Duocarmycin-based prodrugs for cancer prodrug monotherapy

Article abstract of DOI:10.1016/j.bmc.2008.05.009

The synthesis and biological evaluation of novel prodrugs based on the cytotoxic antibiotic duocarmycin SA (1) for a selective treatment of cancer using a prodrug monotherapy (PMT) are described. Transformation of the phenol 8 with the glucuronic acid benzyl ester trichloroacetimidate 9b followed by reaction with DMAI·HCl (10) gives the glucuronide 11b, which is deprotected to afford the desired prodrug 4a containing a glucuronic acid moiety. In addition, the prodrug 4b with a glucuronic methyl ester unit is prepared. The cytotoxicity of the glucuronides is determined using a HTCFA-assay with IC₅₀ values of 610 nM for 4a and 3300 nM for 4b. In the presence of β-glucuronidase, 4a expresses an IC₅₀ value of 0.9 nM and 4b of 2.1 nM resulting in QIC₅₀ values of about 700 for 4a and 1600 for 4b.

Full text of DOI:10.1016/j.bmc.2008.05.009

Bioorganic & Medicinal Chemistry 16 (2008) 6312–6318
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Duocarmycin-based prodrugs for cancer prodrug monotherapy
a
a
a
b
Lutz F. Tietze a, , Heiko J. Schuster , Kianga Schmuck , Ingrid Schuberth , Frauke Alves
*
a Institute of Organic und Biomolecular Chemistry of the Georg-August-University Göttingen, Tammannstraße 2, D-37077 Göttingen, Germany
b Centre of Internal Medicine, Department of Haematology and Oncology of the Georg-August-University of Göttingen, Robert-Koch-Straße 40, 37075 Göttingen, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis and biological evaluation of novel prodrugs based on the cytotoxic antibiotic duocarmycin
SA (1) for a selective treatment of cancer using a prodrug monotherapy (PMT) are described. Transforma-
tion of the phenol 8 with the glucuronic acid benzyl ester trichloroacetimidate 9b followed by reaction
with DMAIꢀHCl (10) gives the glucuronide 11b, which is deprotected to afford the desired prodrug 4a con-
taining a glucuronic acid moiety. In addition, the prodrug 4b with a glucuronic methyl ester unit is pre-
pared. The cytotoxicity of the glucuronides is determined using a HTCFA-assay with IC50 values of 610 nM
for 4a and 3300 nM for 4b. In the presence of b-glucuronidase, 4a expresses an IC50 value of 0.9 nM and
4b of 2.1 nM resulting in QIC50 values of about 700 for 4a and 1600 for 4b.
Received 1 May 2008
Accepted 5 May 2008
Available online 7 May 2008
Keywords:
Antitumor agents
Cancer therapy
Cytotoxicity
Duocarmycin
Glucuronides
Prodrugs
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
b-D-galactosidase the in situ formed seco-drug 3 undergoes a fast
cyclization to give the drug 5 with a spiro-methylcyclopropylcyclo-
hexadienone moiety as pharmacophoric unit.6 Since the prodrug 2
has an IC50 value of 3600 nM in the absence of the cleaving enzyme
and of 0.75 nM in its presence it results in a QIC50 value of about
5000.4 Thus, this substance is superior to almost all compounds de-
scribed so far for the use of ADEPT. Moreover, the in vivo investiga-
tions using mice are highly promising.7
On the other hand, the ADEPT approach has the disadvantage of
being rather costly and must cope with a possible immune re-
sponse of the patients against the used monoclonal antibody and
enzyme.
We therefore also investigated the second approach using the
peptide pentagastrin for targeting8 and the third approach employ-
ing glucuronic acid derivatives of the seco-drug 3. Thus, it has been
known for quite a long time that the concentration of the enzyme
b-glucuronidase is elevated in the extra cellular space of solid ne-
crotic tumours.9 We as one of the first suggested to use this knowl-
edge for the synthesis of non-toxic prodrugs selectively detoxified
by a glucuronic acid moiety.10 This strategy was later introduced
by Bosslet et al.11 as prodrug monotherapy (PMT). Moreover, this
concept is aided by the fact that the activity of b-glucuronidase is
very low at neutral pH12 but elevated at a decreased pH as it is
found in the tumour tissue.13
So far, several glucuronic acid prodrugs have been synthe-
sized14,15 but the resulting QIC50 values are rather low, and more-
over the liberated drugs often show an insufficient cytotoxicity.
We have proposed that in prodrug therapy the IC50 value of the lib-
erated drug should be <10 nM.1b,c Larrick et al. have developed
duocarmycin derivatives containing a glucuronic acid moiety
One of the major problems of the commonly used chemothera-
peutics in cancer therapy is their insufficient differentiation be-
tween normal and cancer cells, which is usually based on the
different proliferation rates of cancer and normal cells. However,
since several normal cells as intestinal, epithelial and bone marrow
cells also show a fast proliferation rate as cancer cells, severe side
effects are often encountered. Some strategies to overcome this
lack of selectivity make use of non-toxic prodrugs that are selec-
tively activated at the tumour-site by conjugates of monoclonal
antibodies and enzymes, which liberate the toxin from the
prodrug.
Another approach is based on conjugates of toxins and peptides,
which bind to tumour-associated receptors. A third way rests upon
enzymes, which are overexpressed in tumours, in combination
with an enzymatically cleavable prodrug.
For the first approach, referred to as ADEPT strategy,1 we have
designed novel duocarmycin-based prodrugs as 2 over the last dec-
ade.2 The natural antibiotic duocarmycin SA (1)3 is a highly potent
cytostatic compound with an IC50 value of about 10 pM against dif-
ferent cancer cell lines and thus one of the strongest anticancer
agents known so far (Scheme 1). The newly developed prodrug
24 contains a benzindole skeleton with a chloroethyl group and a
dimethylaminoethoxyindole side chain (DMAI),5 which is neces-
sary for binding to the minor groove of the DNA as well as a galact-
ose moiety for detoxification. After cleaving off the sugar moiety by
* Corresponding author. Fax: +49 (0) 551 399476.
