 (C-8), 61.47 (OCH3), 66.93 (C-400), 68.62(C-3 00),
0
70.87 (C-200), 71.14 (C-500), 97.62(C-4 ), 99.64/99.74 (C-
100), 106.5 (C-30), 106.8/107.1 (C-7), 113.4/113.5 (C-5),
123.6 (C-3a0), 124.5/124.6 (C–4), 125.6 (C-3a), 125.7/
125.8 (C-7a0), 129.6 (C-20), 133.0 (C-100), 139.0 (C-70),
140.5 (C-60), 144.9 (C-7a), 150.3 (C-50), 157.6 (C-6),
160.2(NCO), 169.4, 170.1, 170.3, 170.4 (Ac–CO); MS
(70 eV, DCI): m/z (%) 764.4 (100) [M+NH4]+, 747.4
(45) [M+H]+.
~
Rf=0.19 (PE/EtOAc=1:2); IR (pellet): ꢀ=3466 (OH),
2938 (aliphat. C–H), 1752 (C¼O, ester), 1630 (C¼O,
amide), 1602, 1490 (C¼C), 1526 (NCO), 1468, 1440,
1412, 1372 (CH3), 1230, 1162, 1110, 1084 (C–O), 950,
870 cmꢁ1 (1,3,4-trisubst.); UV–vis (CH3CN): lmax (lg
e)=207.5 (4.624), 321.0 nm (4.479); 1H NMR
(300 MHz, CDCl3):11 d 2.03, 2.04, 2.06, 2.20 (s; each 3H,
Ac–CH3), 3.66 (mc; 1H, 3-H), 3.78 (mc; 1H, 8-Ha), 3.80
(mc; 1H, 8-Hb), 3.90 (s; 3H, OCH3), 3.94 (s; 3H, OCH3),
4.07 (s; 3H, OCH3), 4.08 (mc; 1H, 600-Ha), 4.13 (mc; 1H,
500-H), 4.30 (dd, J=4.5, 12.5 Hz; 1H, 600-Hb), 4,49 (m;
1H, 2-Ha), 4.61 (m; 1H 2-Hb), 5.39 (t, J=10.0 Hz; 1H,
400-H), 5.45 (dd, J=2.0, 3.5 Hz; 1H, 200-H), 5.57 (m; 2H,
100-H, 300-H), 6.79/6.81 (dd, J=2.5, 8.0 Hz; 1H, 5-H),
6.83 (s; 1H, 40-H), 6.96 (d, J=2.0 Hz; 1H, 30-H), 7.18
(d, J=8.0 Hz; 1H, 4-H), 8.17 (d, J=2.5 Hz; 1H, 7-H),
[(3R/S)-3-Chloromethyl-1-(5,6,7-trimethoxyindol-2-yl-
carbonyl)-2,3-dihydro-1H-indol-6-yl]-2,3,4,6-tetra-O-ace-
tyl-ꢀ-D-glucopyranoside (19b). The primary alcohol 18b
(500 mg, 0.69 mmol) was dissolved in a mixture of di-
chloromethane (5 mL) and tetrachloromethane (25 mL),
and triphenylphosphane (277 mg, 1.37 mmol, 2.0 equiv)
was added. After stirring for 24 h at 50 ꢀC the solvents
were removed in vacuo and the residue was purified by
column chromatography (PE/EtOAc=1:1) to afford
19b (475 mg, contaminated with triphenylphosphane) as
a white solid. Rf=0.25 (PE/EtOAc=1:1); IR (pellet):
ꢀ~=3464 (NH), 3056 (aromat. C–H), 2936, 2854 (ali-
phat. C-H), 1756 (C¼O, ester), 1628 (C¼O, amide),
1594, 1492(C ¼C), 1526 (NCO), 1468, 1440, 1386
(CH3), 1232, 1114, 1068 (C–O), 960, 878 (1,3,4-tri-
9.42(s
;
1H, indole NH); 13C NMR (75 MHz,
b
CDCl3):11 d 20.61, 20.66, 20.84, 21.01 (Ac–CH3), 42.74/
43.82 (C-3), 53.66/53.69 (C-2), 56.20 (OCH3), 61.06
(OCH3), 61.42(OCH ), 62.12 (C-600), 65.06/65.14 (C-8),
