Synthesis and biological evaluation of prodrugs based on the natural antibiotic duocarmycin for use in ADEPT and PMT

Article abstract of DOI:10.1002/chem.201002798

Chemotherapy of malign tumors is usually associated with serious side effects as common anticancer drugs lack selectivity. An approach to deal with this problem is the antibody-directed enzyme prodrug therapy (ADEPT) and the prodrug monotherapy (PMT). Her

Full text of DOI:10.1002/chem.201002798

DOI: 10.1002/chem.201002798
Synthesis and Biological Evaluation of Prodrugs Based on the Natural
Antibiotic Duocarmycin for Use in ADEPT and PMT**
Lutz F. Tietze,* Kianga Schmuck, Heiko J. Schuster, Michael Mꢀller, and
Ingrid Schuberth[a]
Dedicated to Professor Dieter Enders on the occasion of his 65th birthday
Abstract: Chemotherapy of malign
tumors is usually associated with seri-
ous side effects as common anticancer
drugs lack selectivity. An approach to
deal with this problem is the antibody-
directed enzyme prodrug therapy
(ADEPT) and the prodrug monothera-
py (PMT). Herein, the synthesis and
biological evaluation of new glycosidic
prodrugs suitable for both concepts are
described. All prodrugs but one are
stable in human serum and show QIC50
values (IC50 of prodrug/IC50 of prodrug
in the presence of the appropriate gly-
cohydrolase) of up to 6500. This is the
best value found so far for compounds
interacting with DNA.
Keywords: antitumor agents · cyto-
toxicity · duocarmycin · glycosides ·
prodrugs
Introduction
tivity is based on factors (e.g. enzymes) that are overex-
pressed in tumor tissue. For instance, it has been known for
a long time that the concentration of the enzyme b-d-glucu-
ronidase is elevated in the extracellular space of solid necrot-
ic tumors.[2b,3] As one of the first, we developed “nontoxic”
prodrugs bearing a glucuronic acid (GlcA) moiety.[4] A fur-
ther interesting difference between malignant and normal
tissue is the lower pH of cancer cells under hyperglycemic
conditions,[5] which has extensively been exploited by us.[4,6]
In recent years, we have developed several highly promis-
ing compounds based on the natural antibiotic duocarmycin
SA (1a) that are suitable for ADEPT and PMT
(Scheme 1).[7] Duocarmycin SA (1a), found in Streptomyces
sp., is a highly potent cytostatic compound with an IC50
value of about 10 pm against different cancer cell lines.[8]
The molecular structure of 1a shows an indole subunit as
DNA minor groove binding component and a spirocyclopro-
pylcyclohexadienone moiety as a pharmacophoric group
that causes sequence-selective alkylation of N3 of adenine
in AT-rich sites of the minor groove of DNA.[9] With pro-
nounced myelotoxicity, duocarmycin SA (1a) itself is not
applicable in chemotherapy. In our approach we have used
derivatives of the seco-analogue seco-CBI[10] (CBI=cyclo-
propabenzoindole) and methyl-seco-CBI.[11] Thus, we have
prepared (+)-(S)-3a and (+)-(1S,10R)-3b containing a seco-
CBI and an anti-methyl-seco-CBI skeleton, respectively, as
pharmacophoric units connected to DMAI (5-[2-(N,N-dime-
thylamino)ethoxy]-1H-indole)[12] as DNA binding moiety
(Scheme 1). For a selective treatment of cancer, these seco-
One of the major problems in the chemotherapy of cancer
is its so far insufficient selectivity, which is the cause for
severe side effects. This has resulted in the development of
several new approaches to solve this problem in recent
years. One of the most promising concepts is the antibody-
directed enzyme prodrug therapy (ADEPT).[1] Herein, se-
lectivity is achieved by monoclonal antibodies that are con-
jugated to enzymes. After application of such an antibody
conjugate a “nontoxic” prodrug is administered, which is
converted site-selectively by the enzyme in the conjugate to
give a highly cytotoxic compound. This, in principle, would
allow the killing of cancer cells without affecting healthy
tissue.
Another approach for a more selective treatment of
cancer is the prodrug monotherapy (PMT).[2] In contrast to
ADEPT, here an antibody is not necessary since the selec-
[a] Prof. Dr. L. F. Tietze, K. Schmuck, Dr. H. J. Schuster,
Dipl.-Chem. M. Mꢀller, Dr. I. Schuberth
Institut fꢀr Organische und Biomolekulare Chemie
Georg-August-Universitꢁt Gçttingen
Tammannstraße 2, 37077 Gçttingen (Germany)
Fax : (+49)551-399476
E-mail: ltietze@gwdg.de
[**] ADEPT=antibody-directed enzyme prodrug therapy; PMT=pro-
drug monotherapy.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201002798.
1922
ꢂ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2011, 17, 1922 – 1929

FULL PAPER
have
a
different mode of
action. Thus, they do not bind
to DNA, which is understanda-
ble due to the lack of a DNA
binding moiety.
An important objective in de-
signing the prodrugs was the
curtailment of their cell-mem-
brane penetration to avoid a di-
lution effect. Hence, we have
also prepared diglycosidic pro-
drugs by using cellobiose and
lactose that are clearly more
polar than the monoglycosidic
compounds as (+)-(1S,10R)-2b
and, therefore, less able to pass
through the cell membrane.
However, these compounds are
not suitable due to an incom-
plete hydrolysis of the glycosi-
dic bond when using cellula-
se.[7c] Moreover, the employed
anti-methyl-seco-CBI
unit
showed an insufficient stability
in the pharmacokinetic studies.
Here we describe novel di-
Scheme 1. Duocarmycin SA (1a), pseudo-dimeric prodrug 1b, and prodrugs (ꢀ)-(S)-2a and (+)-(1S,10R)-2b
to give the cytotoxic drugs 4a and 4b via the seco-drugs (+)-(S)-3a and (+)-(1S,10R)-3b. Cytotoxic drugs 4a
and 4b share the spirocyclopropylcyclohexadienone moiety with 1a.
saccharide prodrugs with
mannosyl mannoside (ManMan)
and galactosyl galactoside
a
drugs were transformed into the corresponding glycosides as
a
(ꢀ)-(S)-2a and (+)-(1S,10R)-2b with a highly reduced cyto-
toxicity by coupling them with different sugar moieties.[7b–e]
The so-formed prodrugs can selectively be cleaved again by
using the corresponding glycohydrolase or antibody glycohy-
drolase conjugate to liberate the seco-drugs (+)-(S)-3a and
(+)-(1S,10R)-3b, from which the final cytotoxic drugs 4a
and 4b are formed in situ by a rapid Winstein cyclization re-
action. As proposed by us as a requirement for use in
ADEPT, the IC50 values of the seco-drugs are below 10 nm
with 26 pm for (+)-(S)-3a and 750 pm for (+)-(1S,10R)-
3b.[13]
(GalGal) containing the seco-CBI unit, which is more
stable. In addition, we also prepared seco-CBI glucuronides
and a seco-CBI mannoside. In the biological evaluation the
new compounds very well fulfilled our expectation.
Results and Discussion
The synthesis of the desired prodrugs 10a–e was accom-
plished by following a four- and five-step route, respectively,
as depicted in Scheme 2. As starting material, the tert-butox-
The therapeutic window of
these compounds as defined by
their QIC50 values (QIC50 =IC50
of prodrug/IC50 of prodrug and
cleaving enzyme) is very high
with QIC50 values of up to
around 5000 for (+)-(1S,10R)-
2b. They well exceed the QIC50
value of 1000 that was antici-
pated by us as a minimum re-
quirement for ADEPT.[13] Quite
recently, we were able to im-
prove the QIC50 value to almost
1000000.[14] However, the new
pseudo-dimeric
such as 1b (Scheme 1) with (S)-
5 as monomeric unit seem to 30 min–9 h, prep. RP-HPLC (Kromasil 100 C18, not for 10d); e) Pd/C, H2, EtOAc/MeOH 10:1, RT, 6 h.
compounds
Scheme 2. Synthesis of the prodrugs 10a–e (for yields, see Table 1): a) 6a–e (Table 1), BF3·OEt2, CH2Cl2,
ꢀ108C, 3–6.5 h; b) BF3·OEt2, CH2Cl2, RT, 5 h; c) 7, EDC·HCl, DMF, RT, 15–19 h; d) NaOMe, MeOH, RT,
Chem. Eur. J. 2011, 17, 1922 – 1929
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1923

L. F. Tietze et al.
ycarbonyl Boc-protected phenol (ꢀ)-(S)-5[15] was used. For
the transformation into the corresponding prodrugs, (ꢀ)-
(S)-5 was first glycosylated by using the Schmidt proce-
dure[16] with the trichloroacetimidate donors 6a–e (Table 1)
under BF3·OEt2 catalysis in dichloromethane at ꢀ108C.
For the synthesis of the glucuronic acid derivative 10e
one more step was required prior to deacetylation. Thus, the
benzyl ester 8e had to be cleaved, which was accomplished
by using palladium on charcoal under a H2 atmosphere in
ethyl acetate/methanol to give 9 in 86% yield. The final
step in the synthesis of all prodrugs 10 was the solvolysis of
the acetyl protecting groups by using the Zemplꢃn proce-
dure with NaOMe in methanol at room temperature. Subse-
quently, all compounds but 10d were purified by semipre-
parative RP-HPLC employing water and methanol or aceto-
nitrile with 0.05% acetic acid to guarantee high purity for
the in vitro assays. The methyl glucuronide 10d was not
stable during HPLC under these conditions; thus, partial hy-
drolysis of the methyl ester 10d was observed to give the
corresponding acid 10e. Hence, 10d was further purified by
preparative TLC (CH2Cl2/MeOH 5:1).
Table 1. Glycosidic donors 6a–e and yields for the synthesis of prodrugs
10a–e (see Scheme 1).