E-mail address: ltietze@gwdg.de (L.F. Tietze).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.05.009

L. F. Tietze et al. / Bioorg. Med. Chem. 16 (2008) 6312–6318
6313
OMe
OMe
OMe
NH
N
Duocarmycin SA (1)
O
MeO2C
N
H
O
N
N
O
O
Cl
H
2: β-D-galactosidase,
Winstein-cyclisation
H
10
1
NH
NH
N
N
O
O
OR
O
2: β-D-Gal (Prodrug)(+)-(1S
IC50 = 3600 nM
(Seco-Drug)
,
10R)
5 (Drug)
IC50 = 0.75 nM
for 2
QIC50 = 4800
3: R = H
4:
HO
R =
OH
HO
O
CO2R'
a: R' = H
b: R' = Me
Scheme 1. Duocarmycin SA (1) and prodrugs.
which, however, cannot be considered as prodrugs since they eas-
ily form the spirocyclopropylcyclohexadiene moiety as the phar-
macophoric group under physiological conditions. Hence, the
biological investigation gave poor results.16
Here, we describe the novel glucuronic acid containing prodrug
4a, which shows excellent results in HTCFA cell culture
experiments using the human bronchial carcinoma cell line A549
(Scheme 1). In addition, we also prepared the corresponding glucu-
ronic acid methyl ester derivative 4b, which is not cleaved by b-
glucuronidase, but could be transformed in situ into the
corresponding cleavable acid derivative 4a by ubiquitous carboxy-
lesterases.10,17
The synthesis of the prodrug 4a followed the general route
shown in Scheme 2. We started from the known phenol
(1S,10R)-8, which was obtained in a highly selective way from
the known naphthaline derivative 621 and the enantiopure epoxide
(2R,3R)-7.22 Reaction of the phenol (+)-(1S,10R)-8 with the glucu-
ronide trichloroacetimidate 9b23 in the presence of BF3ꢀEt2O in
dichloromethane and molecular sieves (4 Å) at ꢁ10 °C for 3 h gave
the corresponding glucuronide which, however, was not isolated,
but directly transformed into the desired amide 11b. For this
purpose, the primarily formed glucuronide was treated with addi-
tional three equivalents of BF3ꢀEt2O to remove the N-tert-butyloxy-
carbonyl moiety and then reacted with the indole carboxylic acid
DMAIꢀHCl (10) in the presence of EDCꢀHCl in N,N-dimethylformam-
ide for 24 h to give 11b in 43% overall yield.
2. Results and discussion
In a similar way the glucuronic acid methyl ester derivative
11a24 was prepared in 59% yield using the trichloroacetimidate
9a. Under Zemplén deacetylation conditions using sodium methox-
ide in methanol,25 11a and 11b were transformed into the methyl
ester prodrug 4b in 79% and 90% yield, respectively. For the synthe-
sis of the free glucuronic acid prodrug 4a the benzyl ester 11b was
hydrogenated under a hydrogen atmosphere using palladium on
charcoal. The final Zemplén deacetylation led to the desired acid
prodrug 4a in 60% yield over two steps, which was purified by
chromatography on a reversed phase column (Kromasil 100 C18).
The methyl ester trichloroacetimidate 9a was prepared using a
A main problem in the formation of the glucuronic acid deriva-
tive 4a was the proper choice of the glucuronic acid donor. First at-
tempts to prepare the corresponding glucoside followed by
oxidation of the primary hydroxy group of the sugar moiety using
the co-oxidant 2,2,6,6-tetramethylpiperidinyloxyl (TEMPO)18 and
[bis(acetoxy)iodoso]benzene (BAIB), iodosobenzene (PhI@O)/KBr19
or polymer-supported chlorite (PS–O2Cl) were not successful.20
We then employed a route via the glucuronic acid methyl ester
11a, but hydrolysis of the methyl ester moiety in 11a could not be
performed without severe side reactions due to the lability of the
secondary chloride toward basic conditions. On the other hand,
the glucuronic acid benzyl ester derivative 11b could easily be ob-
tained, which allowed the formation of the desired glucuronic acid
prodrug 4a by cleavage of the benzyl ester using a hydrogenolysis
under neutral conditions. Furthermore, 11b could also be em-
ployed for the synthesis of the glucuronic acid methyl ester 4b.
known procedure starting from
D-(+)-glucurono-3,6-lactone in 60%
yield over four steps.26
The first three steps of the synthesis of the benzyl ester trichloro-
acetimidate 9b had first been performed by Vasella et al. in 33% yield
starting from the sodium salt of glucuronic acid.23a We developed a
higher-yielding route to 9b and its intermediate starting from D-glu-

6314
L. F. Tietze et al. / Bioorg. Med. Chem. 16 (2008) 6312–6318
I
Cl
H
H
NHBoc
O
Ref. 22
H
ONs
NBoc
+
H
OBn
(2R,3R)-7
6
OH
8 [(+)-(1 ,10R)]
S
RO
O
O
O
AcO
AcO
N
+
NH
AcO
HO2C
a,b
N
H
·HCl
O
CCl3
9a: R = Me
9b: R = Bn
10: DMAI·HCl
Cl
H
H
O
N
glucuronic acid
prodrug 4a
11b: c,d
N
N
H
O
RO
O
O
11a or 11b: d
AcO
AcO
O
glucuronic acid
methyl ester prodrug 4b
AcO
11a: R = Me
11b: R = Bn
Scheme 2. Synthesis of the glucuronic acid prodrugs 4a and 4b. Reagents and conditions: (a) for 11a: 9a, BF3ꢀEt2O, CH2Cl2, MS (4 Å), ꢁ18 to ꢁ10 °C, 3 h, then BF3ꢀEt2O, rt, 3 h;
for 11b: 9b, BF3ꢀEt2O, CH2Cl2, MS (4 Å), ꢁ15 °C, 3.5 h, then BF3ꢀEt2O, rt, 4.5 h; (b) 10, EDCꢀHCl, DMF, rt, 24 h, 11a: 59%, 11b: 43%; (c) Pd/C, H2, EtOAc/ MeOH (1:10), rt, 12 h, 90%;
(d) NaOMe, MeOH, rt, 60 min, then HOAc/MeOH; for 4a: after RP-HPLC (Kromasil 100 C18), 60% over two steps; for 4b: from 11a: 79%, from 11b: 90%.