3
65.95 (C-400), 66.86 (C-300), 69.01 (C-200), 69.34 (C-500),
95.71 (C-40), 97.57 (C-100), 106.4 (C-30), 107.1 (C-7),
112.1/112.2 (C-5), 123.6 (C-3a0), 124.4/124.5 (C-4),
125.4 (C-3a), 126.1 (C-7a0), 129.8 (C-20), 136.8 (C-70),
ꢁ1
subst.), 752cm
(Cl); UV–vis (CH3CN): lmax (lg
1
e)=195.0 (4.786), 265.0 (3.809), 320.0 nm (4.400); H
NMR (500 MHz, CDCl3):11 d 2.03, 2.04, 2.07, 2.08 (s;
each 3H, Ac–CH3), 3.57 (dd, J=8.5, 10.5 Hz; 1H, 8-Ha),
3.81 (dd, J=4.5, 8.5 Hz; 1H, 8-Hb), 3.88 (m; 2H, 3-H,
500-H), 3.91 (s; 3H, OCH3), 3.95 (s; 3H, OCH3), 4.08 (s;
3H, OCH3), 4.18 (dd, J=2.0, 12.5 Hz; 1H, 600-Ha), 4.28
(dd, J=5.0, 12.5 Hz; 1H, 600-Hb), 4.48 (dd, J=4.5, 10.5
Hz; 1H, 2-Ha), 4.66 (dd, J=9.5, 10.5 Hz; 1H, 2-Hb),
5.17 (m; 2H, 100-H, 200-H), 5.29 (m; 1H, 300-H, 400-H),
6.76 (dd, J=2.5, 8.0 Hz; 1H, 5-H), 6.87 (s; 1H, 40-H),
6.96 (d, J=2.0 Hz; 1H, 30-H), 7.18 (d, J=8.0 Hz; 1H, 4-
140.4 (C-60), 145.1/145.2(C-7a), 150.0 (C-5 ), 155.8 (C-
0
6), 160.1 (NCO), 169.7, 169.9, 170.0, 170.6 (Ac–CO);
MS (70 eV, DCI): m/z (%) 746.6 (100) [M+NH4]+,
729.6 (25) [M+H]+.
[(3R/S)-3-Chloromethyl-1-(5,6,7-trimethoxyindol-2-yl-
carbonyl)-2,3-dihydro-1H-indol-6-yl)-2,3,4,6-tetra-O-ace-
tyl-ꢀ-D-galactopyranoside (19a). The primary alcohol
18a (122 mg, 0.167 mmol) was dissolved in a mixture of
dichloromethane (2mL) and tetrachloromethane (5
mL), and triphenylphosphane (101 mg, 0.501 mmol, 3.0
equiv) was added. After stirring for 8 h at 50 ꢀC the
solvents were removed in vacuo and the residue was
purified by column chromatography (PE/EtOAc=1:1)
to afford 19a (103 mg, 0.138 mmol, 82%) as a white
H), 8.07 (sb; 1H, 7-H), 9.42(s ; 1H, indole NH); 13C
b
NMR (75 MHz, CDCl3):11 d 20.50, 20.60, 20.64, 21.04
(Ac–CH3), 42.90/43.03 (C-3), 46.88/47.02 (C-2), 54.81/
54.87 (C-600), 56.25 (OCH3), 61.08 (OCH3), 61.45 (C-8),
61.54 (OCH3), 67.94 (C-400), 70.97 (C-300), 71.86 (C-200),
0
71.91 (C-500), 97.72(C-4 ), 98.67 (C-100), 106.2/106.3 (C-
solid. Rf=0.30 (PE/EtOAc=1:1); IR (pellet): ꢀ=3460
7), 106.6 (C-30), 113.4 (C-5), 123.5/123.6 (C-3a0), 124.5/
124.7 (C-4), 125.7/125.8 (C-7a0), 129.6 (C-3a), 133.2 (C-
20), 138.0 (C-70), 140.7/140.8 (C-60), 144.9 (C-7a), 150.2
(C-50), 157.4 (C-6), 160.4 (NCO), 169.3, 170.1, 170.7,
171.1 (Ac–CO); MS (70 eV, DCI): m/z (%) 764.4 (100)
[M+NH4]+, 747.4 (10) [M+H]+.
