Synthesis of the first spacer containing prodrug of a duocarmycin analogue and determination of its biological activity

Article abstract of DOI:10.1039/b925070k

The synthesis of the first spacer containing, duocarmycin analogue prodrug 11 was realised, its biological properties evaluated and compared to its counterpart prodrug 2 without a spacer unit. The synthesis comprises the manufacture of the new acetylated

Full text of DOI:10.1039/b925070k

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Synthesis of the first spacer containing prodrug of a duocarmycin analogue
and determination of its biological activity†‡
Heiko J. Schuster,a Birgit Krewer,a J. Marian von Hof,a Kianga Schmuck,a Ingrid Schuberth,a Frauke Alvesb
and Lutz F. Tietze*a
Received 30th November 2009, Accepted 26th January 2010
First published as an Advance Article on the web 17th February 2010
DOI: 10.1039/b925070k
The synthesis of the first spacer containing, duocarmycin analogue prodrug 11 was realised, its
biological properties evaluated and compared to its counterpart prodrug 2 without a spacer unit. The
synthesis comprises the manufacture of the new acetylated derivatives 19 and 20b of two double spacer
systems, their activation and coupling to the pharmacophoric seco-drug (+)-3. Unprecedented
biological results were found as the new prodrug 11 showed a fairly low QIC50 value of 20, but on the
other hand a high stability and very low DNA alkylation efficiency. These findings indicate a changed
cytostatic mode of action induced by the self-immolative spacer moiety which was employed.
Introduction
One of the major goals in modern chemotherapy of malignant
tumours is the development of more selective anticancer drugs to
circumvent severe side effects caused by an insufficient differenti-
ation between normal and tumour cells. A promising concept to
overcome this problem is the antibody-directed enzyme prodrug
therapy, which was first described by Bagshawe and is commonly
referred to as ADEPT.1,2 Selectivity in this approach is achieved by
the use of conjugates of an enzyme which is capable of activating
the prodrug and a monoclonal antibody which selectively binds
to tumour associated antigens. In that way it is possible to restrict
the release of the active drug predominantly to the tumour tissue.
The highly potent natural antibiotic duocarmycin SA (1, Fig. 1)
with an IC50 value of 10 pM (L1210)3 formed the basis of
glycosidic prodrugs which were introduced by our group for a
use in ADEPT.4 Seco-analogues of 1 such as 3 were successfully
detoxified by transforming their phenolic hydroxyl group into
glycosides as e.g. in the b-galactoside 2 (Fig. 1).5 By enzymatic
cleavage of the glycosidic bond in 2 seco-structure 3 is released,
which under physiological conditions subsequently undergoes a
fast Winstein-cyclisation under loss of HCl. The active drug which
is formed in such a cyclisation reaction contains a DNA alkylating
spiro-cyclopropylcyclohexadienone moiety as in 1.
A necessary requirement for a successful application is a
sufficient difference in cytotoxicity of the prodrugs and the
corresponding drugs which we have defined as QIC50 (QIC50
IC50 of prodrug/IC50 of prodrug in the presence of the cleaving
enzyme). Minimum requirements for suitable prodrugs are a QIC50
=
Fig. 1 Duocarmycin SA (1), prodrugs 2 and 4 for ADEPT and/or PMT
as well as seco-drug 3.