HO
O
O
BnO
BnO
O
O
O
O
a,b
c,d
AcO
AcO
HO
HO
AcO
AcO
OAc
NH
OH
AcO
HO
AcO
13
O
CCl3
12
9b
Scheme 3. Novel route to the trichloroacetimidate 9b. Reagents and conditions: (a) Ac2O, I2, rt, 2 h, 99%; (b) BnOH, CH2Cl2, rt, 16 h, 65%; (c) NH2NH2ꢀHOAc, DMF, rt, 2.5 h, 90%
of 14; (d) CCl3CN, PS-DBU, CH2Cl2, rt, 1 h, 85%.
curonic acid (12) itself (Scheme 3). In the first step, a mixed anhy-
dride was formed with simultaneous peracetylation of the hydroxy
groups using acetic acid anhydride in the presence of catalytic
amounts of iodine. This is then directly converted into the peracety-
lated benzyl ester 13 by reaction with benzyl alcohol within 16 h in
65% yield over two steps. It follows an anomeric deprotection to give
14 by employing hydrazine acetate in DMF in 90% yield. The
transformation into the glucuronic acid donor 9b containing a tri-
chloroacetimidate moiety was accomplished by using trichloroace-
tonitrile and polymer-supported DBU27,28 as base avoiding a
purification step of the highly sensitive trichloroacetimidate moiety.
This route allows the synthesis of 9b in 46% over four steps.
The cytotoxicity of the glucuronides was determined using a
HTCFA-assay with IC50 values of 610 nM for 4a and 3300 nM for
4b (Fig. 1). In the presence of b-glucuronidase 4a expresses an
IC50 value of 0.9 nM and 4b a value of 2.1 nM resulting in QIC50 val-
ues of about 700 for 4a and 1600 for 4b. As expected, the IC50 value
of 4b in the presence of glucuronidase is lower than the value ob-
tained for 4a; however, the cytotoxicity is still quite high indicating
that the glucuronic acid methyl ester was cleaved in the cell cul-
ture medium by a present carboxylesterase. It is important to note
that the necessary amount of b-D-glucuronidase for the cleavage of
the glucuronide moiety in 4a and 4b is rather low and comes up to
only four fishman units.
3. Conclusion
We have synthesized the novel glucuronic acid prodrug 4a as
detoxified analogue of the cytotoxic antibiotic duocarmycin SA
for a selective treatment of cancer using the prodrug monotherapy
with a high QIC50 value of 700. Furthermore, we were able to show
that also the glucuronic methyl ester 4b with an even higher QIC50
value of 1600 might be also useable since it will be cleaved in situ
by ubiquitous carboxylesterases.
4. Experimental
4.1. General
All reactions were performed in flame-dried glassware under
an atmosphere of argon. Solvents were dried and purified accord-
ing to the method defined by Perrin and Armarego. Commercial

L. F. Tietze et al. / Bioorg. Med. Chem. 16 (2008) 6312–6318
6315
5.19–5.29 (m, 2H, 3-H, 4-H), 5.74 (d, J = 7.7 Hz, 1H, 1-H), 7.29–7.38
(m, 5H, Ph-H); dC (75.5 MHz, CDCl3) = 20.21, 20.51, 20.74 (4ꢂ
COCH3), 67.98 (OCH2Ph), 68.80 (C-4), 70.11 (C-3), 71.87 (C-2),
72.94 (C-5), 91.28 (C-1), 128.6, 128.7, 128.8 (Ph-Co, Ph-Cm, Ph-
Cp), 134.5 (Ph-Ci), 166.3, (C-6), 168.8, 169.2, 169.3, 169.9 (4ꢂ
COCH3); C21H24O11 (452.41).