~
(NH), 2992, 2936, 2836 (aliphat. C–H), 1728 (C¼O,
ester), 1624 (C¼O, amide), 1598, 1492(C ¼C), 1526
(NCO), 1464, 1442, 1412, 1388 (CH3), 1234, 1196, 1176,
1110, 1084 (C–O), 946, 826 (1,3,4-trisubst.), 748
cmꢁ1(Cl); UV–vis (CH3CN): lmax (lg e)=207.0 (4.898),

1938
L. F. Tietze et al. / Bioorg. Med. Chem. 9 (2001) 1929–1939
[(3R/S)-3-Chloromethyl-1-(5,6,7-trimethoxyindol-2-yl-
carbonyl)-2,3-dihydro-1H-indol-6-yl]-2,3,4,6-tetra-O-ace-
tyl - ꢁ-D-mannopyranoside (19c). The primary alcohol
18c (200 mg, 0.28 mmol) was dissolved in a mixture of
dichloromethane (2mL) and tetrachloromethane (10
mL), and triphenylphosphane (112mg, 0.55 mmol, 2.0
equiv) was added. After stirring for 8 h at 50 ꢀC the
solvents were removed in vacuo and the residue was
purified by column chromatography (PE/EtOAc=1:1)
to afford 19c (202 mg, 0.27 mmol, 98%) as a white solid.
(C-400), 72.21 (C-300), 74.81 (C-200), 76.75 (C-500), 99.26
(C-40), 102.9 (C-100), 107.7 (C-7), 108.1 (C-30), 113.6/
113.7 (C-5), 125.1 (C-3a0), 125.8 (C-4), 126.7 (C-7a0),
0
127.0 (C-3a), 131.4 (C-20), 140.2(C-7 ), 141.5 (C-60),
145.6/145.7 (C-7a), 151.0 (C-50), 159.3 (C-6), 162.3
(NCO); MS (70 eV, DCI): m/z (%) 596.3 (100)
[M+NH4]+, 560.3 (50) [M+2H]+.
[(3R/S)-3-Chloromethyl-1-(5,6,7-trimethoxyindol-2-yl-
carbonyl)-2,3-dihydro-1H-indol-6-yl]-ꢀ-D-glucopyrano-
side (17b). Glucoside 19b (475 mg, max. 0.63 mmol,
slightly contaminated with triphenylphosphane) was
dissolved in methanol (10 mL) and treated with a solu-
tion of sodium methoxide in methanol (5.4 M, 22 mL,
0.12mmol, 0.2equiv). The mixture was stirred for 2h at
rt before methanol (10 mL) was added, and the solution
was neutralized with acidic duolite. Evaporation and
purification by column chromatography (EtOAc/
MeOH=12:1) afforded 17b (209 mg, 0.36 mmol, 60%
from 18b) as a white powder. Rf=0.59 (EtOAc/
~
Rf=0.29 (PE/EtOAc=1:1); IR (pellet): ꢀ=3466 (NH),