aInstitut for Organic and Biomolecular Chemistry, Georg-August-University
Go¨ttingen, Tammannstrasse 2, 37077, Go¨ttingen, Germany. E-mail: ltietze@
gwdg.de; Fax: +49(0)551-399476
value of > 1000 combined with an IC50 value of the liberated drugs
of < 10 nm.6
bCentre of Internal Medicine, Department of Haematology and Oncology of
the Georg-August-University of Go¨ttingen, Robert-Koch-Straße 40, 37075,
Go¨ttingen, Germany
For prodrug 2, a QIC50 value of 4800 and an IC50 value
of the corresponding drug 3 of 750 pm was achieved. This
is to date the highest QIC50 value reported for a prodrug
suitable for ADEPT. However, we recently described a related
prodrug that revealed a detoxification which was similarly effective
(QIC50 = 3500), but whose corresponding seco-drug showed a
† Dedicated to Professor Saverio Florio on the occasion of his 70th
birthday
‡ Electronic supplementary information (ESI) available: Synthesis of
compound 14 and 1H and 13C NMR spectra of all compounds. See DOI:
10.1039/b925070k
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30–40 times increased cytotoxicity (IC50 = 16–26 pm) as compared
to 3.7
cytotoxicity of such prodrugs as compared to the corresponding
drugs. These issues have so far not been addressed and since
the galactosidic prodrug 2 has thoroughly been investigated we
aimed at the synthesis of the spacer analogues 11 and 12 for
comparison (Fig. 3). As a spacer we chose the one in prodrug 6
and a combination of 6 and 7 (Fig. 2) with a b-D-galactose moiety
as blocking group and a nitro group at the aromatic system in
ortho-position to the glycosidic bond. The nitro group decreases
the pKa of the phenol formed after the enzymatic cleavage of the
phenyl glycoside and hence improves the rate of self-immolation.
Furthermore, good results for the stability of this spacer unit were
reported.13 However, Schmidt et al. pointed out that an ortho-nitro
group in the spacer might facilitate decomposition after enzymatic
sugar cleavage.12a
Further, we have also reported the synthesis and biological
evaluation of the glucuronic acid derivative 4 that is not only
applicable in ADEPT but also in prodrug monotherapy (PMT),8
since the activating enzyme b-D-glucuronidase occurs in elevated
concentrations in the extra-cellular space of solid tumours.
An undesired consequence of the direct linkage of a carbohy-
drate moiety to sterically demanding pharmacophores could be
a reduced enzyme accessibility of the carbohydrates resulting in
a lower rate of enzymatic hydrolysis. Thus, in some cases, e.g.
using b-D-glucosides, the cytoxicity of the prodrug in the presence
of the enzyme b-D-glucosidase was reduced which indicates an
incomplete cleavage of the glycosidic bond.9 One possibility to
overcome this problem is the introduction of a spacer between
the seco-drug or the active drug and the detoxifying unit. The
work in the field of tumour-selective prodrugs which contain
spacer units was pioneered by Scheeren et al. and Monneret
et al. who introduced and evaluated various self-immolative spacer
systems 5–10 in combination with analogues of the cytostatic
antitumour drug paclitaxel (Fig. 2).10 Despite recent advances in
linker-equipped cytostatics with protease cleavable peptide-linkers
by Senter et al.,11 we focused on self-immolative spacers.
Important general requirements for prodrugs which contain
spacer units are a swift enzymatic cleavage of the blocking group
and a successive complete self-immolation to release the drug.
Although good results for these parameters were achieved, the
resulting QIC50 of prodrugs 5–10 range only from 1 to 722,12 which
might not be sufficient for a selective treatment of cancer as we
pointed out.6
Results and discussion
Synthesis
For the synthesis of 11 and 12 acetobromogalactose was coupled
with commercially available 4-hydroxy-3-nitro-benzaldehyde (13,
Scheme 1). Best results were obtained using a phase-transfer
Michael glycosidation14 leading to 14 in 80% yield after crystalli-
sation. The reduction of 14 using sodium borohydride to give the
alcohol 15 according to the procedure of Farquhar et al.15 required
some optimisation as the intermediately formed alcoholate caused
a partial migration of the acetyl groups. However, by addition of
silica gel and acidic ion exchange resin a reproducible yield of
98% could be obtained. The transformation of the benzyl alcohol
15 into the reactive but at room temperature stable carbonate 16
was achieved using para-nitrochloroformate (p-NCF). Compound
16 was then coupled with the mono protected diamine 1810e,16 in
the presence of catalytic amounts of 4-dimethylamino pyridine
(DMAP) to afford the carbamate 20a in 88% yield (based on 15:
Essential issues in this approach are 1) the sufficient stability of
the prodrug containing spacer units, 2) the improvement of the rate
of the enzymatic cleavage, 3) an increased cytotoxity of the prodrug
in the presence of the enzyme and 4) the dramatic reduction of the
Fig. 2 Paclitaxel prodrugs with self-immolative spacer units by Monneret et al. and Scheeren et al. (Drug = Paclitaxel based drugs).