2
1
0
10
10
10
-1
-2
-3
10
10
10
4.3. Benzyl (2,3,4-tri-O-acetyl-a/b-D-glucopyran)uronate (14)
To a stirred solution of 13 (1.94 g, 4.29 mmol, 1.00 equiv) in
DMF (20 mL) was added hydrazine acetate (503 mg, 5.47 mmol,
1.28 equiv) and stirring was continued for 2.5 h at 25 °C. After dilu-
tion with EtOAc (150 mL) the solution was washed with ice-water
(60 mL). The water phase was extracted with EtOAc (2ꢂ 25 mL),
and the combined organic layers were washed successively with
satd NaHCO3 solution (2ꢂ 70 mL), ice-water (70 mL), 1 N HCl solu-
tion (70 mL), and brine (70 mL). After drying (Na2SO4), removal of
the solvent in vacuo, and column chromatography on silica gel
(pentane/EtOAc = 1.5:1) 14 was obtained as a colorless solid
(1.58 g, 3.85 mmol, 90%) at a a/b ratio of 6:1; Rf = 0.35 (pentane/
EtOAc = 1:1); a-anomer: dH (300 MHz, CDCl3): 1.71, 1.96, 2.03
(3ꢂ s, 9H, 3ꢂ COCH3), 4.27 (sbr, 1H, OH), 4.62 (d, J = 10.2 Hz, 1H,
5-H), 4.84 (dd, J = 10.3, 3.5 Hz, 1H, 2-H), 5.05 (d, J = 12.0 Hz, 1H,
OCH2Ph), 5.10 (t, J = 10.0 Hz, 3-H), 5.19 (d, J = 11.9 Hz, 1H, OCH2Ph),
5.55 (d, J = 4.0 Hz, 1H, 1-H), 5.56 (t, J = 9.8 Hz, 1H, 4-H), 7.28–7.43
(m, 5H, Ph-H); dC (75.5 MHz, CDCl3): 20.27, 20.62, 20.65 (3ꢂ
COCH3), 67.91 (OCH2Ph), 67.99 (C-2), 69.16, 69.39 (C-3, C-4),
70.62 (C-5), 90.19 (C-1), 128.6, 128.7, 128.7 (Ph-Co, Ph-Cm, Ph-
Cp), 134.5 (Ph-Ci), 168.0 (C-6), 169.6, 170.0, 170.1 (3ꢂ COCH3);
0,01 0,1
1
10 100 1000 10000
c (substance) / nM L–1
IC50 610 nM L–1
IC50 0.9 nM L–1
Glucuronide 4a without enzyme
Glucuronide 4a with 209 µU mL–1
β-D-glucuronidase
QIC50 = 700
Methylester-glucuronide 4b without enzyme IC50 3300 nM L–1
IC50 2.1 nM L–1
Methylester-glucuronide 4b with 209 µU mL–1
β-D-glucuronidase
QIC50 = 1600
Figure 1. HTCFA assay of the cytotoxicity of 4a and 4b against human bronchial
carcinoma cells of line A549.
reagents were used without further purification. Thin-layer chro-
matography (TLC) was carried out on precoated Alugram SIL G/
UV254 (0.25 mm) plates from Macherey-Nagel & Co. Column chro-
matography was carried out on silica gel 60 from Merck with par-
ticle size 0.063–0.200 mm for normal pressure and 0.020–
0.063 mm for flash chromatography (P = pentane). IR spectra were
determined on a Bruker Vektor 22, UV–vis spectra on a Perkin-
Elmer Lambda 2, and mass spectra on a Varian MAT 311A, Varian
MAT 731 for EI-HRMS, and a Bioapex fourier transformation ion
cyclotron resonance mass spectrometer for ESI-HRMS.
C19H22O10 (410.37).
4.4. Benzyl (2,3,4-tri-O-acetyl-a-D-glucopyran)uronate
trichloroacetimidate (9b)
To a solution of 14 (1.00 g, 2.44 mmol, 1.00 equiv) in CH2Cl2
(35.0 mL) was added polymer-supported DBU (0.50 mmol/g,
2.44 g, 1.22 mmol 0.50 equiv) and trichloroacetonitrile (1.76 g,
1.22 mL, 12.1 mmol, 5.00 equiv). After stirring at 25 °C for 60 min
the suspension was filtered over Celite, and the solvent removed
in vacuo. The residue was taken up in toluene and the toluene dis-
tilled off in vacuo (3ꢂ 15 mL) to give the clean trichloroacetimi-
date 9b as slightly yellow syrup (1.15 g, 2.07 mmol, 85%), which
was used for the next step without further purification; Rf = 0.49
(pentane/EtOAc = 3:1); dH (300.0 MHz, CDCl3): 1.79, 2.02, 2.04,
(3ꢂ s, 9H, 3ꢂ COCH3), 4.54 (d, J = 10.5 Hz, 1H, 5-H), 5.15 (s, 2H,
OCH2Ph), 5.16 (dd, J = 9.8, 4.1 Hz, 1H, 2-H), 5.27 (t, J = 9.8 Hz, 1H,
4-H), 5.62 (t, J = 9.8 Hz, 1H, 3-H), 6.66 (d, J = 3.9 Hz, 1H, 1-H),
7.37 (s, 5H, Ph-H), 8.75 (s, 1H, NH); dC (75.8 MHz, CDCl3): 20.23,
20.39, 20.63 (3ꢂ COCH3), 68.07 (CH2Ph), 68.94 (C-2), 69.00, 69.38
(C-3, C-4), 70.49 (C-5), 91.76 (CCl3), 92.58 (C-1), 128.6, 128.7,
128.8 (Ph-Co, Ph-Cm, Ph-Cp), 134.5 (Ph-Ci), 160.5 (C@NH),166.6,
169.3, 169.6, 169.7 (4ꢂ COCH3); C21H22Cl3NO10 (554.76).
1H NMR spectra were recorded either on a Varian UNITY-
300 MHz, Varian Inova 500 MHz, or Varian Inova 600 MHz. 13C
NMR spectra were recorded at 75, 125, or 150 MHz. Spectra were
taken at room temperature except otherwise stated in deuterated
solvents as indicated using the solvent peak as internal standard.