2940 (aliphat. C–H), 1752 (C¼O, ester), 1630 (C¼O,
amide), 1604, 1490 (C¼C), 1526 (NCO), 1468, 1442,
1412, 1388 (CH3), 1232, 1126, 1110, 1084 (C–O), 944,
870 (1,3,4-trisubst.), 750 cmꢁ1 (Cl); UV–vis (CH3CN):
1
lmax (lg e)=207.0 (4.612), 319.5 nm (4.407); H NMR
(300 MHz, CDCl3):11 d 2.00/2.01, 2.02, 2.04, 2.18, (s;
each 3H, Ac–CH3), 3.54 (mc; 1H, 3-H), 3.80 (mc; 2H, 8-
H2), 3.88 (s; 3H, OCH3), 3.91 (s; 3H, OCH3), 4.03 (mc;
1H, 600-Ha), 4.04 (s; 3H, OCH3), 4.10 (mc; 1H, 500-H),
4.27 (dd, J=4.5, 12.5 Hz; 1H, 600-Hb), 4,45 (m; 1H, 2-
Ha), 4.63 (m; 1H 2-Hb), 5.27 (t, J=10.0 Hz; 1H, 400-H),
5.42(dd, J=2.0, 3.5 Hz; 1H, 200-H), 5.54 (m; 2H, 100-H,
300-H), 6.81 (dd, J=2.5, 8.0 Hz; 1H, 5-H), 6.83 (s; 1H,
40-H), 6.92(d, J=2.0 Hz; 1H, 30-H), 7.15 (d, J=8.0 Hz;
1H, 4-H), 8.14 (sb; 1H, 7-H), 9.39 (sb; 1H, indole NH);
13C NMR (75 MHz, CDCl3):11 d 20.56, 20.59, 20.76,
20.93 (Ac–CH3), 42.93 (C-3), 46.66 (C-2), 54.59 (C-600),
~
MeOH=12:1); IR (pellet): ꢀ=3404 (OH), 3060 (aro-
mat. C–H), 2928 (aliphat. C–H), 1628 (C¼O, amide),
1602, 1490 (C¼C), 1526 (NCO), 1464, 1440, 1414, 1390
(CH3), 1308 (OH), 1236, 1192, 1116, 1074 (C–O), 904,
826 (1,3,4-trisubst.), 750 cmꢁ1 (Cl); UV–vis (CH3CN):
lmax (lg e)=195.0 (4.773), 265.5 (3.690), 320.0 (4.250),
1
272.5 nm (3.721); H NMR (300 MHz, DMSO-d6):11
d
3.13–3.25 (m; 4H, 3-H, 200-H, 300-H, 400-H), 3.49 (mc; 1H,
8-Ha), 3.65 (mc; 1H, 8-Hb), 3.78 (s; 3H, OCH3), 3.79 (s;
3H, OCH3), 3.81 (m; 2H, 600-H2), 3.92(s; 3H, OCH ),
56.13 (OCH3), 61.02(OCH ), 61.35 (OCH3), 62.04 (C-
3
8), 65.87 (C-400), 68.74 (C-300), 69.02(C-2 00), 69.24 (C-
500), 95.79/95.64 (C-40), 97.51 (C-100), 106.4 (C-30), 107.0/
107.1 (C-7), 112.2/112.3 (C-5), 123.5 (C-3a0), 124.5 (C-
3a), 125.2/125.5 (C-4), 126.9 (C-7a0), 129.5 (C-20), 136.8
(C-70), 140.5 (C-60), 144.8/144.9 (C-7a), 150.0 (C-50),
156.1 (C-6), 160.1 (NCO), 169.6, 169.8, 169.9, 170.5
(Ac–CO); MS (70 eV, DCI): m/z (%) 764.4 (100)
[M+NH4]+.