Fig. 3 Prodrugs 11 and 12 as Duocarmycin SA analogues with the glycosidic spacers.
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Scheme 1 Formation of the activated carbamates with the synthesis of 21–24. Reagents conditions: a) a-D-Acetobromogalactose, BnEt3NBr, NaOH,
H2O/CH3Cl, reflux, 3 h, 80%; b) NaBH4, IR120 H+, silica gel, CH3Cl/i-PrOH (4 : 1), 0 ◦C, 1.5 h, 98%; c) para-nitrophenylchloroformate (p-NCF) , pyridine,
CH2Cl2, 0 ◦C, 2 h, (91% when isolated); d) N-methyl-2-aminoethanol (17), DMAP, CH2Cl2, 0 → 25 ◦C, 2 h, 72%; e) N-Boc-N,N-dimethylaminoethane
(18), DMAP, CH2Cl2, 0 → 25 ◦C, 4.5 h, 88%; f) 3M HCl in EtOAc, 0 → 25 ◦C, 30 min, 98%; g) Phosgene, Et3N, CH2Cl2, 0 ◦C, 1 h, 21: 97%, 22: 85%;
p-NCF, pyridine, CH2Cl2, 0 →25 ◦C, 30 min, 92%; h) p-NCF, pyridine, CH2Cl2, 0 → 25 ◦C, 23: 6 h, 98%, 24: 30 min, 92%.
81% yield) (Scheme 1). Alternatively, a one pot procedure starting
from 15 gave 20a in a slightly increased yield of 88% and cleavage of
the tert-butyloxycarbonyl moiety in 20a with anhydrous 3 M HCl
in ethyl acetate led to the hydrochloride 20b in almost quantitative
yield.12b Similarly, coupling of 16 with the amino alcohol 17 gave
19 in 79% yield (based on 15: 75%). The one pot procedure
starting from 15 led to 19 in a slightly decreased yield of 72%
yield compared to the two step transformation.
For the formation of the prodrugs 11 and 12 containing either
a carbamate or a carbonate moiety, compounds 20b and 19
were transformed into the activated chlorides 21 and 22 and the
activated p-nitrophenylcarbamate 23 and p-nitrophenylcarbonate
24. In the first case, 20b and 19 were treated with phosgene in
the presence of triethylamine to give 21 and 22 in 97% and 85%
yield, respectively. In the second case, reaction of 20b and 19 with
para-nitrochloroformate gave 23 in 98% yield and 24 in 92% yield.
The structure determination of all compounds by NMR spec-
troscopy was straight forward; however, it should be mentioned
that especially the NMR spectra of 21 and 22 indicate the existence
of four conformational isomers even at elevated temperatures.
The coupling of compounds 21–24 with the enantiopure
anti-methyl-seco-CBI-DMAI (+)-(1S,10R)-3, whose enantiopure
synthesis has recently been described by us,17 were performed in
DMF under DMAP catalysis (Scheme 2). The reaction of the
carbamoyl chloride 21 with 3 led to 25 in 38% yield; contrary,
using the p-nitrocarbamate 23, the desired compound could not
be obtained despite several variations of the reaction conditions.
On the other hand, using the p-nitrocarbonate 24, product 26
was obtained in 58% yield, whereas in this case the corresponding
chloride 22 led to 26 in only 27% yield. The final deacetylation
step of 25 using Zemple´n conditions led readily to the desired
prodrug 11 in 79% yield after purification. Unfortunately, we
were not able to perform the deacetylation of 26 using the same
conditions as well as other methods, e.g. 1% HCl in MeOH.18
In all attempts either a decomposition or no conversion was
observed.