Product
Glycosidic donor
Coupling of 5, 6, Deacetylation
and
(d) [%]
7 (a–c) [%]
10a
62
62
83
The structures of the molecules and especially the config-
uration at the glycosidic linkage could be determined by
1H NMR spectroscopy for all compounds but the mannosyl
mannoside 10b. Though, an a-glycosidic bond had to be ex-
pected according to the mechanism of the glycosidation, a
clear cut evidence of the configuration as a at both anome-
ric centers was obtained by a 2D-TOCSY-NMR spectro-
scopic experiment (total correlation spectroscopy) giving the
traces as described for a-d-mannoses and a-l-rhamnoses
10b
96
1
(mixing time: 100 ms).[18] Besides, the JC,H coupling constant
10c
10d
49
39
90
93
was measured to be 170 (C-1’’’) and 167 Hz (C-1’’’’) corre-
sponding to an a-configuration as stated by Pedersen and
Jennings.[19]
All new prodrugs were analyzed with regard to their sta-
bility in undiluted human serum (Table 2). Over a time span
of 24 h, the prodrug-serum mixtures were incubated at 378C
and substrates and metabolites were examined by HPLC-
MS. The stability of the prodrugs 10a–c and 10e was very
high in the serum showing almost no changes within 24 h.
Important to note, the new prodrugs containing the seco-
CBI unit have a much better stability in human serum than
10e
48
77[a]
the corresponding compounds with
a seco-methyl-CBI
[a] Yield for Bn and OAc deprotection (two steps).
moiety. Only prodrug 10d showed a change in human
serum; however, here the chloromethyl group was not al-
tered as in the case of the seco-methyl-CBI compounds but
the ester group of the glucuronic acid methyl ester moiety
was cleaved. Obeying a first-order reaction, it is hydrolyzed
to give the corresponding GlcA prodrug 10e with a rate
constant of k=3.9ꢁ0.2ꢄ10ꢀ5 sꢀ1 and a half-life of t1/2 =4.9ꢁ
0.3 h (Table 2).
The necessary disaccharide donors 6a (GalGal) and 6b
(ManMan) as well as 6c–e were prepared according to liter-
ature-known procedures with slight variations. Thus, for the
formation of the trichloroacetimidates we used trichloroace-
tonitrile
ACHTUNGTRENNUNG
and
polymer-supported
1,8-diazabicyclo-
[5.4.0]undec-7-ene (DBU),[17] which allowed us to improve
the yields considerably. Moreover, the obtained products
were so clean that a chromatographic purification was un-
necessary.
After the glycosylation, the N-Boc protecting group was
removed in a one-pot protocol by adding three equivalents
of BF3·OEt2 and keeping the reaction mixture for 5 h at RT.
Subsequent amidation of the obtained secondary amine with
DMAI (7) under EDC·HCl activation in DMF gave the ace-
tylated prodrugs 8a–e in reasonable yields over three steps
(Table 1).
In vitro cytotoxicity: The in vitro cytotoxicities of the novel
prodrugs and of the corresponding seco-drug (Table 2) were
determined on human bronchial carcinoma cells A549 em-
ploying a colony-forming test that reflects the proliferation
capacity of single cells (Table 2). These assays also give evi-
dence of the efficiency of drug formation from the prodrug
and whether an undesired suicide mechanism inactivating
the enzyme takes place. If the IC50 value of the prodrug in
the presence of the enzyme is similar to that of the corre-
sponding seco-drug, one can assume that the enzymatic
1924
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ꢂ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2011, 17, 1922 – 1929

Synthesis and Biological Evaluation of Prodrugs Based on Duocarmycin
FULL PAPER
Table 2. In vitro cytotoxicity[a] and stability of the prodrugs (ꢀ)-(S)-2a, 10a–e, and the seco-drug (+)-(S)-3a.
[f]
Prodrug
Detoxifying unit[b]
IC50 without
IC50 with
QIC50
Stability over
24 h [%][g]
enzyme [nm]
enzyme [pm]
(+)-(S)-3a
(ꢀ)-(S)-2a
10a
10b
10c
–
0.026
56.3
130
183
42.5
57.0
–
–
–
–
100
100
b-d-galactose
16[c]
20[c]
1250[d]
60[d]
56[e]
3500
6500
150
700
1020
b-d-galactosyl-(1!6)-b-d-galactose
a-d-mannosyl-(1!6)-a-d-mannose
a-d-mannose
b-d-glucuronic acid
methyl ester
100
10d
98% conversion to 10e[h]
10e
b-d-glucuronic acid
6.20
13[e]
480
100
[a] Determined by HTCFA test; see the Experimental Section. [b] Carbohydrate moiety on enantiopure (ꢀ)-(S)-seco-CBI pharmacophor. [c] Enzyme:
4 UmLꢀ1 b-d-galactosidase. [d] Enzyme: 0.4 UmLꢀ1 a-d-mannosidase. [e] Enzyme: 4 UmLꢀ1 b-d-glucuronidase. [f] QIC50 =IC50 of prodrug/IC50 of pro-
drug in presence of cleaving enzyme. [g] Incubation in human serum at 378C; see the Experimental Section. [h] First-order reaction, [S]=[S]0·eꢀkt, k=
3.9ꢁ0.2ꢄ10ꢀ5 sꢀ1, t1/2 =4.9ꢁ0.3 h, R2 =0.984.
cleavage is fast and the enzyme is not deactivated by the
product formed. For the assay, the cells were exposed to var-
ious concentrations of each prodrug 10a–e in the absence
and presence of the corresponding enzyme in serum-free
UC medium (UltraCulture medium).
QIC50 =6500 the GalGal prodrug 10a shows the best selec-
tivity of all prodrugs prepared so far based on compounds
interacting with DNA; thus, 10a is very suitable for in vivo
studies.
As anticipated, the IC50 values of the diglycosidic pro-
drugs 10a and 10b are higher than those found for the mono-
glycosidic compounds 2a and 10c with IC50 =130 and IC50 =
183 nm, respectively, relative to IC50 =56.3 and IC50 =
42.5 nm, respectively. This might be due to a reduced intake
of the diglycosidic relative to the monoglycosidic prodrugs
due to a higher polarity of the former. The enzymatic cleav-
age of the GalGal moiety in 10a seems to be very fast when
using b-d-galactosidase from E. coli since the IC50 value of
10a in the presence of the enzyme corresponds very well to
that of the seco-drug 2a. This results in a QIC50 value of
6500, which is so far the best result for this family of com-
pounds interacting with DNA. In contrast, prodrug 10b
seems to be less suitable since the enzymatic cleavage of the
a-d-mannose moieties when using a-d-mannosidase is
rather slow resulting in lower cytotoxicity. Here, either the
prodrug 10b is not a good substrate for the enzyme or
indeed a suicide mechanism might be operating. A similar
effect was found for the monoglycosidic compound 10c con-
taining a mannose residue.
Experimental Section
General: All reactions were carried out under argon in flame-dried glass-
ware. Solvents were distilled prior to use by common laboratory methods
or used from commercial sources in p.a. grade and stored over molecular
sieves. All reagents purchased from commercial sources were used with-
out further purification. TLC was performed on silica gel 60 F254 plates
from Merck and silica gel 60 (0.032–0.063 mm, Merck) was used for
column chromatography. Vanillin in methanolic sulfuric acid was used as
the staining reagent for TLC. Preparative HPLC for purification of the
target compounds was performed on a Jasco system, equipped with two
solvent pumps PU-2087 PLUS, UV detector UV-2075 PLUS, and a semi-
preparative RP column Kromasil 100 C18 (250ꢄ20 mm, 7 mm, Jasco).
UV spectra were taken with a Perkin–Elmer Lambda 2 spectrometer. IR
spectra were recorded as KBr pellets with a Bruker IFS 25 spectrometer.
Optical rotations were measured on a Perkin–Elmer 241 polarimeter in
the solvent indicated. 1H and 13C NMR spectra were recorded with Mer-
cury-300, Unity-300, Inova-500, Inova-600 (Varian) spectrometers. Spec-
tra were taken at room temperature or 358C in deuterated solvents as in-
dicated by using the solvent peak as an internal standard. For 13C num-
bering see the Supporting Information. Mass spectra (ESI) were obtained
from an ion-trap mass spectrometer LCQ (Finnigan) and a TOF spec-
trometer micrOTOF (Bruker). HRMS was performed with 7 T FTICR-
MS APEX IV equipment (Bruker) and micrOTOF (Bruker). The purities
and stabilities of the prodrugs were analyzed by HPLC-MS by using ESI
mass spectrometry with the ion-trap mass spectrometer LCQ (Finnigan).
The HPLC system comprises of solvent pump Rheos 400, degasser ERC-
3415a (Flux Instruments), autosampler 851 (Jasco), and a diode array de-
tector (Thermo). The column was a Synergi Max-RP C12 (150ꢄ2 mm,
4 mm, phenomenex). Mobile phases were water (A) and methanol (B)
(hypergrade for LC-MS, Merck) with 0.05% formic acid (Roth) each.
The flow was 300 mLminꢀ1 with the following gradient: 0–15 min: 70A/
30B!0A/100B, 15–22 min: 0A/100B, 22–23 min: 0A/100B!70A/30B,
23–29 min: 70A/30B. Pooled human serum was obtained from the Klini-
kum Gçttingen, dept. of transfusion medicine, and used without dilution.
A highly promising compound for PMT seems to be 10d
bearing a b-d-methyl glucuronide moiety with a QIC50 value
of 1020. Its primary cytotoxicity is much lower than that of
the prodrug 10e containing a b-d-glucuronide moiety.
Conclusion
Five new glycosidic prodrugs based on duocarmycin SA that
differ in the detoxifying sugar moiety have been prepared
for application in ADEPT and PMT. All compounds except
10d are stable in human serum, which is essential for appli-
cation in vivo. The ester 10d showing a good QIC50 value of
1020 can act as a “double-prodrug” as it is hydrolyzed to
give the corresponding glucuronic acid prodrug 10e in
serum and during cell culture experiments, which then can
be activated by the enzyme b-d-glucuronidase. With a
General procedure
A for the synthesis of the trichloroacetimidate
donors: Polymer-supported DBU[17] (1.26 mmolgꢀ1, 0.50 equiv) and tri-
chloroacetonitrile (4.0–10.0 equiv) were added to
a solution of the
anomeric deprotected sugar (1.0 equiv) in dry CH2Cl2 (8–13 mLmmolꢀ1).
After stirring for 1–3.5 h at room temperature, the brown suspension was
filtered over Celite to separate the polymer and the Celite was washed
with CH2Cl2. The solvent was removed at room temperature in vacuo to
Chem. Eur. J. 2011, 17, 1922 – 1929
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1925

L. F. Tietze et al.
give the clean trichloroacetimidate as a slightly yellow syrup. Yields: 6a:
97%, 6b: 95%, 6c: 78%, 6d: 82%, 6e: 85%.