3
3.94 (m; 1H, 500-H), 4.27 (mc; 1H, 2-Ha), 4.58 (mc; 1H,
2-Hb), 5.74 (mc; 1H, 100-H), 6.76/6.77 (dd, J=2.5, 8.0
Hz; 1H, 5-H), 6.93 (s; 1H, 40-H), 6.98 (d, J=2.0 Hz; 1H,
30-H), 7.31 (d, J=8.0 Hz; 1H, 4-H), 7.71 (sb; 1H, 7-H);
13C NMR (125 MHz, CD3OD):11 d 43.66 (C-3), 56.07/
56.18 (C-2), 56.67/56.69 (OCH3), 61.72(OCH ), 61.88
3
(OCH3), 62.48 (C600, C-8), 71.13 (C-400, C-300), 74.81 (C-
200), 77.90 (C-500), 99.18 (C-40), 102.3 (C-100), 107.7 (C-7),
108.1 (C-30), 113.5/113.6 (C-5), 125.0 (C-3a0), 125.8/
125.9 (C-4), 127.0 (C-7a0), 127.1/127.2 (C-3a), 131.4 (C-
[(3R/S)-3-Chloromethyl-1-(5,6,7-trimethoxyindol-2-yl-
carbonyl)-2,3-dihydro-1H-indol-6-yl]-ꢀ-D-galactopyrano-
side (17a). Galactoside 19a (93 mg, 0.13 mmol) was dis-
solved in methanol (2mL) and treated with a solution
of sodium methoxide in methanol (5.4 M, 5 mL, 0.025
mmol, 0.2equiv). The mixture was stirred for 2h at rt
before methanol (2mL) was added, and the solution
was neutralized with acidic duolite. Evaporation and
purification by column chromatography (EtOAc/
MeOH=12:1) afforded 17a (69 mg, 0.12mmol, 95%) as
a white powder. Rf=0.48 (EtOAc/MeOH=12:1); IR
(pellet): ꢀ~=3420 (OH), 2936 (aliphat. C–H), 1628
(C¼O, amide), 1526 (NCO), 1492 (C¼C), 1466, 1442,
0
20), 140.2(C-7 ), 141.5 (C-60), 145.6/145.7 (C-7a), 151.0
(C-50), 159.2(C-6), 162.3 (NCO); MS (70 eV, DCI): m/z
(%) 596.5 (100) [M+NH4]+, 560.5 (50) [M+2H]+,
180.1 (65) [Glc]+.
[(3R/S)-3-Chloromethyl-1-(50,60,70-trimethoxyindol-20-yl-
carbonyl)-2,3-dihydro-1H-indol-6-yl]-ꢁ-D-mannopyrano-
side (17c). Mannoside 19c (151 mg, 0.20 mmol) was
dissolved in methanol (5 mL) and treated with a solu-
tion of sodium methoxide in methanol (5.4 M, 8 mL,
0.04 mmol, 0.2equiv). The mixture was stirred for 2h at
rt before methanol (5 mL) was added, and the solution
was neutralized with acidic duolite. Evaporation and
purification by column chromatography (EtOAc/
MeOH=12:1) afforded 17c (116 mg, 0.20 mmol, 99%)
as a white powder. Rf=0.52(EtOAc/MeOH=12:1); IR
1414, 1392(CH ), 1236, 1196, 1106, 1076 (C–O), 950,
3
868 (1,3,4-trisubst.), 750 cmꢁ1 (Cl); UV–vis (CH3CN):
1
lmax (lg e)=207.0 (4.596), 320.0 nm (4.421); H NMR
(300 MHz, CD3OD):11 d 3.39–3.76 (m; 9H, 3-H, 8-H2,
200-H, 300-H, 400-H, 500-H, 600-H), 3.76/3.77 (s; 3H,
OCH3), 3.78 (s; 3H, OCH3), 3.93 (s; 3H, OCH3), 4.29
(mc; 1H, 2-Ha), 4.50 (mc; 1H, 2-Hb), 4.69 (mc; 1H, 100-
H), 6.75/6.76 (dd, J=2.5, 8.0 Hz; 1H, 5-H), 6.86/6.87 (s;
1H, 40-H), 6.90/6.91 (s; 1H, 30-H), 7.16 (d, J=8.0 Hz;
1H, 4-H), 7.72(s b; 1H, 7-H); 13C NMR (125 MHz,
CD3OD):11 d 43.67 (C-3), 56.20 (C-2), 56.74 (OCH3),
~
(pellet): ꢀ=3422 (OH), 2936, 2840 (aliphat. C–H), 1628
(C¼O, amide), 1528 (NCO), 1492 (C¼C), 1466, 1442,
1414, 1392(CH ), 1308 (OH), 1236, 1196, 1110, 1086
3
(C–O), 912, 824 (1,3,4-trisubst.), 750 cmꢁ1 (Cl); UV–vis
(CH3CN): lmax (lg e)=207.0 (4.400), 320.5 nm (4.365);
1H NMR (500 MHz, CD3OD):11 d 3.58–3.68 (mc; 2H, 3-