Surprisingly, besides a hydrolysis of the carbonate moiety in
the case of the decomposition, a cleavage of the normally stable
amide bond between the dimethylaminoethoxyindole carboxylic
acid moiety and the anti-methyl-seco-CBI-unit was observed.
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Scheme 2 Coupling of 21–24 with the seco-drug 25. Reagents and conditions: a) DMAP, DMF, 0 → 25 ◦C, 4 h, 27: 38%, 28: 58%; b) NaOMe, MeOH,
0 → 25 ◦C, 3 h, 11: 79%.
Biological evaluation
On the other hand the high cytotoxicity of prodrug 11 could also
be explained by a direct alkylation of the cellular DNA by the
prodrug. To exclude the last possibility we performed additional
experiments determining the DNA alkylationefficiency ofprodrug
11 in comparison to the corresponding seco-drug 3 and also of
prodrug 2.
Stability. The stability of prodrug 11 in UltraCultureTM cell
culture medium was determined using HPLC-MS. Prodrug 11
was stable over 24 h at pH 7.4 and 37 ◦C and no cleavage of any
of the carbamate bonds and thus no generation of the cytotoxic
drug was observed.
DNA alkylation studies. For the DNA alkylation studies
of 11, 2 and 3, synthetic double-stranded oligonucleotides in
combination with high-resolution electrospray mass spectrometry
was used, a method which was among others recently established in
our group in order to investigate these compounds interaction with
DNA.19 For this purpose, the compounds were incubated together
with DNA for 24 h in a 1 : 1 and 3 : 1 molecular ratio in water as
solvent. The subsequent electrospray ionization Fourier transform
ion cyclotron resonance mass spectrometry (ESI-FTICR-MS)
measurements were carried out directly without chromatographic
purification of the reaction mixture and enrichment of the alky-
lated DNA.20 Whereas seco-drug 3 showed a very high alkylation
efficiency towards N-3 of adenine in one of the two double strands
of the oligonucleotide already at a ratio of 1 : 1, only a small
alkylation tendency could be observed for prodrug 2, and no
alkylation at all was found for prodrug 11. Furthermore, even at a
higher prodrug to DNA ratio of 3 : 1 prodrug 11 did not alkylate
the DNA. Since prodrug 11 is more toxic than prodrug 2 but at
the same time shows a decreased reactivity against DNA, a direct
alkylation of DNA by 11 can be excluded as reason for its higher
cytotoxicity as compared to 2. Besides the already mentioned
possibility of an intramolecular activation of prodrug 11, a cell
membrane disintegration effect or a general toxicity of 11 caused
by its structure are also possible.
Cytotoxicity. The cytotoxicity of prodrug 11 in the presence
and in the absence of b-D-galactosidase was determined using a
humantumourcolonyformingability(HTCFA)-assaythatreflects
the proliferation capacity of single cells and human bronchial
carcinoma cells of line A549.
For prodrug 11 an IC50 value of 29 nM was determined in
the absence of the enzyme, whereas in the presence of b-D-
galactosidase a slight increase of the cytotoxicity of 11 with an
IC50 value of 1.3 nM was found. From these data, a QIC50 value
of approximately 20 results. Since the IC50 of the prodrug in the
presence of the enzyme is almost identical to the IC50 of the seco-
drug 3 (0.75 nM), an efficient cleavage of the glycosidic bond
and a self-immolation of the spacer moiety can be assumed.
Furthermore, an inhibition of the enzyme by the formed drug
and the products from the spacer in a suicide mechanism can be
excluded.
In comparison to prodrug 2 the QIC50 of 11 is surprisingly
low. As 11 was shown to be perfectly stable over 24 h in cell
culture medium and no free seco-drug and hence no drug was
generated, we assume that the spacer moiety somehow facilitates
the cellular uptake of the prodrug. The uptake is then followed
by an intracellular cleavage of the glycosidic bond going along
with an alkylation of cellular DNA by the drug which is released.