4.2. Benzyl (1,2,3,4-tetra-O-acetyl-b-
D-glucopyran)uronate (13)
To a stirred suspension of -glucuronic acid (7.50 g, 38.6 mmol,
D
1.00 equiv) in acetic anhydride (113 mL, 122 g, 1.20 mol) was
slowly added at room temperature iodine (530 mg, 1.93 mmol,
0.05 equiv) and stirring was continued for 2 h. The solvent was re-
moved in vacuo, the residue taken up in toluene followed by distil-
lation in vacuo (3ꢂ 200 mL) to remove the iodine and traces of
acetic acid to give the analytically pure mixed anhydride as slightly
yellow solid (15.5 g, 38.4 mmol, 99%). Then, a solution of 10.4 g
(25.8 mmol, 1.00 equiv) of the mixed anhydride in CH2Cl2
(70 mL) and benzyl alcohol (5.01 g, 4.80 mL, 46.7 mmol,
1.80 equiv) was stirred for 16 h at 25 °C. The solution was diluted
with EtOAc (250 mL), washed with 1 M Na2S2O3 solution
(100 mL), 1 M HCl solution (100 mL), satd NaHCO3 solution
(100 mL), and brine (100 mL). The organic phase was dried
(MgSO4), the solvent removed in vacuo and the residue purified
by column chromatography on silica gel (pentane/EtOAc = 2:1) to
give 13 as a colorless solid (6.76 g, 14.9 mmol, 65%) which can be
recrystallised from EtOH. Rf = 0.53 (pentane/EtOAc = 1:1); dH
(300 MHz, CDCl3) = 1.76, 1.96, 2.01, 2.09 (4ꢂ s, 12H, 4ꢂ COCH3),
4.19 (dd, J = 9.5, 4.5 Hz, 1H, 5-H), 5.08 (d, J = 12.0 Hz, 1H, OCH2Ph),
5.11 (dd, J = 9.0, 7.7 Hz, 1H, 2-H), 5.13 (d, J = 12.0 Hz, 1H, OCH2Ph),
4.5. (+)-Benzyl {(1S,10 R)-1-(10-chloroethyl)-3-[(5-(2-(N,N-
dimethylamino)ethoxy)indol-2-yl)carbonyl]-1,2-dihydro-3H-
benz[e]indol-5-yl]}-2,3,4-tri-O-acetyl-b-D-glucopyranuronate
(11b)
A
suspension of the trichloroacetimidate 9b (646 mg,
1.16 mmol, 1.25 equiv), phenol (+)-(1S,10R)-8 (325 mg, 938 lmol,
1.00 equiv) and molecular sieves 4 Å (1.75 g) in CH2Cl2 (42.0 mL)
was stirred at 25 °C for 30 min. The mixture was cooled to
ꢁ18 °C and BF3ꢀOEt2 (59.4 lL, 468 lmol, 0.50 equiv) in CH2Cl2
(1.0 mL) was added dropwise, then, the mixture was allowed to
warm to ꢁ10 °C. After stirring for 3 h at this temperature addi-
tional BF3ꢀOEt2 (200 lL, 1.58 mmol, 2.00 equiv) in CH2Cl2 (7.0 mL)

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L. F. Tietze et al. / Bioorg. Med. Chem. 16 (2008) 6312–6318
was added, the suspension warmed to 25 °C, stirred for 3 h and the
reaction quenched by filtration over a Celite pad. The solvent was
removed in vacuo and the resulting salt dried under high vacuum
for 1 h to give a foam which was dissolved in DMF (60.0 mL). To the
obtained solution was added at 0 °C DMAIꢀHCl (10) (400 mg,
1.41 mmol, 1.50 equiv) and EDCꢀHCl (539 mg, 2.81 mmol,
3.00 equiv) and stirring was continued at 25 °C for 24 h. Then,
EtOAc (70 mL) was added and the mixture washed with ice-water
(70 mL) and satd NaHCO3 solution (25 mL). The layers were sepa-
rated and the water phase reextracted with EtOAc (4ꢂ 100 mL).
The combined organic layers were washed with brine (4ꢂ 60
mL), dried over MgSO4 and the solvent removed in vacuo. Column
chromatography on silica gel (CH2Cl2/MeOH = 10:1) yielded the
acetylated prodrug 11b as colorless solid (352 mg, 40.4 lmol,
(d, J = 1.3 Hz, 1H, H-40), 7.39 (d, J = 8.9 Hz, 1H, H-70), 7.44, 7.57
(2ꢂ mc, 2H, H-7, H-8), 7.97 (d, J = 8.4 Hz, 1H, H-9), 8.17 (sbr, 1H,
H-4), 8.34 (d, J = 8.4 Hz, 1H, H-6), 11.70 (s, 1H, NH); dC
(125.7 MHz, DMSO-d6, 35 °C): 23.34 (11-CH3), 44.76 (N(CH3)2),
45.89 (C-1), 51.93 (C-2), 57.12 (C-200), 61.21 (C-10), 65.35 (C-100),
71.70 (C-3000), 73.05 (C-2000), 75.24 (C-4000), 76.03 (C-5000), 101.2 (C-
1000), 101.6 (C-4), 103.4 (C-40), 105.4 (C-30), 113.1 (C-70), 115.8 (C-
60), 119.1 (C-5a), 122.9, 123.3, 123.7 (C-6, C-7, C-9, C-9b), 127.3,
127.4 (C-8, C-3a0), 129.5, 130.9, 131.7 (C-20, C-7a0, C-9a), 141.9
(C-3a), 152.7, 153.2 (C-5, C-50), 160.0 (NC@O), 171.9 (C-6000); UV/
vis (MeOH): kmax (lg e): 209.5 nm (1.6010), 240.5 (1.4051), 299.5
(1.2945), 330.0 (1.3307); IR (KBr): v ¼ 3406 cmꢁ1, 1615, 1592,
~
1516, 1465, 1415, 1290, 1267, 1234, 1179, 1061, 762; MS (ESI):
m/z: calcd for C33H37ClN3O9 [M+H]+: 654.2 (100) [M+H]+; HRMS
(ESI): m/z: calcd for C33H37ClN3O9 [M+H]+: 654.22128; found:
654.22095.