(C-7a’), 141.7 (C-3a), 152.7 (C-5), 152.8 (C-5’), 160.2 (C=O), 168.7, 169.1,
169.2 (2 signals), 169.3, 169.5 ppm (2 signals; 7ꢄC(O)CH3); IR (KBr):
n˜ =2942, 1753, 1627, 1519, 1462, 1370, 1221, 1068, 760 cmꢀ1
; UV
General procedure B for the glycosylation, Boc-deprotection, and DNA-
binder coupling: A mixture of the phenol (ꢀ)-(S)-5 (1.00 equiv), trichloro-
acetimidate 6 (1.18–1.45 equiv), and freshly activated molecular sieves
4 ꢅ (1.80–2.40 gmmolꢀ1) in dry CH2Cl2 (33–46 mLmmolꢀ1) was stirred
for 30 min at RT. After cooling to the indicated temperature, the pre-
cooled promoter BF3·OEt2 (0.5 equiv) in dry CH2Cl2 (10.5 mLmmolꢀ1
BF3·OEt2) was added dropwise and the mixture stirred at ꢀ108C for the
given time. To remove the Boc-protecting group, an excess of BF3·OEt2
(3.0 equiv) in CH2Cl2 (1.5 mLmmolꢀ1 BF3·OEt2) was added, the mixture
warmed to room temperature, and stirred for 5 h. The reaction mixture
was transferred into a second flask by transfer cannula to separate the
molecular sieves, which were then thoroughly washed with CH2Cl2. The
solvent was removed under reduced pressure and the resulting foam
dried under high vacuum for 1 h. The formed salt was dissolved in DMF
(45 mLmmolꢀ1), the stirred solution cooled to 08C, and EDC·HCl
(3.0 equiv) followed by DMAI·HCl (7) (1.5 equiv) were added. After stir-
ring at RT for 15–19 h, the mixture was diluted with EtOAc
(70 mLmmolꢀ1), water (70 mLmmolꢀ1), and saturated NaHCO3 solution
(55 mLmmolꢀ1), the phases separated, and the water layer extracted
again with EtOAc (4ꢄ110 mLmmolꢀ1). The combined organic layers
were washed with brine (2ꢄ90 mLmmolꢀ1), dried over MgSO4, and the
solvent removed under reduced pressure. The crude material was purified
by column chromatography on silica gel (CH2Cl2/MeOH 10:1) to yield
the acetylated prodrugs 8.
(MeOH): lmax (lg e)=202.5 (4.7014), 298.5 (4.4572), 332.5 nm (4.443);
HRMS (ESI): m/z: calcd for C52H60ClN3O20: 1082.3532; found: 1082.3537
[M+H]+.
Compound 10a: According to the general procedure C, a solution of the
acetylated prodrug 8a (131 mg, 121 mmol, 1.0 equiv) in MeOH (18 mL)
was treated with NaOMe (67.1 mL, 362 mmol, 30% solution in MeOH,
3.0 equiv) for 1 h at room temperature. Workup and purification by RP-
HPLC with water (A), MeOH (B)+0.05% acetic acid as the eluent (gra-
dient: 0–15 min: 70A/30B!0A/100B, 15–20 min: 0A/100B, 20–21 min:
0A/100B!70A/30B, 21–26 min: 70A/30B, flow: 18 mLminꢀ1; l=254 nm;
injection volume: 0.8 mL; tR =7.0 min) gave 10a as a slightly ocher solid
(78.9 mg, 100 mmol, 83%). Rf =0.15 (MeOH/H2O 5:1, 0.5% HOAc); a=
1
ꢀ14.18 (c=0.21 in DMSO); H NMR (600 MHz, [D6]DMSO): d=2.25 (s,
6H; NMe2), 2.66 (t, J=5.9 Hz, 2H; 2’’-H2), 3.29 (dd, J=9.5, 3.4 Hz, 1H;
3’’’’-H), 3.36–3.41 (m, 2H; 2’’’’-H, 5’’’’-H), 3.48 (dd, J=9.6, 2.8 Hz, 1H;
3’’’-H), 3.54 (mc, J=10.9, 6.2 Hz, 2H; 6’’’’-Ha, 6’’’’-Hb), 3.62 (d, J=3.3 Hz,
1H; 4’’’’-H), 3.65 (dd, J=10.2, 5.6 Hz, 1H; 6’’’-Ha), 3.79 (mc, 2H; 2’’’-H,
5’’’-H), 3.84 (d, J=2.8 Hz, 1H; 4’’’-H), 3.91 (dd, J=11.2, 7.4 Hz, 1H; 10-
Ha), 3.98 (dd, J=10.2, 6.6 Hz, 1H; 6’’’-Hb), 4.04–4.09 (m, 3H; 10-Hb, 1’’-
H2), 4.17 (d, J=7.6 Hz, 1H; 1’’’’-H), 4.30 (mc, 1H; 1-H), 4.60 (dd, J=
10.8, 1.7 Hz, 1H; 2-Ha), 4.83 (t, J=10.0 Hz, 1H; 2-Hb), 4.92 (d, J=
7.3 Hz, 1H; 1’’’-H), 5.32 (brs, 1H; OH), 6.93 (dd, J=8.9, 2.4 Hz, 1H; 6’-
H), 7.13 (d, J=1.6 Hz, 1H; 3’-H), 7.18 (d, J=2.3 Hz, 1H; 4’-H), 7.44 (mc,
2H; 7-H, 7’-H), 7.58 (t, J=7.6 Hz, 1H; 8-H), 7.93 (d, J=8.4 Hz, 1H; 9-
H), 8.28 (brs, 1H; 4-H), 8.36 (d, J=8.5 Hz, 1H; 6-H), 11.46 ppm (d, J=
1.2 Hz, 1H; NH); 13C NMR (125 MHz, [D6]DMSO): d=41.2 (C-1), 45.5
(NMe2), 47.4 (C-10), 54.9 (C-2), 57.7 (C-2’’), 60.5 (C-6’’’’), 66.2 (C-1’’),
67.2 (C-6’’’), 67.9 (C-4’’’), 68.1 (C-4’’’’), 70.3 (C-2’’’), 70.4 (C-2’’’’), 72.8 (C-
3’’’), 73.1 (C-3’’’’), 73.5 (C-5’’’), 75.1 (C-5’’’’), 102.1 (C-4), 102.3 (C-1’’’),
103.0 (C-4’), 103.4 (C-1’’’’), 105.4 (C-3’), 113.1 (C-7’), 115.8 (C-6’), 118.1
(C-9b), 122.6 (C-9), 122.9 (C-5a), 123.2 (C-6), 123.7 (C-7), 127.3 (2 sig-
nals; C-8, C-3a’), 129.3 (C-9a), 130.5 (C-2’), 131.5 (C-7a’), 141.8 (C-3a),
152.8 (C-5’), 153.6 (C-5), 160.2 ppm (C=O); IR (KBr): n˜ =3386, 1589,
1516, 1462, 1413, 1267, 1075, 761 cmꢀ1; UV (MeOH): lmax (lg e)=206.5
(4.629), 300.0 (4.4603), 336.5 nm (4.4265); HRMS (ESI): m/z: calcd for
C38H46ClN3O13: 810.2611; found: 810.2629 [M+Na]+.
General procedure C for the Zemplꢁn deacetylation: At 08C a solution
of the acetylated prodrug (1.0 equiv) in MeOH was treated with a
NaOMe solution (25 or 30% in MeOH, 0.5–3.0 equiv) and the mixture
stirred at RT until complete conversion was observed (TLC control). The
mixture was neutralized with acidic ion-exchange resin Amberlite IR120
or 1m methanolic acetic acid, respectively. After evaporation of the sol-
vent, the crude material was purified by RP-HPLC unless otherwise
stated.