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NH4Cl solution (10 mL) and transferred to a separatory funnel
with CH2Cl2–pentane (1 : 2, 15 mL). After phase separation, the
aqueous layer was extracted again with CH2Cl2–pentane (1 : 2,
15 mL), the combined organic layers were washed with ice-water
(10 mL) and brine (10 mL), filtered over cotton wool and the
solvents were removed. Purification by column chromatography
on silica (toluene–MeOH = 6 : 1) gave the benzyl alcohol 15 as
colourless solid (327 mg, 0.66 mmol, 97%). Rf 0.37 (toluene–
MeOH = 6 : 1), 0.15 (EtOAc–pentane = 1 : 1); [a]2D3 = +54.8 (c =
1.0, CHCl3); lmax (CH3CN)/nm = 213.5, 257.5 and 314.5 (lg e
Conclusion
We have prepared the novel prodrug 11 containing a spacer unit
with a galactoside moiety as blocking group, which is based on
the seco-form of an analogue of the highly cytotoxic antibiotic
duocarmycin SA. It was shown that the introduction of the
self-immolative spacer has no negative influence on the prodrug
stability or its enzyme accessibility. However, the compound has
a quite high cytotoxicity which is reflected by its low QIC50
value of only 20, thus, in the low range of the earlier reported
ones of this type. Investigations of the alkylation efficiency of
11 using oligonucleotides attempting to explain these findings
show a very low tendency for a direct DNA alkylation. The
comparable high cytoxicity of 11 is therefore considered to either
result from a intracellular activation after penetration of the
compound through the cell membrane, a disintegration of the
cell membrane or a general toxicity of 11 due to a changed
biological mechanism. The results clearly indicate that in the future
development of spacer units and prodrugs containing spacers, a
careful investigation of the influence of the spacer unit on the
bioactivity will be necessary.
1.2078, 0.4980 and 0.2546); nmax/cm-1 3584, 3492, 2888, 1745,
˜
1624, 1580, 1533, 1499, 1437, 1371, 1240, 1129, 1073, 1045, 952,
913, 837, 713, 595; 1H-NMR (599.7 MHz, CDCl3): d = 1.98, 2.04,
2.09, 2.15 (4 ¥ s, zus. 12 H, 4 ¥ COCH3), 2.22 (sbr, 1 H, OH), 4.04
(dt, J = 7.0, 6.2, 2.0 Hz, 1 H, H-5), 4.13 (dd, J = 11.5, 6.2 Hz,
1 H, H-6a), 4.22 (dd, J = 11.5, 7.0 Hz, 1 H, H-6b), 4.69 (s, 2 H,
ArCH2OH), 5.02 (d, J = 7.9 Hz, 1 H, H-1), 5.07 (dd, J = 10.5,
3.1 Hz, 1 H, H-3), 5.43 (dd, J = 3.3, 1.0 Hz, 1 H, H-4), 5.49 (dd,
J = 10.5, 8.0 Hz, 1 H, H-2), 7.31(d, J = 8.6 Hz, 1 H, H-12), 7.48
(dd, J = 8.6, 2.2 Hz, 1 H, H-11), 7.77 (d, J = 2.2 Hz, 1 H, H-9);
13C-NMR (150.7 MHz, CDCl3): d = 20.5, 20.6 (4 ¥ COCH3), 61.3
(C-6), 63.3 (ArCH2OH), 66.7 (C-4), 67.8 (C-2), 70.5 (C-3), 71.3
(C-5), 100.8 (C-1), 119.9 (C-12), 123.2 (C-9), 131.7 (C-11), 137.2
(C-10), 141.2 (C-7), 148.4 (C-8), 169.5, 170.1, 170.2, 170.3 (4 ¥
COCH3); C21H25NO13 (499.42).