20
43%); Rf = 0.44 (CH2Cl2/MeOH = 10:1); ½aꢃD +12.1° (c 0.87, MeOH);
dH (600 MHz, DMSO-d6, 35 °C): 1.65 (d, J = 6.7 Hz, 3H, 11-H3),
1.83, 2.01, 2.03 (3ꢂ s, 9H, 3ꢂ COCH3), 2.28 (s, 6H, N(CH3)2), 2.70
(t, J = 5.8 Hz, 2H, H2-200), 4.09 (t, J = 5.8 Hz, 2H, 100-H2), 4.28 (dt,
J = 9.4, 2.3 Hz, 1H, 1-H), 4.64 (dd, J = 10.0, 2.0 Hz, 1H, 2a-H), 4.78
(mc, 2H, 2b-H, 5000-H), 4.81 (dq, J = 6.6, 2.3 Hz, 1H, 10-H), 5.13, 5.
16 (2ꢂ d, J = 12.3 Hz, 2H, OCH2Ph), 5.20 (t, J = 9.6 Hz, 1H, 4000-H),
5.34 (dd, J = 9.6, 7.7 Hz, 1H, 2000-H), 5.57 (t, J = 9.6 Hz, 1H, 3000-H),
5.79 (d, J = 7.7 Hz, 1H, 1000-H), 6.93 (dd, J = 8.9, 2.3 Hz, 1H, 60-H),
7.19 (sbr, 2H, 30-H, 40-H), 7.26–7.38 (mc, 5H, Ph-H), 7.41 (d,
J = 8.9 Hz, 1H, 70-H), 7.47, 7.59 (2ꢂ t, J = 7.7 Hz, 2H, 7-H, 8-H),
8.00 (d, J = 8.9 Hz, 2H, 6-H, 9-H), 8.25 (sbr, 1H, 4-H), 11.61 (s, 1H,
NH); dc (150.8 MHz, DMSO-d6, 35 °C): 19.99, 20.20, 20.28 (3ꢂ
COCH3), 23.31 (11-CH3), 45.34 (N(CH3)2), 45.93 (C-1), 52.06 (C-2),
57.65 (C-200), 61.18 (C-10), 66.08 (C-100), 66.96 (OCH2Ph), 69.08
(C-4000), 70.70 (C-2000), 70.96 (C-3000), 71.28 (C-5000), 98.71 (C-1000),
103.0 (C-4), 103.3 (C-40), 105.5 (C-30), 113.2 (C-70), 115.9 (C-60),
120.6 (C-5a), 122.1 (C-6), 122.7 (C-9b), 123.2 (C-9), 124.4 (C-7),
127.5 (C-8, C-3a0), 128.2, 128.3, 128.4 (5ꢂ Ph-Co, Ph-Cm, Ph-Cp),
129.5, 130.7, 131.8 (C-20, C-7a0, C-9a), 134.9 (Ph-Ci), 141.8 (C-3a),
152.4, 152.9 (C-5, C-50), 160.2 (NC@O), 166.4 (C-6000), 169.0, 169.2,
4.7. Chromatographic purification of crude (4a)
A
solution of 30.0 mg of crude 4a in 4.00 mL CH3CN/
H2O = 1:3 + 0.05% HOAc was separated (injection volume
0.80 mL) by semipreparative RP-HPLC (Kromasil 100 C18,
250 ꢂ 20 mm, particle size: 7 lm, gradient from 20% to 25% CH3CN
in H2O + 0.05% HOAc within 5 min, then CH3CN/H2O = 1:3 + 0.05%
HOAc, flow: 12 mL minꢁ1; UV-detector: k = 299 nm, Jasco-module)
to provide pure 4a (tR = 15.8 min).
4.8. (+)-Methyl {(1S,10R)-1-(10-chloroethyl)-3-[(5-(2-(N,N-
dimethylamino)ethoxy)indol-2-yl)carbonyl]-1,2-dihydro-3H-
benz[e]indol-5-yl]}-2,3,4-tri-O-acetyl-b-D-glucopyranuronate
(11a)
A suspension of the trichloroacetimidate 9a (109 mg, 227 lmol,
1.05 equiv), phenol (+)-(1S,10R)-8 (75.0 mg, 216 lmol, 1.00 equiv)
and molecular sieves 4 Å (450 mg) in CH2Cl2 (10.0 mL) was stirred
at 25 °C for 30 min. The mixture was cooled to ꢁ20 °C and BF3ꢀOEt2
(13.7 lL, 108 lmol, 0.50 equiv) in CH2Cl2 (150 lL) was added drop-
wise. After stirring for 3.5 h, additional BF3ꢀOEt2 (82.2 lL, 648 lmol,
3.00 equiv) in CH2Cl2 (2.00 mL) was added, the suspension warmed
to 25 °C, and stirring was continued for 4.5 h. The reaction mixture
was filtered over a Celite pad, the filtrate evaporated in vacuo and
the resulting salt dried under high vacuum for 1 h to give a foam
which was dissolved in DMF (60.0 mL). To the obtained solution
were added at 0 °C DMAIꢀHCl (10) (92.3 mg, 324 lmol, 1.50 equiv)
and EDCꢀHCl (124 mg, 648 lmol, 3.00 equiv) and the mixture was
stirred at 25 °C for 24 h. Work-up and purification was performed
as described for 11b to give the acetylated prodrug 11a as colorless
solid (109 mg, 146 lmol, 68%). Rf = 0.34 (CH2Cl2/MeOH = 10:1);
169.4 (3ꢂ COCH3); IR (KBr): v ¼ 3418 cmꢁ1, 2937, 1759, 1626,
~
1517, 1461, 1412, 1216, 1038, 758; MS (ESI): m/z: calcd for
C
46H49ClN3O12 [M+H]+: 870.3 (100) [M+H]+, 1740.8 (12) [2M+H]+;
868.4 (100) [MꢁH]ꢁ, 1739.1 (72) [2MꢁH]ꢁ; HRMS (ESI): m/z: calcd
for C46H49ClN3O12 [M+H]+: 870.29993; found: 870.30003.