Compound 8a: According to general procedure B, a stirred mixture of
the trichloroacetimidate 6a (200 mg, 256 mmol, 1.22 equiv), phenol (ꢀ)-
(1S)-5 (70 mg, 210 mmol, 1.0 equiv), and molecular sieves 4 ꢅ (450 mg) in
CH2Cl2 (8 mL) were treated with BF3·OEt2 (13.3 mL, 105 mmol, in
CH2Cl2, 0.5 equiv) at ꢀ168C and stirring was continued at ꢀ108C for
3.5 h. Additional BF3·OEt2 (79.7 mL, 629 mmol, in 0.9 mL CH2Cl2,
3.0 equiv) was added and the mixture kept at RT for 5 h. Evaporation
and subsequent reaction with DMAI·HCl (7) (89.6 mg, 315 mmol,
1.5 equiv) and EDC·HCl (121 mg, 629 mmol, 3.0 equiv) in DMF (10 mL)
for 16 h yielded crude material that was purified by column chromatogra-
phy to afford the peracetylated prodrug 8a (140 mg, 130 mmol, 62%) as a
pale-yellow solid. Rf =0.39 (CH2Cl2/MeOH 10:1); a=+4.38 (c=0.21 in
MeOH); 1H NMR (600 MHz, [D6]DMSO): d=1.88 1.89, 1.90, 1.96 (4ꢄs,
4ꢄ3H; 4ꢄC(O)CH3), 2.06 (s, 6H; 2ꢄC(O)CH3), 2.17 (s, 3H; C(O)CH3),
2.26 (s, 6H; NMe2), 2.68 (t, J=5.8 Hz, 2H; 2’’-H), 3.72 (mc, 1H; 6’’’-Ha),
3.78 (mc, 1H; 6’’’-Hb), 3.94 (dd, J=11.2, 6.9 Hz, 1H; 10-Ha), 3.98–4.06
(m, 3H; 6’’’’-Ha, 6’’’’-Hb), 4.08 (t, J=5.9 Hz, 2H; 10-Hb, 1’’-H), 4.14 (t, J=
6.7 Hz, 1H; 5’’’’-H), 4.33 (mc, 1H; 1-H), 4.38 (mc, 1H; 5’’’-H), 4.60 (dd,
J=10.9, 2.0 Hz, 1H; 2-Ha), 4.71 (d, J=8.0 Hz, 1H; 1’’’’-H), 4.83 (t, J=
10.0 Hz, 1H; 2-Hb), 4.87 (dd, J=10.3, 8.0 Hz, 1H; 2’’’’-H), 5.02 (dd, J=
10.4, 3.6 Hz, 1H; 3’’’’-H), 5.21 (d, J=3.4 Hz, 1H; 4’’’’-H), 5.34 (d, J=
2.0 Hz, 1H; 4’’’-H), 5.36–5.43 (m, 2H; 2’’’-H, 3’’’-H), 5.56 (d, J=6.6 Hz,
1H; 1’’’-H), 6.93 (dd, J=8.9, 2.4 Hz, 1H; 6’-H), 7.11 (d, J=1.7 Hz, 1H;
3’-H), 7.18 (d, J=2.3 Hz, 1H; 4’-H), 7.39 (d, J=8.9 Hz, 1H; 7’-H), 7.48
(ddd, J=8.1, 6.8, 0.8 Hz, 1H; 7-H), 7.60 (ddd, J=8.1, 6.9, 1.1 Hz, 1H; 8-
H), 7.96 (d, J=8.4 Hz, 1H; 9-H), 8.01 (d, J=8.5 Hz, 1H; 6-H), 8.24 (brs,
1H; 4-H), 11.57 ppm (d, J=1.6 Hz, 1H; NH); 13C NMR (125 MHz,
[D6]DMSO): d=20.2 (4 signals; 4ꢄC(O)CH3), 20.3 (2ꢄC(O)CH3), 20.5
(C(O)CH3), 41.1 (C-1), 45.4 (NMe2), 47.4 (C-10), 54.9 (C-2), 57.7 (C-2’’),
61.0 (C-6’’’’), 64.8 (C-6’’’), 66.1 (C-1’’), 66.6 (C-4’’’), 67.0 (C-4’’’’), 68.3 (C-
2’’’’), 68.6 (C-2’’’), 69.8 (C-5’’’’), 70.0 (C-3’’’), 70.2 (C-3’’’’), 70.5 (C-5’’’),
99.0 (C-1’’’’), 99.6 (C-1’’’), 103.1 (C-4’), 103.4 (C-4), 105.2 (C-3’), 113.0 (C-
7’), 115.7 (C-6’), 119.4 (C-9b), 122.0 (C-6), 122.7 (C-5a), 122.9 (C-9),
124.3 (C-7), 127.3 (C-3a’), 127.5 (C-8), 129.4 (C-9a), 130.6 (C-2’), 131.5
Compound 8b: According to general procedure B, a stirred mixture of
the trichloroacetimidate 6b (220 mg, 281 mmol, 1.25 equiv), phenol (ꢀ)-
(1S)-5 (75 mg, 225 mmol, 1.0 equiv), and molecular sieves 4 ꢅ (500 mg) in
CH2Cl2 (8 mL) was treated with BF3·OEt2 (14.3 mL, 113 mmol, in 1.2 mL
CH2Cl2, 0.5 equiv) at ꢀ168C and stirring was continued at ꢀ108C for
3.5 h. Additional BF3·OEt2 (85.5 mL, 675 mmol, in 1.0 mL CH2Cl2,
3.0 equiv) was added and the mixture kept at RT for 5 h. Evaporation
and subsequent reaction with DMAI·HCl (7) (96.0 mg, 338 mmol,
1.5 equiv) and EDC·HCl (129 mg, 675 mmol, 3.0 equiv) in DMF (10 mL)
for 17.5 h gave crude material that was purified by column chromatogra-
phy to afford the peracetylated prodrug 8b (150 mg, 139 mmol, 62%) as
a pale-yellow solid. Rf =0.32 (CH2Cl2/MeOH 10:1); a=+74.08 (c=0.2 in
MeOH); 1H NMR (600 MHz, [D6]DMSO): d=1.89, 1.92, 1.95, 2.05, 2.07
(2 signals), 2.19 (7ꢄs, 7ꢄ3H; 7ꢄC(O)CH3), 2.25 (s, 6H; NMe2), 2.67 (t,
J=5.8 Hz, 2H; 2’’-H2), 3.55 (dd, J=11.5, 2.6 Hz, 1H; 6’’’-Ha), 3.71 (dd,
J=11.5, 3.6 Hz, 1H; 6’’’-Hb), 3.83 (ddd, J=9.6, 5.4, 2.5 Hz, 1H; 5’’’’-H),
3.91–3.96 (m, 2H; 10-Ha, 6’’’’-Ha), 4.07 (mc, 5H; 10-Hb, 1’’-H2, 5’’’-H, 6’’’’-
Hb), 4.33 (mc, 1H; 1-H), 4.60 (dd, J=10.8, 1.9 Hz, 1H; 2-Ha), 4.83 (t, J=
10.0 Hz, 1H; 2-Hb), 4.86 (d, J=1.3 Hz, 1H; 1’’’’-H), 5.04 (t, J=10.0 Hz,
1H; 4’’’’-H), 5.10 (dd, J=10.2, 3.4 Hz, 1H; 3’’’’-H), 5.15 (dd, J=3.4,
1.6 Hz, 1H; 2’’’’-H), 5.46 (t, J=10.1 Hz, 1H; 4’’’-H), 5.52 (dd, J=3.2,
2.0 Hz, 1H; 2’’’-H), 5.61 (dd, J=10.2, 3.3 Hz, 1H; 3’’’-H), 5.91 (s, 1H; 1’’’-
H), 6.92 (dd, J=8.9, 2.4 Hz, 1H; 6’-H), 7.08 (d, J=1.8 Hz, 1H; 3’-H),
7.17 (d, J=2.3 Hz, 1H; 4’-H), 7.39 (d, J=8.9 Hz, 1H; 7’-H), 7.54 (ddd,
J=8.1, 7.0, 0.8 Hz, 1H; 7-H), 7.63 (ddd, J=8.1, 7.0, 1.0 Hz, 1H; 8-H),
7.99 (d, J=8.4 Hz, 1H; 9-H), 8.16 (d, J=8.4 Hz, 1H; 6-H), 8.25 (brs,
1H; 4-H), 11.50 ppm (d, J=1.6 Hz, 1H; NH); 13C NMR (125 MHz,
[D6]DMSO): d=20.2, 20.3 (2 signals; 3ꢄC(O)CH3), 20.4 (2 signals; 3ꢄ
C(O)CH3), 20.5 (C(O)CH3), 41.1 (C-1), 45.4 (NMe2), 47.4 (C-10), 54.9
(C-2), 57.7 (C-2’’), 61.7 (C-6’’’’), 65.0 (C-4’’’), 65.1 (C-4’’’’), 65.4 (C-6’’’),
1926
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ꢂ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2011, 17, 1922 – 1929

Synthesis and Biological Evaluation of Prodrugs Based on Duocarmycin
FULL PAPER
66.1 (C-1’’), 67.8 (C-5’’’’), 68.3 (C-2’’’’), 68.5 (C-3’’’’), 68.6 (C-2’’’), 68.7 (C-
3’’’), 69.8 (C-5’’’), 95.4 (C-1’’’), 96.8 (C-1’’’’), 101.7 (C-4), 103.1 (C-4’),
105.2 (C-3’), 112.9 (C-7’), 115.7 (C-6’), 119.0 (C-9b), 121.8 (C-6), 122.3
(C-5a), 123.1 (C-9), 124.4 (C-7), 127.3 (C-3a’), 127.6 (C-8), 129.5 (C-9a),
130.6 (C-2’), 131.4 (C-7a’), 141.8 (C-3a), 151.0 (C-5), 152.8 (C-5’), 160.0
(C=O), 169.1 (2 signals; 3ꢄC(O)CH3), 169.3, 169.4, 169.6 ppm (2 signals;
4ꢄC(O)CH3); IR (KBr): n˜ =2945, 1752, 1627, 1519, 1462, 1371, 1225,
1085, 1048, 759 cmꢀ1; UV (MeOH): lmax (lg e)=205.5 (4.6778), 298.5
3’), 113.1 (C-7’), 115.9 (C-6’), 119.0 (C-9b), 121.9 (C-6), 122.3 (C-5a),
123.1 (C-9), 124.5 (C-7), 127.4 (C-3a’), 127.7 (C-8), 129.6 (C-9a), 130.7
(C-2’), 131.6 (C-7a’), 141.9 (C-3a), 151.2 (C-5), 153.0 (C-5’), 160.1 (C=O),
169.4, 169.6, 169.7, 169.8 ppm (4ꢄC(O)CH3); IR (KBr): n˜ =2952, 1752,
1627, 1518, 1462, 1397, 1228, 1050, 760 cmꢀ1; UV (MeOH): lmax (lg e)=
204.5 (4.6945), 299.0 (4.5186), 335.0 nm (4.5033); HRMS (ESI): m/z:
calcd for C40H44ClN3O12: 794.2686; found: 794.2676 [M+H]+.
Compound 10c: By following general procedure C, a solution of the ace-
tylated prodrug 8c (170 mg, 214 mmol, 1.0 equiv) in MeOH (9 mL) was
treated with NaOMe (40 mL, 214 mmol, 30% solution in MeOH,
1.0 equiv) for 30 min at RT. Workup and purification by RP-HPLC with
water (A), MeOH (B)+0.05% acetic acid as the eluent (gradient: 0–
15 min: 70A/30B!0A/100B, 15–20 min: 0A/100B, 20–21 min: 0A/100B!