Experimental
General
All reactions were performed in flame-dried glassware under an
atmosphere of argon. Solvents were dried and purified according
to the method defined by Perrin and Armarego. Commercial
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
chromatography (CC) was carried out on silica gel 60 from
Merck with particle size 0.063–0.200 mm for normal pressure
and 0.020–0.063 mm for flash chromatography. IR spectra were
determined on a Bruker Vektor 22 as KBr-pellets, UV-vis spectra
on a Perkin-Elmer Lambda 2, and mass spectra on a Bruker Apex
IV Fourier transform ion cyclotron resonance mass spectrometer
for ESI-HRMS. 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 stated
otherwise) in deuterated solvents as indicated using the solvent
peak as internal standard. The spectra of compounds 14–16, 19–
24 and the prodrug 11 and the new procedure for 14 can be found
in the ESI.‡
2,3,4,6-Tetra-O-acetyl-[2-nitro-4-(4-nitrophenoxycarbonyloxy-
methyl)phenyl]-b-D-galactopyranoside (16). To a solution of the
benzyl alcohol 15 (400 mg, 0.80 mmol, 1.0 equiv.) and pyridine
(126 mg, 129 ml, 1.59 mmol, 2.0 equiv.) in CH2Cl2 (20.0 ml) at
0 ◦C p-nitrophenylchloroformate (320 mg, 1.59 mmol, 2.0 equiv.)
◦
was added and the mixture was stirred for 2 h at 0 C. Silica gel
(500 mg) was added directly and the solvents were removed. From
the residue the target molecule 16 (484 mg, 0.73 mmol, 91%) was
obtained by column chromatography on silica (gradient: pentane-
EtOAc = 2 : 1 → 1 : 2) as colourless foam. Rf 0.45 (EtOAc–
pentane = 1 : 1) ; 1H-NMR (300.1 MHz, CDCl3): d = 2.03, 2.08,
2.14, 2.20 (4 ¥ s, zus. 12 H, 4 ¥ COCH3), 4.08–4.21 (m, 2 H, H-5,
H-6b), 4.27 (dd, J = 11.0, 6.8 Hz, 1 H, H-6a), 5.13 (d, J = 7.3 Hz, 1
H, H-1), 5.13 (dd, J = 10.3, 2.4 Hz, 1 H, H-3), 5.30 (s, 2 H, H-10),
5.49 (dd, J = 3.4, 0.7 Hz, 1 H, H-4), 5.56 (dd, J = 10.5, 7.4 Hz, 1 H,
H-2), 7.39 (d, J = 9.3 Hz, 2 H, H-16, H-20), 7.42 (d, J = 8.8 Hz,
1 H, H-11), 7.63 (dd, J = 8.7, 2.2 Hz, 1 H, H-12), 7.92 (d, J =
2.2 Hz, 1 H, H-9), 8.29 (d, J = 9.3 Hz, 2 H, H-17, H-19). 13C-NMR
(75.5 MHz, CDCl3); d = 20.48, 20.55, 20.58 (4 ¥ COCH3), 61.23
(C-6), 66.58 (C-4), 67.66 (C-2), 68.76 (C-13), 70.36 (C-3), 71.42
(C-5), 100.5 (C-1), 119.6 (C-8), 121.6 ( C-16, C-20), 125.3 ( C-17
,
For stability measurements by HPLC-MS the used column was
a Phenomenex Synergi Max-RP C12 (150 mm ¥ 2 mm, particle
size 4 mm).
C-19), 125.4 (C-11), 130.1 (C-10), 133.8 (C-9), 141.1 (C-12), 145.4
(C-18), 149.6 (C-7), 152.2 (C-15), 155.2 (C-14), 169.3, 170.0, 170.1,
170.2 (4 ¥ COCH3); m/z (ESI) 703.10223 (M+ + K, C28H28N2O17K
requires 703.10196), 682.2 (17%), 687.1 (100), 703.1, 1351.3
Synthesis of the spacer basic unit
2,3,4,6-Tetra-O-acetyl-[2-nitro-4-(hydroxymethyl)phenyl]-b-D-
galactopyranoside (15). Benzaldehyde 14 (335 mg, 0.67 mmol,
1.0 equiv.) was dissolved in degassed CHCl3 (5.0 mL) and iPrOH
(1.1 mL), silica gel (42–60 mesh, 800 mg) and ion exchanger IR-120
H+-form (~10 mg) added and the mixture cooled to 0 ◦C. Within
30 min freshly powdered NaBH4 (50.1 mg, 1.35 mmol, 2.0 equiv.)
was added portionwise and stirring continued at 0 ◦C for further
1.5 h. The reaction was quenched by addition of ice-cold sat.