4.6. (ꢁ)-{(1S,10R)-1-(10-Chloroethyl)-3-[(5-(2-(N,N-
dimethylamino)ethoxy)indol-2-yl)carbonyl]-1,2-dihydro-3H-
benz[e]indol-5-yl]}-b-D-glucopyranuronate (4a)
A suspension of 11b (150 mg, 172 lmol, 1.00 equiv) and palla-
dium on charcoal (10 %, 104 mg, 98.0 lmol, 0.57 equiv of Pd) in
MeOH/EtOAc (66.0 mL, 1:10) was stirred under a H2-atmosphere
for 12 h at normal pressure and 25 °C. Filtration over Celite, wash-
ing with MeOH and evaporation of the solvent yielded an analyti-
cally pure colorless solid (121 mg, 155 lmol, 90%; Rf = 0.79, CH2Cl2/
MeOH = 3:1). The crude salt (121 mg) was dissolved in MeOH
(40.0 mL) and a NaOMe solution (30% in MeOH, 70.0 lL, 366 lmol,
2.50 equiv) added dropwise. After stirring at 25 °C for 2 h the solu-
tion was neutralized by addition of HOAc. Removal of the solvent
under high vacuum gave the crude prodrug, which was purified
by reversed phase column chromatography to give 4a as a colorless
solid (61.0 mg, 93.0 lmol, 60%). Rf = 0.12 (CH2Cl2/MeOH = 1:1.5);
20
½aꢃD +1.6° (c 0.1, MeOH); dH (300 MHz, DMSO-d6, 35 °C): 1.64 (d,
J = 6.7 Hz, 3H, 11-H3), 2.01, 2.02 (3ꢂ s, 9H, 3ꢂ COCH3), 2.37 (s,
6H, N(CH3)2), 2.84 (t, J = 5.7 Hz, 2H, 200-H2), 3.67 (s, 3H, OCH3),
4.14 (t, J = 5.7 Hz, 2H, 100-H2), 4.26 (dd, J = 8.9, 2.4 Hz, 1H, 1-H),
4.63 (dd, J = 11.4, 1.5 Hz, 1H, 2a-H), 4.71–4.83 (m, 3H, 2b-H, 5000-H,
10-H), 5.15 (t, J = 9.7 Hz, 1H, 4000-H), 5.32 (dd, J = 9.7, 7.8 Hz, 1H,
2000-H), 5.60 (t, J = 9.6 Hz, 1H, 3000-H), 5.80 (d, J = 7.8 Hz, 1H, 1000-H),
6.94 (dd, J = 9.0, 2.3 Hz, 1H, 60-H), 7.18, 7.18 (2ꢂ sbr, 2H, 30-H, 40-
H), 7.41 (d, J = 9.0 Hz, 1H, 70-H), 7.47 (t, J = 7.4 Hz, 1H, 7-H), 7.60
(t, J = 7.6 Hz, 1H, 8-H), 7.99, 8.01 (2ꢂ d, J = 8.1 Hz, 2H, 6-H, 9-H),
8.21 (s, 1H, 4-H), 11.63 (s, 1H, NH); dC (125.7 MHz, DMSO-d6,
35 °C): 20.14, 20.24, 20.29 (3ꢂ COCH3), 23.32 (11-CH3), 45.02
(N(CH3)2), 45.88 (C-1), 52.11 (C-2), 52.53 (OCH3), 57.36 (C-200),
61.24 (C-10), 65.66 (C-100), 69.06 (C-4000), 70.70 (C-2000, C-3000), 71.09
(C-5000), 98.40 (C-1000), 102.6 (C-4), 103.4 (C-40), 105.5 (C-30), 113.2
(C-70), 115.9 (C-60), 120.5 (C-5a), 122.0 (C-6), 122.6 (C-9b), 123.3
(C-9), 124.4 (C-7), 127.4, 127.6 (C-8, C-3a0), 129.5, 130.7, 131.4
(C-20, C-7a0, C-9a), 141.7 (C-3a), 152.4, 152.8 (C-5, C-50), 160.1
20
½aꢃD ꢁ5.6° (c 0.6, DMSO); dH (600 MHz, DMSO-d6, 35 °C): 1.65 (d,
J = 6.6 Hz, 3H, H3-11), 2.39 (s, 6H, N(CH3)2), 2.83–2.88 (mc, 2H,
H2-200), 3.36 (d, J = 9.1 Hz, 1H, H-5000), 3.37–3.43 (mc, 1H, H-3000),
3.48 (t, J = 8.1 Hz, 1H, H-2000), 3.63–3.70 (mc, 1H, H-4000), 4.00 (sbr
,
1H, OH), 4.12 (t, J = 5.7 Hz, 2H, H2-100), 4.25 (dt, J = 9.5, 2.4 Hz, 1H,
H-1), 4.62 (mc, 1H, H-2a), 4.73 (t, J = 9.7 Hz, 1H, H-2b), 4.81 (dq,
J = 6.4, 2.4 Hz, 1H, H-10), 5.34 (mc, 1H, H-1000), 5.47 (sbr, 2H, 2ꢂ
OH), 6.92 (dd, J = 8.9, 2.2 Hz, 1H, H-60), 7.15 (sbr, 1H, H-30) 7.18

L. F. Tietze et al. / Bioorg. Med. Chem. 16 (2008) 6312–6318
6317
(NC@O), 167.0 (C-6000), 169.2, 169.3, 169.4 (3ꢂ COCH3); UV/vis
(MeOH): kmax (lg e): 205.0 nm (1.5788), 299.0 (1.3633), 334.0
The IC50 values are based on the relative clone forming rate,
which was determined according to the following formula: relative
clone forming rate [%] = 100 ꢂ (number of clones counted after
exposure)/(number of clones counted in the control).