70A/30B, 21–26 min: 70A/30B, flow: 18 mLminꢀ1; l=254 nm; injection
volume: 1 mL; tR =8.4 min) gave 10c as a slightly ocher solid (120 mg,
192 mmol, 90%). Rf =0.20 (MeOH/H2O 5:1, 0.5% HOAc); a=+68.68
(c=0.45 in DMSO); 1H NMR (600 MHz, [D6]DMSO): d=2.25 (s, 6H;
NMe2), 2.66 (t, J=5.9 Hz, 2H; 2’’-H2), 3.43 (mc, J=9.4, 5.0, 2.2 Hz, 1H;
5’’’-H), 3.50 (dd, J=11.8, 5.1 Hz, 1H; 6’’’-Ha), 3.57 (dd, J=11.8, 2.1 Hz,
1H; 6’’’-Hb), 3.64 (t, J=9.5 Hz, 1H; 4’’’-H), 3.89–3.94 (m, 2H, 10-Ha; 3’’’-
H), 4.03–4.09 (m, 4H; 10-Hb, 1’’-H2, 2’’’-H), 4.29 (mc, 1H; 1-H), 4.59 (dd,
J=10.8, 1.9 Hz, 1H; 2-Ha), 4.84 (t, J=10.0 Hz, 1H; 2-Hb), 5.68 (s, 1H;
1’’’-H), 6.93 (dd, J=8.9, 2.4 Hz, 1H; 6’-H), 7.11 (d, J=1.7 Hz, 1H; 3’-H),
7.18 (d, J=2.2 Hz, 1H; 4’-H), 7.40 (d, J=8.9 Hz, 1H; 7’-H), 7.47 (t, J=
7.6 Hz, 1H; 7-H), 7.58 (t, J=7.6 Hz, 1H; 8-H), 7.93 (d, J=8.4 Hz, 1H; 9-
H), 8.14 (d, J=8.4 Hz, 1H; 6-H), 8.23 (brs, 1H; 4-H), 11.58 ppm (brs,
1H; NH); 13C NMR (125 MHz, [D6]DMSO): d=41.1 (C-1), 45.5 (NMe2),
47.5 (C-10), 55.1 (C-2), 57.8 (C-2’’), 60.7 (C-6’’’), 66.2 (C-1’’), 66.5 (C-4’’’),
70.1 (C-2’’’), 71.0 (C-3’’’), 75.4 (C-5’’’), 98.5 (C-1’’’), 101.0 (C-4), 103.2 (C-
4’), 105.3 (C-3’), 113.1 (C-7’), 115.9 (C-6’), 117.8 (C-9b), 122.4 (C-6),
122.7 (C-5a), 123.0 (C-9), 124.0 (C-7), 127.4 (C-3a’), 127.5 (C-8), 129.6
(C-9a), 130.8 (C-2’), 131.6 (C-7a’), 142.1 (C-3a), 151.9 (C-5), 153.0 (C-5’),
160.2 ppm (C=O); IR (KBr): n˜ =3385, 1611, 1514, 1461, 1415, 1264, 1181,
1065, 973, 755 cmꢀ1; UV (MeOH): lmax (lg e)=204.5 (4.6462), 300.0
(4.4595), 336.5 nm (4.4273); HRMS (ESI): m/z: calcd for C32H36ClN3O8:
626.2264; found: 626.2263 [M+H]+.
(4.5086), 335.0 nm (4.4884); HRMS (ESI): m/z: calcd for C52H60ClN3O20
:
1082.3531; found: 1082.3557 [M+H]+.
Compound 10b: By following general procedure C, a solution of the ace-
tylated prodrug 8b (133 mg, 123 mmol, 1.0 equiv) in MeOH (10 mL) was
treated with NaOMe (68.5 mL, 370 mmol, 30% solution in MeOH,
3.0 equiv) and the mixture stirred for 2 h at RT. Workup and purification
by RP-HPLC with water (A), MeOH (B)+0.05% acetic acid as the
eluent (gradient: 0–15 min: 70A/30B!0A/100B, 15–20 min: 0A/100B,
20–21 min: 0A/100B!70A/30B, 21–26 min: 70A/30B, flow: 18 mLminꢀ1
;
l=254 nm; injection volume: 0.8 mL; tR =8.0 min) gave 10b as a slightly
ocher solid (93.2 mg, 118 mmol, 96%). Rf =0.15 (MeOH/H2O 5:1, 0.5%
HOAc); a=+72.68 (c=0.17 in DMSO); 1H NMR (600 MHz,
[D6]DMSO): d=2.24 (s, 6H; NMe2), 2.66 (t, J=5.8 Hz, 2H; 2’’-H2), 3.35–
3.40 (m, 2H; 4’’’’-H, 5’’’’-H), 3.43 (dd, J=11.4, 5.1 Hz, 1H; 6’’’’-Ha), 3.46
(d, J=10.9 Hz, 2H; 6’’’-Ha, 3’’’’-H), 3.51 (dd, J=3.1, 1.5 Hz, 1H; 2’’’’-H),
3.58 (mc, 2H; 5’’’-H, 6’’’’-Hb), 3.70 (t, J=9.5 Hz, 1H; 4’’’-H), 3.74 (dd, J=
11.2, 4.9 Hz, 1H; 6’’’-Hb), 3.89–3.94 (m, 2H; 10-Ha, 3’’’-H), 4.03–4.09 (m,
4H; 10-Hb, 1’’-H2, 2’’’-H), 4.30 (mc, 1H; 1-H), 4.51 (d, J=1.2 Hz, 1H;
1’’’’-H), 4.58 (dd, J=10.8, 2.0 Hz, 1H; 2-Ha), 4.85 (t, J=9.9 Hz, 1H; 2-
Hb), 5.62 (s, 1H; 1’’’-H), 6.92 (dd, J=8.9, 2.4 Hz, 1H; 6’-H), 7.10 (s, 1H;
3’-H), 7.17 (d, J=2.2 Hz, 1H; 4’-H), 7.41 (d, J=8.9 Hz, 1H; 7’-H), 7.47
(t, J=7.7 Hz, 1H; 7-H), 7.58 (t, J=7.5 Hz, 1H; 8-H), 7.93 (d, J=8.3 Hz,
1H; 9-H), 8.13 (d, J=8.4 Hz, 1H; 6-H), 8.24 (s, 1H; 4-H), 11.61 ppm
(brs, 1H; NH); 13C NMR (125 MHz, [D6]DMSO): d=41.1 (C-1), 45.5
(NMe2), 47.5 (C-10), 54.9 (C-2), 57.8 (C-2’’), 61.0 (C-6’’’’), 65.0 (C-6’’’),
66.1 (C-4’’’), 66.2 (C-1’’), 66.9 (C-4’’’’), 70.0 (C-2’’’), 70.1 (C-2’’’’), 70.8 (C-
3’’’’), 71.1 (C-3’’’), 73.2 (C-5’’’), 73.4 (C-5’’’’), 98.9 (C-1’’’), 99.3 (C-1’’’’),
101.6 (C-4), 103.1 (C-4’), 105.2 (C-3’), 113.0 (C-7’), 115.6 (C-6’), 117.9 (C-
9b), 122.3 (C-6), 122.7 (C-5a), 122.9 (C-9), 123.9 (C-7), 127.3 (2 signals)
(C-8, C-3a’), 129.5 (C-9a), 130.8 (C-2a’), 131.5 (C-7a’), 141.9 (C-3a), 151.8
(C-5), 152.8 (C-5’), 160.1 ppm (C=O); IR (KBr): n˜ =3383, 1580, 1516,
1462, 1413, 1065, 972, 760 cmꢀ1; UV (MeOH): lmax (lg e)=202.0 (5.3727),
203.0 (4.7069), 300.0 (4.4287), 335.5 nm (4.4016); HRMS (ESI): m/z:
calcd for C38H46ClN3O13: 788.2792; found: 788.2788 [M+H]+.
Compound 8d: According to general procedure B, a stirred mixture of
the trichloroacetimidate 6d (170 mg, 355 mmol, 1.18 equiv), phenol (ꢀ)-
(1S)-5 (100 mg, 300 mmol, 1.0 equiv), and molecular sieves 4 ꢅ (550 mg)
in CH2Cl2 (10 mL) was treated with BF3·OEt2 (19.0 mL, 150 mmol, in
1.6 mL CH2Cl2, 0.5 equiv) at ꢀ188C and stirring was continued at ꢀ108C
for 4 h. Additional BF3·OEt2 (114 mL, 900 mmol, in 1.3 mL CH2Cl2,
3.0 equiv) was added and the mixture kept at RT for 5 h. Evaporation
and subsequent reaction with DMAI·HCl (7) (128 mg, 150 mmol,
1.5 equiv) and EDC·HCl (172 mg, 900 mmol, 3.0 equiv) in DMF (14 mL)
for 19 h after purification yielded 8d (89.5 mg, 155 mmol, 39%) as a
slightly ocher solid. Rf =0.35 (CH2Cl2/MeOH 10:1); 1H NMR (600 MHz,
[D6]DMSO): d=2.02, 2.03 (2 signals; 3ꢄs, 3ꢄ3H; 3ꢄC(O)CH3), 2.25 (s,
6H; NMe2), 2.67 (t, J=5.8 Hz, 2H; 2’’-H2), 3.67 (s, 3H; OCH3), 3.93 (dd,
J=11.2, 7.0 Hz, 1H; 10-Ha), 4.03–4.10 (m, 3H; 10-Hb, 1’’-H2), 4.31 (mc,
1H; 1-H), 4.60 (dd, J=10.8, 2.0 Hz, 1H; 2-Ha), 4.74 (d, J=9.8 Hz, 1H;
5’’’-H), 4.83 (t, J=10.0 Hz, 1H; 2-Hb), 5.15 (t, J=9.6 Hz, 1H; 4’’’-H), 5.34
(dd, J=9.7, 7.8 Hz, 1H; 2’’’-H), 5.60 (t, J=9.6 Hz, 1H; 3’’’-H), 5.81 (d,
J=7.8 Hz, 1H; 1’’’-H), 6.93 (dd, J=8.9, 2.4 Hz, 1H; 6’-H), 7.11 (d, J=
1.7 Hz, 1H; 3’-H), 7.18 (d, J=2.3 Hz, 1H; 4’-H), 7.41 (d, J=8.9 Hz, 1H;
7’-H), 7.48 (ddd, J=8.1, 6.9, 0.9 Hz, 1H; 7-H), 7.60 (ddd, J=8.1, 7.0,
1.1 Hz, 1H; 8-H), 7.96 (d, J=8.4 Hz, 1H; 9-H), 7.99 (d, J=8.5 Hz, 1H;
6-H), 8.23 (s, 1H; 4-H), 11.61 ppm (d, J=1.5 Hz, 1H; NH); 13C NMR
(125 MHz, [D6]DMSO): d=20.2, 20.3 (2 signals; 3ꢄC(O)CH3), 41.1 (C-
1), 45.5 (NMe2), 47.4 (C-10), 52.5 (OCH3), 55.0 (C-2), 57.7 (C-2’’), 66.2
(C-1’’), 69.0 (C-4’’’), 70.6 (2 signals; C-2’’’, C-3’’’), 71.0 (C-5’’’), 98.2 (C-
1’’’), 102.4 (C-4), 103.1 (C-4’), 105.2 (C-3’), 113.0 (C-7’), 115.8 (C-6’),
119.4 (C-9b), 121.8 (C-6), 122.4 (C-5a), 122.9 (C-9), 124.3 (C-7), 127.3 (C-