H, 8-Ha), 3.71–3.80 (mc; 4H, 8-Hb, 600-H2, 500-H), 3.84
61.72(OCH ), 61.88 (OCH3), 62.39 (C-600, C-8), 70.41
3

L. F. Tietze et al. / Bioorg. Med. Chem. 9 (2001) 1929–1939
1939
(s; 3H, OCH3), 3.85 (m; 1H, 400-H), 3.86 (s; 3H, OCH3),
3.90 (dd, J=2.0, 10.0 Hz; 1H, 300-H), 4.00 (m; 1H, 200-
H), 4.35 (mc; 1H, 2-Ha), 4.56 (mc; 1H, 2-Hb), 5.47 (d,
J=1.5 Hz; 1H, 100-H), 6.86 (dd, J=1.5, 8.0 Hz; 1H, 5-
H), 6.90 (s; 1H, 40-H), 6.97 (s; 1H, 30-H), 7.24 (d, J=8.0
Hz; 1H, 4-H), 7.91 (sb; 1H, 7-H); 13C NMR (125 MHz,
CD3OD): d 43.67 (C-3), 56.20 (C-2), 56.74 (OCH3),
(EC 3.2.1.23, Grade X), b-d-glucosidase (EC 3.2.1.21)
or a-d-mannosidase (EC 3.2.1.25) (all purchased from
Sigma Germany, Deisenhofen, Germany) to the cells
during incubation with the substances.
Results are presented as means of at least three separate
experiments.
61.72(OCH ), 61.88 (OCH3), 62.39 (C-600, C-8), 70.41
3
(C-400), 72.21 (C-300), 74.81 (C-200), 76.75 (C-500), 99.26
(C-40), 102.9 (C-100), 107.7 (C-7), 108.1 (C-30), 113.6/
113.7 (C-5), 125.1 (C-3a0), 125.8 (C-4), 126.7 (C-7a0),
Acknowledgements
0
127.0 (C-3a), 131.4 (C-20), 140.2(C-7 ), 141.5 (C-60),
145.6/145.7 (C-7a), 151.0 (C-50), 159.3 (C-6), 162.3
(NCO); MS (70 eV, DCI): m/z (%) 596.3 (100)
[M+NH4]+, 560.3 (50) [M+2H]+.
This research was supported by the Deutsche For-
schungsgemeinschaft (Sonder-forschungsbereich 416),
and the Fonds der Chemischen Industrie. F.H. thanks
the Fonds for a PhD fellowship. The authors would like
to thank Angela Rubeling and Stefanie Heidrich for
their excellent work in the cytotoxicity tests.
Cell culture
Human bronchial carcinoma cells of line A549 (ATCC
CCL 185) were kindly provided by the Institut fur Zell-
biologie, Universitat Essen, and were maintained as
exponentially growing cultures at 37 ꢀC and 7.5% CO2
in air in DMEM (Dulbecco’s modified Eagle’s medium,
Biochrom, Berlin, Germany) supplemented with 10%
fetal calf serum (heat-inactivated for 30 min at 56 ꢀC,
GibcoBRL, Karlsruhe, Germany), 44 mM NaHCO3
(Biochrom, Berlin, Germany) and 4 mM l-Glutamine
(GibcoBRL, Karlsruhe, Germany).
References and Notes
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Adherent cells of line A549 were sown in triplicate in six
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105 cells per cavity. Culture medium was sucked off
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incubation medium to a final concentration of DMSO
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The relative clone forming rate was determined accord-
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11. Compounds 16–19 were obtained as mixtures of diaster-
eomers. The NMR spectroscopical data refer to these mix-
tures.
relative clone forming rate ð%Þ
number of clones counted after exposure
¼
ꢀ 100
number of clones counted in the control
Liberation of the drugs from their glycosidic prodrugs
was achieved by addition of 0.4 U/mL b-d-galactosidase