(1.3523); IR (KBr): v ¼ 2928 cmꢁ1, 2759, 1626, 1516, 1462, 1414,
~
1217, 1054, 764; C36H44ClN3O12 (745.85).
Liberation of the drugs from their glycosidic prodrugs was
4.9. (ꢁ)-Methyl {(1S,10R)-1-(10-chloroethyl)-3-[(5-(2-(N,N-
dimethylamino)ethoxy)indol-2-yl)carbonyl]-1,2-dihydro-3H-
benz[e]indol-5-yl]}-b-D-glucopyranuronate (4b)
achieved by addition of 4.0 U mLꢁ1b-
D-galactosidase (EC 3.2.1.23,
Grade X, purchased from Sigma Germany, Deisenhofen, Germany)
to the cells during incubation with the substances.
To a stirred solution of 11a (25.0 mg, 33.4 lmol, 1.00 equiv) in
MeOH (10.0 mL) were added dropwise NaOMe in MeOH (30% in
MeOH, 0.90 mg, 3.20 lL, 16.8 lmol, 0.50 equiv in 1.00 mL MeOH)
and stirring was continued at 25 °C for 2 h. Column chromatogra-
phy on silica gel (CH2Cl2/ MeOH = 1:1) and filtration through a
membrane filter gave 4b as a colorless solid (17.6 mg, 26.4 lmol,
Acknowledgments
This research was supported by the Deutsche Forschungsgeme-
inschaft and the Fonds der chemischen Industrie. H.J.S. thanks the
Friedrich-Ebert-Stiftung for a Ph.D. scholarship.
20
79%); Rf = 0.29 (MeOH/CH2Cl2 = 1:1); ½aꢃD ꢁ8.2° (c 0.6, MeOH); dH
References and notes
(600 MHz, DMSO-d6, 35 °C): 1.65 (d, J = 6.6 Hz, 3H, 11-H3), 2.25
(s, 6H, N(CH3)2), 2.67 (t, J = 5.7 Hz, 2H, 200-H2), 3.38 (t, J = 9.0 Hz,
1H, 3000-H), 3.47–3.57 (m, 1H, 2000-H, 4000-H), 3.68 (s, 3H, OCH3),
3.97 (d, J = 9.3 Hz, 1H, 5000-H), 4.08 (t, J = 5.9 Hz, 2H, 100-H2), 4.26
(dd, J = 9.4, 2.1 Hz, 1H, 1-H), 4.62 (dd, J = 10.9, 2.2 Hz, 1H, 2b-H),
4.75 (t, J = 10.3 Hz, 1H, 2a-H), 4.81 (dq, J = 6.5, 2.3 Hz, 1H, 10-H),
5.11 (d, J = 7.5 Hz, 1H, 1000H), 5.38, 5.62 (2ꢂ sbr, 3H, 3ꢂ OH), 6.92
(dd, J = 8.8, 2.4 Hz, 1H, 60-H), 7.17, 7.18 (2ꢂ d, J = 2.2 Hz, 2H, 30-H,
40-H), 7.40 (d, J = 8.9 Hz, 1H, 70-H), 7.45, 7.58 (2ꢂ t, J = 7.5 Hz, 2H,
7-H, 8-H), 7.97 (d, J = 8.3 Hz, 1H, 9-H), 8.16 (s, 1H, 4-H), 8.33 (d,
J = 8.4 Hz, 1H, 6-H), 11.59 (s, 1H, NH); dC (125.7 MHz, DMSO-d6,
35 °C): 23.36 (11-CH3), 45.49 (N(CH3)2), 45.93 (C-1), 51.92
(OCH3), 52.02 (C-2), 57.77 (C-200), 61.31 (C-10), 65.66 (C-100),
69.06 (C-4000), 70.70 (C-2000, C-3000), 71.09 (C-5000), 101.5 (C-4), 102.1
(C-1000), 103.3 (C-40), 105.5 (C-30), 113.2 (C-70), 115.9 (C-60), 120.5
(C-5a), 123.0 (C-9, C-9b), 123.3 (C-6), 123.9 (C-7), 127.4, 127.5
(C-8, C-3a0), 129.5, 130.8, 131.7 (C-20, C-7a0, C-9a), 141.9 (C-3a),
153.0, 153.1 (C-5, C-50), 160.1 (NC@O), 169.0 (C-6000); HRMS (ESI):
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668.23791.
C
34H39ClN3O9 [M+H]+: 668.23748; found:
4.10. Cell culture
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Human bronchial carcinoma cells of line A549 (ATCC CCL 185)
were kindly provided by the Institut für Zellbiologie, Universität
Essen, and were maintained as exponentially growing cultures at
37 °C and 7.5% CO2 in air in Dulbecco’s modified Eagle’s medium
(DMEM) (Biochrom, Berlin, Germany) supplemented with 10% fetal
calf serum (heat-inactivated for 30 min at 56 °C, Gibco–BRL,
Karlsruhe, Germany), 44 mM NaHCO3 (Biochrom, Berlin, Germany)
and 4 mM L-glutamine (Gibco–BRL, Karlsruhe, Germany).
4.11. In vitro cytotoxicity assays
Adherent cells of line A549 were sown in triplicate in six-
multiwell plates at concentrations of 102–105 cells per cavity.
Culture medium was sucked off after 24 h and cells were
washed in the incubation medium Ultraculture (UC, serum-free
special medium, purchased from BioWhittaker Europe, Verviers,
Belgium). Incubation with compounds 4a and 4b was then per-
formed in Ultraculture medium at various concentrations 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.
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cells were washed with fresh medium. Cultivation was done 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.

6318
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