3a’), 127.6 (C-8), 129.3 (C-9a), 130.5 (C-2’), 131.5 (C-7a’), 141.7 (C-3a),
152.3 (C-5), 152.8 (C-5’), 160.0 (C(O)N), 166.8 (C-6’’’), 169.0 (2 signals),
169.1 ppm (3ꢄC(O)CH3); IR (KBr): n˜ =1759, 1626, 1517, 1462, 1413,
1218, 1040, 757 cmꢀ1; UV (MeOH): lmax (lg e)=204.0 (4.1497), 298.5
Compound 8c: According to general procedure B, a stirred solution of
the mannose trichloroacetimidate 6c (386 mg, 782 mmol, 1.45 equiv),
phenol (ꢀ)-(1S)-5 (180 mg, 539 mmol, 1.0 equiv), and molecular sieves
4 ꢅ (1.30 g) in CH2Cl2 (25 mL) was treated with BF3·OEt2 (34.0 mL,
270 mmol, in 2.8 mL CH2Cl2, 0.5 equiv) at ꢀ208C and the mixture stirred
at ꢀ108C for 6.5 h. Additional BF3·OEt2 (205 mL, 1.62 mmol, in 2.4 mL
CH2Cl2, 3.0 equiv) was added and the mixture kept at RT for 5 h. Evapo-
ration and subsequent reaction with DMAI·HCl (7) (230 mg, 809 mmol,
1.5 equiv) and EDC·HCl (310 mg, 1.62 mmol, 3.0 equiv) in DMF (25 mL)
for 16 h after purification gave prodrug 8c (209 mg, 263 mmol, 49%) as a
pale-yellow solid. Rf =0.38 (CH2Cl2/MeOH 10:1); a=+67.48 (c=0.5 in
MeOH); 1H NMR (600 MHz, [D6]DMSO): d=1.87 (s, 3H; C(O)CH3),
2.04 (2 signals, 2ꢄs, 2ꢄ3H; 2ꢄC(O)CH3), 2.19 (s, 3H; C(O)CH3), 2.24
(s, 6H; NMe2), 2.66 (t, J=5.8 Hz, 2H; 2’’-H2), 3.95 (dd, J=11.3, 6.6 Hz,
1H; 10-Ha), 3.96 (dd, J=12.4, 2.6 Hz, 1H; 6’’’-Ha), 4.04–4.08 (m, 3H; 10-
Hb, 1’’-H2), 4.10 (dd, J=5.8, 2.6 Hz, 1H; 5’’’-H), 4.21 (dd, J=12.3, 5.7 Hz,
1H; 6’’’-Hb), 4.34 (mc, 1H; 1-H), 4.60 (dd, J=10.8, 2.1 Hz, 1H; 2-Ha),
4.82 (t, J=10.0 Hz, 1H; 2-Hb), 5.26 (t, J=10.0 Hz, 1H; 4’’’-H), 5.55 (dd,
J=3.5, 1.8 Hz, 1H; 2’’’-H), 5.57 (dd, J=10.0, 3.5 Hz, 1H; 3’’’-H), 5.88 (s,
1H; 1’’’-H), 6.93 (dd, J=8.9, 2.4 Hz, 1H; 6’-H), 7.10 (d, J=1.9 Hz, 1H;
3’-H), 7.17 (d, J=2.3 Hz, 1H; 4’-H), 7.40 (d, J=8.9 Hz, 1H; 7’-H), 7.54
(t, J=7.6 Hz, 1H; 7-H), 7.63 (t, J=7.7 Hz, 1H; 8-H), 7.99 (d, J=8.4 Hz,
1H; 9-H), 8.15 (d, J=8.4 Hz, 1H; 6-H), 8.29 (brs, 1H; 4-H), 11.55 ppm
(d, J=1.5 Hz, 1H; NH); 13C NMR (125 MHz, [D6]DMSO): d=20.1, 20.3,
20.4, 20.5 (4 x C(O)CH3), 41.1 (C-1), 45.5 (NMe2), 47.5 (C-10), 54.9 (C-
2), 57.8 (C-2’’), 61.7 (C-6’’’), 65.2 (C-4’’’), 66.3 (C-1’’), 68.6 (2 signals; C-
2’’’, C-3’’’), 69.0 (C-5’’’), 95.7 (C-1’’’), 101.8 (C-4), 103.2 (C-4’), 105.3 (C-
(3.932), 335.0 nm (3.9196); HRMS (ESI): m/z: calcd for C39H42ClN3O12
:
780.2530; found: 780.2525 [M+H]+.
Chem. Eur. J. 2011, 17, 1922 – 1929
ꢂ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
1927

L. F. Tietze et al.
Compound 10d: By following general procedure C, a solution of 8d
(30 mg, 38.5 mmol, 1.0 equiv) in MeOH (12 mL) was treated with
NaOMe (4.4 mL, 19.2 mmol, 25% solution in MeOH, 0.5 equiv) and
stirred for 9 h at room temperature. After workup and purification by
column chromatography (CH2Cl2/MeOH 1:1) compound 10d (23.5 mg,
35.9 mmol, 93%, 99% purity (LC-MS)) was obtained as a slightly ocher
solid. Rf =0.23 (CH2Cl2/MeOH 1:1); a=ꢀ24.88 (c=0.20 in DMSO);
1H NMR (600 MHz, [D6]DMSO): d=2.31 (s, 6H; NMe2), 2.75 (mc, 2H;
2’’-H2), 3.40 (t, J=9.0 Hz, 1H; 3’’’-H), 3.46–3.55 (m, 2H; 2’’’-H, 4’’’-H),
3.68 (s, 3H; OCH3), 3.93 (dd, J=11.1, 7.3 Hz, 1H; 10-Ha), 3.97 (d, J=
9.6 Hz, 1H; 5’’’-H), 4.06 (dd, J=11.1, 2.9 Hz, 1H; 10-Hb), 4.10 (t, J=
5.7 Hz, 2H; 1’’-H2), 4.31 (mc, 1H; 1-H), 4.59 (d, J=10.8 Hz, 1H; 2-Ha),
4.82 (t, J=10.1 Hz, 1H; 2-Hb), 5.12 (d, J=7.7 Hz, 1H; 1’’’-H), 5.28, 5.40,
5.61 (3ꢄbrs, 3ꢄ1H; 3ꢄOH), 6.93 (dd, J=8.9, 2.2 Hz, 1H; 6’-H), 7.10 (s,
1H; 3’-H), 7.18 (d, J=1.7 Hz, 1H; 4’-H), 7.41 (d, J=8.9 Hz, 1H; 7’-H),
7.45 (t, J=7.6 Hz, 1H; 7-H), 7.59 (t, J=7.6 Hz, 1H; 8-H), 7.93 (d, J=
8.3 Hz, 1H; 9-H), 8.16 (brs, 1H; 4-H), 8.33 (d, J=8.5 Hz, 1H; 6-H),
11.62 ppm (s, 1H; NH); 13C NMR (125 MHz, [D6]DMSO): d=41.2 (C-1),
45.2 (NMe2), 47.4 (C-10), 51.9 (OCH3), 54.9 (C-2), 57.5 (C-2’’), 65.8 (C-
1’’), 71.4 (C-4’’’), 73.0 (C-2’’’), 75.3 (C-3’’’), 75.5 (C-5’’’), 101.3 (C-1’’’),
102.0 (C-4), 103.2 (C-4’), 105.2 (C-3’), 113.1 (C-7’), 115.7 (C-6’), 118.4 (C-
9b), 122.7 (C-9), 122.8 (C-5a), 123.1 (C-6), 123.8 (C-7), 127.3 (C-3a’),
127.5 (C-8), 129.4 (C-9a), 130.7 (C-2’), 131.6 (C-7a’), 141.8 (C-3a), 152.7
(C-5’), 153.0 (C-5), 160.0 (C(O)N), 168.8 ppm (C-6’’’); IR (KBr): n˜ =
3386, 1745, 1625, 1516, 1462, 1414, 1267, 1178, 1065, 759 cmꢀ1; UV
(MeOH): lmax (lg e)=271.0 (5.0708), 334.0 nm (4.3873); HRMS (ESI): m/
z: calcd for C33H36ClN3O9: 654.2213; found: 654.2212 [M+H]+.
(128 mg, 167 mmol, 86%) was obtained as a slightly yellow solid. Rf =0.33
(CH2Cl2/MeOH 3:1); a=+34.78 (c=0.20 in MeOH); 1H NMR
(600 MHz, [D6]DMSO): d=1.92, 1.98 (2 signals; 3ꢄs, 3ꢄ3H; 3ꢄ
C(O)CH3), 2.27 (s, 6H; NMe2), 2.68 (t, J=5.8 Hz, 2H; 2’’-H2), 3.91 (dt,
J=19.4, 9.7 Hz, 1H; 10-Ha), 4.03–4.12 (m, 4H; 10-Hb, 1’’-H2, 5’’’-H), 4.31
(mc, 1H; 1-H), 4.60 (dd, J=10.8, 2.0 Hz, 1H; 2-Ha), 4.82 (t, J=10.0 Hz,
1H; 2-Hb), 5.14 (t, J=9.7 Hz, 1H; 4’’’-H), 5.26 (dd, J=9.8, 7.9 Hz, 1H;
2’’’-H), 5.41 (t, J=9.6 Hz, 1H; 3’’’-H), 5.60 (d, J=7.8 Hz, 1H; 1’’’-H), 6.91
(dd, J=8.9, 2.4 Hz, 1H; 6’-H), 7.11 (d, J=1.7 Hz, 1H; 3’-H), 7.17 (d, J=
2.3 Hz, 1H; 4’-H), 7.40 (d, J=8.9 Hz, 1H; 7’-H), 7.45 (ddd, J=8.1, 6.9,
1.0 Hz, 1H; 7-H), 7.58 (ddd, J=8.2, 6.9, 1.1 Hz, 1H; 8-H), 7.95 (d, J=
8.4 Hz, 1H; 9-H), 8.01 (d, J=8.4 Hz, 1H; 6-H), 8.23 (brs, 1H; 4-H),
11.80 ppm (brs, 1H; NH); 13C NMR (125 MHz, [D6]DMSO): d=20.3,
20.4, 20.6 (3ꢄC(O)CH3), 41.1 (C-1), 45.4 (NMe2), 47.4 (C-10), 54.9 (C-2),
57.7 (C-2’’), 66.1 (C-1’’), 70.4 (C-4’’’), 71.1 (C-2’’’), 72.2 (C-3’’’), 74.5 (C-
5’’’), 98.2 (C-1’’’), 102.1 (C-4), 103.1 (C-4’), 105.3 (C-3’), 113.0 (C-7’),
115.8 (C-6’), 118.8 (C-9b), 122.0 (C-6), 122.6 (C-5a), 122.8 (C-9), 124.1
(C-7), 127.3 (C-3a’), 127.5 (C-8), 129.3 (C-9a), 130.6 (C-2’), 131.6 (C-7a’),
141.8 (C-3a), 152.6 (C-5), 152.7 (C-5’), 160.0 (C(O)N), 168.3 (C-6’’’),
168.6, 169.1, 169.2 ppm (3ꢄC(O)CH3); IR (KBr): n˜ =2945, 1756, 1623,
1516, 1463, 1415, 1229, 1038, 761 cmꢀ1; UV (MeOH): lmax (lg e)=206.0
(4.6307), 298.5 (4.425), 334.0 nm (4.4237); HRMS (ESI): m/z: calcd for
C38H40ClN3O12: 764.2228; found: 764.2226 [MꢀH]ꢀ.
Compound 10e: By following general procedure C, a solution of the ace-
tylated prodrug 9 (50 mg, 65.3 mmol, 1.0 equiv) in MeOH (10 mL) was
treated with NaOMe (37.3 mL, 163 mmol, 25% solution in MeOH,
2.5 equiv) and stirred for 3 h at room temperature. After workup and pu-
rification by RP-HPLC with water (A), CH3CN (B)+0.05% acetic acid
as the eluent (gradient: 0–10 min: 80A/20B!60A/40B, 10–15 min: 60A/
40B, 15–16 min: 60A/40B!80A/20B, 16–22 min: 80A/20B, flow:
12 mLminꢀ1; l=299 nm; injection volume: 0.8 mL; tR =11.8 min) the
prodrug 10e was obtained as a colorless solid (37.4 mg, 58.4 mmol, 89%).
Rf =0.12 (CH2Cl2/MeOH 1:5); a=ꢀ23.28 (c=0.2 in DMSO); 1H NMR
(600 MHz, [D6]DMSO): d=2.37 (s, 6H; NMe2), 2.82 (s, 2H; 2’’-H2), 3.29
(mc, 1H; 5’’’-H), 3.35 (t, J=8.9 Hz, 1H; 3’’’-H), 3.40 (t, J=9.1 Hz, 1H;
4’’’-H), 3.48 (mc, 1H; 2’’’-H), 3.68 (brs, 1H; OH), 3.92 (dd, J=11.2,
7.5 Hz, 1H; 10-Ha), 4.06 (dd, J=11.2, 3.2 Hz, 1H; 10-Hb), 4.11 (mc, J=
10.1, 4.9 Hz, 2H; 1’’-H2), 4.31 (mc, 1H; 1-H), 4.59 (dd, J=8.8, 1.8 Hz,
1H; 2-Ha), 4.80 (t, J=10.0 Hz, 1H; 2-Hb), 5.05 (d, J=7.0 Hz, 1H; 1’’’-H),
5.16, 5.49 (2ꢄbrs, 2ꢄ1H; 2ꢄOH), 6.92 (dd, J=8.9, 2.4 Hz, 1H; 6’-H),
7.09 (s, 1H; 3’-H), 7.18 (d, J=2.2 Hz, 1H; 4’-H), 7.39 (d, J=8.9 Hz, 1H;
7’-H), 7.44 (ddd, J=8.1, 6.8, 1.0 Hz, 1H; 7-H), 7.58 (ddd, J=8.1, 6.8,
1.1 Hz, 1H; 8-H), 7.93 (d, J=8.4 Hz, 1H; 9-H), 8.17 (brs, 1H; 4-H), 8.33
(d, J=8.5 Hz, 1H; 6-H), 11.70 ppm (s, 1H; NH); 13C NMR (125 MHz,
[D6]DMSO): d=41.2 (C-1), 45.0 (NMe2), 47.4 (C-10), 54.8 (C-2), 57.3 (C-
2’’), 65.5 (C-1’’), 71.6 (C-4’’’), 73.0 (C-2’’’), 75.2 (C-5’’’), 75.9 (C-3’’’), 100.9
(C-1’’’), 101.5 (C-4), 103.2 (C-4’), 105.1 (C-3’), 113.0 (C-7’), 115.7 (C-6’),
118.0 (C-9b), 122.6 (C-9), 122.8 (C-5a), 123.1 (C-6), 123.7 (C-7), 127.3 (C-
3a’), 127.4 (C-8), 129.4 (C-9a), 130.7 (C-2’), 131.6 (C-7a’), 141.8 (C-3a),
152.6 (C-5’), 153.2 (C-5), 160.0 (C(O)N), 170.1 ppm (C-6’’’); IR (KBr):
n˜ =3386, 1615, 1516, 1462, 1399, 1290, 1233, 1179, 1063, 759 cmꢀ1; UV
(MeOH): lmax (lg e)=209.0 (4.6106), 239.0 (4.4127), 299.5 (4.3174),
329.5 nm (4.3425); HRMS (ESI): m/z: calcd for C32H34ClN3O9: 638.1911;
found: 638.1915 [MꢀH]ꢀ.
Compound 8e: According to general procedure B, a stirred mixture of
the trichloroacetimidate 6e (312 mg, 562 mmol, 1.25 equiv), phenol (ꢀ)-
(1S)-5 (150 mg, 449 mmol, 1.0 equiv), and molecular sieves 4 ꢅ (820 mg)
in CH2Cl2 (20 mL) was treated with BF3·OEt2 (28.0 mL, 225 mmol, in
2.4 mL CH2Cl2, 0.5 equiv) at ꢀ188C and stirring was continued at ꢀ108C
for 3 h. Additional BF3·OEt2 (171 mL, 1.35 mmol, in 2.0 mL CH2Cl2,
3.0 equiv) was added and the mixture kept at RT for 5 h. Evaporation
and subsequent reaction with DMAI·HCl (7) (192 mg, 674 mmol,
1.5 equiv) and EDC·HCl (259 mg, 1.35 mmol, 3.0 equiv) in DMF (20 mL)
for 15 h yielded the peracetylated prodrug 8e (185 mg, 216 mmol, 48%)
as slightly
a ocher solid after purification. Rf =0.38 (CH2Cl2/MeOH
10:1); a=ꢀ4.78 (c=0.22 in CHCl3); 1H NMR (600 MHz, CDCl3): d=
1.85, 2.04, 2.05 (3ꢄs, 3ꢄ3H; 3ꢄC(O)CH3), 2.39 (s, 6H; NMe2), 2.81 (t,
J=5.6 Hz, 2H; 2’’-H2), 3.40 (t, J=11.0 Hz, 1H; 10-Ha), 3.95 (dd, J=11.4,
3.1 Hz, 1H; 10-Hb), 4.13 (t, J=5.7 Hz, 2H; 1’’-H2), 4.15–4.19 (m, 1H; 1-
H), 4.35 (d, J=8.8 Hz, 1H; 5’’’-H), 4.66 (t, J=9.6 Hz, 1H; 2-Ha), 4.78
(dd, J=10.6, 1.7 Hz, 1H; 2-Hb), 5.10 (d, J=2.1 Hz, 2H; OCH2Ph), 5.37
(t, J=9.2 Hz, 1H; 3’’’-H), 5.43 (t, J=9.4 Hz, 1H; 4’’’-H), 5.43 (d, J=
7.3 Hz, 1H; 1’’’-H), 5.51 (dd, J=8.9, 7.7 Hz, 1H; 2’’’-H), 7.01 (d, J=
1.5 Hz, 1H; 3’-H), 7.02 (dd, J=9.0, 2.3 Hz, 1H; 6’-H), 7.13 (d, J=2.0 Hz,
1H; 4’-H), 7.23–7.27 (m, 3H; 3ꢄPh-H), 7.27–7.32 (m, 3H; 7’-H, 2ꢄPh-
H), 7.40 (t, J=7.6 Hz, 1H; 7-H), 7.54 (t, J=7.5 Hz, 1H; 8-H), 7.69 (d,
J=8.3 Hz, 1H; 9-H), 8.13 (d, J=8.5 Hz, 1H; 6-H), 8.35 (brs, 1H; 4-H),
9.36 ppm (brs, 1H; NH); 13C NMR (125 MHz, CDCl3): d=20.4, 20.7 (2
signals; 3ꢄC(O)CH3), 43.2 (C-1), 45.9 (NMe2), 46.1 (C-10), 54.9 (C-2),
58.4 (C-2’’), 66.5 (C-1’’), 67.7 (OCH2Ph), 69.2 (C-4’’’), 70.8 (C-2’’’), 71.9
(C-3’’’), 72.7 (C-5’’’), 99.1 (C-1’’’), 102.2 (C-4), 103.5 (C-4’), 105.9 (C-3’),
112.6 (C-7’), 117.3 (C-6’), 118.7 (C-9b), 122.0 (C-9), 123.3 (C-6), 123.5 (C-
5a), 124.6 (C-7), 127.9 (C-8), 128.2 (C-3a’), 128.3, 128.4 (2 signals; Ph-Co,
Ph-Cm, Ph-Cp), 129.6 (C-9a), 130.4 (C-2’), 131.2 (C-7a’), 134.7 (Ph-Ci),
141.7 (C-3a), 153.4 (C-5), 153.7 (C-5’), 160.3 (C(O)N), 166.1 (C-6’’’),
169.2, 169.3, 169.8 ppm (3ꢄC(O)CH3); IR (KBr): n˜ =1759, 1626, 1516,
Serum stability studies: Preincubated human serum (495 mL, 378C) was
treated with the appropriate prodrug (10a–e, 5.00 mL, 240 nmol, 48.0 mm
stock solution in DMSO) to give a 480 mm reaction mixture at a final
DMSO concentration of 1%. The solution was mixed for 5 s by using a
vortex mixer and a sample of 40 mL (19.2 nmol prodrug) was taken im-
mediately. Protein precipitation was initiated by adding an acetonitrile
working solution of the standard 11 (100 mL, 4.80 nmol, 48 mm solution in
CH3CN, for structure see the Supporting Information) and mixing for
15 s. After centrifugation at 14000 Uminꢀ1, 48C, 5 min) the supernatant
was analyzed by HPLC-MS (see General Information). The remaining
reaction mixture was incubated for 24 h at 378C. Further samples were
taken at t=0.5, 1.0, 1.5, 2.0, 3.0, 4.0, 5.0, 6.0, 24.0 h and workup as well as
HPLC-MS were done as described. Concerning interpretation, for all in-
vestigated times the area under the curve (AUC) of the corresponding
ion currents of substrate and metabolite(s) was determined. The integrals
1461, 1396, 1216, 1039, 755 cmꢀ1
; UV (MeOH): lmax (lg e)=299.0
(3.8214), 334.0 nm (3.796); HRMS (ESI): m/z: calcd for C45H46ClN3O12
:
856.2843; found: 856.2866 [M+H]+.
Compound 9:
A mixture of the benzyl-protected acid 8e (167 mg,
195 mmol, 1.0 equiv) and palladium on charcoal (10%, 125 mg, 117 mmol,
0.6 equiv of Pd) in MeOH/EtOAc (66.0 mL, 1:10) was stirred under a H2
atmosphere (1 atm) at RT for 6 h. Filtration over Celite, washing with
MeOH, and evaporation of the solvent yielded the crude product, which
was then purified by column chromatography on silica gel (CH2Cl2/
MeOH 3:1). After membrane filtration, the debenzylated compound 9
1928
www.chemeurj.org
ꢂ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2011, 17, 1922 – 1929

Synthesis and Biological Evaluation of Prodrugs Based on Duocarmycin
FULL PAPER
were normalized by referencing to the AUC of the standard 11. The rela-
tive AUCs were plotted against time and the rate constant determined
by using OriginPro 8G and Dynafit 3.[20]
[6] a) L. F. Tietze, Nachr. Chem. Tech. Lab. 1988, 36, 728–737; b) L. F.
Tietze, M. Neumann, T. Mçllers, R. Fischer, K.-H. Glꢀsenkamp,
M. F. Rajewsky, E. Jꢁhde, Cancer Res. 1989, 49, 4179–4184; c) E.
Jꢁhde, K.-H. Glꢀsenkamp, I. Klꢀnder, D. F. Hꢀlser, L. F. Tietze,
M. F. Rajewsky, Cancer Res. 1989, 49, 2965–2972; d) L. F. Tietze, M.
Beller, R. Fischer, M. Lçgers, E. Jꢁhde, K. Glꢀsenkamp, M. F. Ra-
jewsky, Angew. Chem. 1990, 102, 812–813; Angew. Chem. Int. Ed.
Engl. 1990, 29, 782–783.
[7] a) L. F. Tietze, B. Krewer, Anti-Cancer Agents Med. Chem. 2009, 9,
304–325; b) H. J. Schuster, B. Krewer, J. M. von Hof, K. Schmuck, I.
Schuberth, F. Alves, L. F. Tietze, Org. Biomol. Chem. 2010, 8, 1833–
1842; c) L. F. Tietze, H. J. Schuster, B. Krewer, I. Schuberth, J. Med.
Chem. 2009, 52, 537–543; d) L. F. Tietze, H. J. Schuster, K.
Schmuck, I. Schuberth, F. Alves, Bioorg. Med. Chem. 2008, 16,
6312–6318; e) L. F. Tietze, F. Major, I. Schuberth, Angew. Chem.
2006, 118, 6724–6727; Angew. Chem. Int. Ed. 2006, 45, 6574–6577;
f) L. F. Tietze, B. Krewer, J. M. von Hof, H. Frauendorf, I. Schu-
berth, Toxins 2009, 1, 134–150.
In vitro cytotoxicity assays: Adherent cells of line A549 (human bron-
chial carcinoma cells) were sown in triplicate in 6 multiwell plates at con-
centrations of 102, 103, and 104 cells per cavity. Culture medium was
sucked off after 24 h and cells were washed in the incubation medium Ul-
traCulture (UC, serum-free special medium, Lonza). Incubation with
compounds 10a–e was then performed in UltraCulture medium at 6–8
various concentrations for 24 h. All substances were used as freshly pre-
pared solutions in DMSO (Merck, Darmstadt, Germany) diluted with in-
cubation medium to a final DMSO concentration 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 done at 378C and 7.5%
CO2 in air for 9–10 days. The medium was removed and the clones were
dried, stained with Lçfflerꢆs methylene blue (Merck, Darmstadt, Germa-
ny) and counted macroscopically. The IC50 values are based on the rela-
tive clone forming rate, which was determined according to the following
formula: relative clone forming rate [%]=100ꢄ(number of clones count-
ed after exposure)/(number of clones counted in the control). Liberation
of the drugs from their glycosidic prodrugs was achieved by addition of
0.4 UmLꢀ1 a-d-mannosidase (EC 3.2.1.24, 1 U=1 mmolminꢀ1, Sigma),
4.0 UmLꢀ1 b-d-galactosidase (EC 3.2.1.23, 1 U=1 mmolminꢀ1, Sigma),
[8] M. Ichimura, T. Ogawa, K. Takahashi, E. Kobayashi, I. Kawamoto,
T. Yasuzawa, I. Takahashi, H. Nakano, J. Antibiot. 1990, 43, 1037–
1038.
[9] L. H. Hurley, C.-S. Lee, J. P. McGovren, M. A. Warpehoski, M. A.
Mitchell, R. C. Kelly, P. A. Aristoff, Biochemistry 1988, 27, 3886–
3892.
4 UmLꢀ1 b-d-glucuronidase (EC 3.2.1.31, 1 U=0.0523 nmolminꢀ1
,
[10] a) D. L. Boger, I. Takayoshi, R. J. Wysocki Jr. , S. A. Munk, J. Am.
Chem. Soc. 1989, 111, 6461–6463; b) D. L. Boger, I. Takayoshi, P. A.
Kitos, O. Suntornwat, J. Org. Chem. 1990, 55, 5823–5832.
[11] L. F. Tietze, T. Herzig, A. Fecher, F. Haunert, I. Schuberth, Chem-
BioChem 2001, 2, 758–765.
[12] J. B. J. Milbank, M. Tercel, G. Atwell, W. R. Wilson, A. Hogg, W. A.
Denny, J. Med. Chem. 1999, 42, 649–658.
[13] L. F. Tietze, T. Herzig, T. Feuerstein, I. Schuberth, Eur. J. Org.
Chem. 2002, 1634–1645.
Sigma) to the cells during incubation with the substances.
Acknowledgements
The work was supported by the Deutsche Forschungsgemeinschaft and
the Fonds der Chemischen Industrie. K.S. thanks the Fonds der Chemi-
schen Industrie and H.J.S. the Friedrich-Ebert-Stiftung for Ph.D. scholar-
ships. We are also grateful to the Klinikum Gçttingen for provision of
human serum.
[14] L. F. Tietze, J. M. von Hof, M. Mꢀller, B. Krewer, I. Schuberth,
Angew. Chem. 2010, 122, 7494–7497; Angew. Chem. Int. Ed. 2010,
49, 7336–7339.
[15] L. F. Tietze, J. M. v. Hof, B. Krewer, M. Mꢀller, F. Major, H. J.
Schuster, I. Schuberth, ChemMedChem 2008, 3, 1946–1955.
[16] a) R. R. Schmidt, Angew. Chem. 1986, 98, 213–236; Angew. Chem.
Int. Ed. Engl. 1986, 25, 212–235; b) W. Dullenkopf, J. C. Castro-Pal-
omino, L. Manzoni, R. R. Schmidt, Carbohydr. Res. 1996, 296, 135–
147.
[17] M. Tomoi, Y. Kato, H. Kakiuchi, Makromol. Chem. 1984, 185,
2117–2124.
[18] K. Gheysen, C. Mihai, K. Conrath, J. C. Martins, Chem. Eur. J. 2008,
14, 8869–8878.
[1] a) K. D. Bagshawe, Br. J. Cancer 1987, 56, 531–532; b) L. F. Tietze,
K.-H. Glꢀsenkamp, unpublished work; c) K. D. Bagshawe, Curr.
Drug Targets 2009, 10, 152–157; d) K. D. Bagshawe, Expert Rev. An-
ticancer Ther. 2006, 6, 1421–1431; e) L. F. Tietze, T. Feuerstein,
Curr. Pharm. Des. 2003, 9, 2155–2175.
[2] a) K. Bosslet, J. Czech, D. Hoffmann, Tumor Target. 1995, 1, 45–50;
b) K. Bosslet, R. Straub, M. Blumrich, J. Czech, M. Gerken, B.
Sperker, H. K. Kroemer, J.-P. Gesson, M. Koch, C. Monneret,
Cancer Res. 1998, 58, 1195–1201.
[3] a) T. A. Connors, M. E. Whisson, Nature 1966, 210, 866–867; b) A.
Sinhababu, D. Thakker, Adv. Drug Delivery Rev. 1996, 19, 241–273.
[4] L. F. Tietze, R. Seele, B. Leiting, T. Krach, Carbohydr. Res. 1988,
180, 253–262.
[19] a) K. Bock, C. Pedersen, J. Chem. Soc. Perkin Trans. 2 1974, 293–
297; b) F. Michon, J. R. Brisson, R. Roy, F. E. Ashton, H. J. Jennings,
Biochemistry 1985, 24, 5592–5598.
[20] P. Kuzmic, Anal. Biochem. 1996, 237, 260–273.
[5] a) O. Warburg, On the Metabolism of Tumors, Constable, London,
1930; b) R. K. Jain, Spektrum der Wissenschaft 1994, 9, 48–55; c) P.
Vaupel, F. Kallinowski, P. Okunieff, Cancer Res. 1989, 49, 6449–
6465.
Received: September 29, 2010
Published online: January 7, 2011
Chem. Eur. J. 2011, 17, 1922 – 1929
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