Synthesis of the aminoalcohol spacer prodrug
N,N-Methyl-[4-(2,3,4,6-tetra-O-acetyl-b-D-galactopyranosyl]-
3-nitrobenzyl-oxycarbonyl]-2-aminoethanol (19). To a solution of
benzylalcohol 15 (300 mg, 0.60 mmol, 1.0 equiv.) in CH2Cl2
pyridine (71.3 mg, 72.7 mL, 0.90 mmol, 1.5 equiv.) followed by
one portion of p-nitrophenylchloroformate (60.5 mg, 0.30 mmol,
1.5 equiv.) was added at 0 ◦C and stirring continued for 30 min at
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◦
0 ◦C and another 1.5 h at ambient temperature. After cooling to
0 ◦C, liquid N-methyl-2-aminoethanol (17) (63.7 mg, 0.34 mmol,
1.7 equiv.) and DMAP (41.5 mg, 0.34 mmol, 1.7 equiv.) were
added and stirring at ambient temperature was continued for 2 h.
The reaction was stopped through addition of silica gel (1.38 g)
and removal of the solvents under reduced pressure. After column
chromatography on silica (gradient: EtOAc–pentane = 1 : 1 →
2 : 1→ 4 : 1) the aminoalcohol 19 (258 mg, 0.43 mmol, 72%) was
obtained as colourless syrup. Rf 0.20 (EtOAc–pentane = 4 : 1);
[a]2D3 = +45.0 (c = 0.5, CHCl3) ; lmax (CH3CN)/nm = 214.0,
255.0, 309.0 and 429.5 (lg e 1.2243, 0.5020, 0.2498 and 0.1354);
˜
914, 822, 768, 590; H-NMR (599.7 MHz, CDCl3): d = 1.97,
2.03, 2.08, 2.14 (4 ¥ s, 12 H, 4 ¥ COCH3), 2.78, 2.96 (sbr, 3 H,
NCH3), 3.41 (t, J = 5.2 Hz, 2 H, H2-15), 3.65–3.79 (m, 2 H, H2-
16), 4.05 (t, J = 6.5 Hz, 1 H, H-5), 4.12 (dd, J = 11.4, 6.1 Hz,
1 H, H-6a), 4.21 (dd, J = 11.4, 7.0 Hz, 1 H, H-6b), 5.04 (d, J =
8.2 Hz, 1 H, H-1), 5.06 (dd, J = 10.6, 3.2 Hz, 1 H, H-3) 5.07 (sbr,
2 H, H-13), 5.42 (dd, J = 3.3, 0.7 Hz, 1 H, H-4), 5.49 (dd, J =
10.2, 8.2 Hz, 1 H, H-2), 7.30 (d, J = 8.4 Hz, 1 H, H-12), 7.48
(dd, J = 8.6, 2.2 Hz, 1 H, H-11), 7.77 (sbr, 1 H, H-9); 13C-NMR
(125.7 MHz, CDCl3): d = 20.48, 20.55, 20.56, 20.58 (4 ¥ COCH3),
34.47, 35.25, 35.54 (NCH3), 50.78, 51.82 (C-15), 60.39, 60.95 (C-
16), 61.25 (C-6), 65.19, 65.37 (C-13), 66.63 (C-4), 67.72 (C-2), 70.43
(C-3), 71.31 (C-5), 100.5 (C-1), 119.6 (C-12), 124.5 (C-9), 133.0,
133.1 ( C-10, C-11), 141.0 (C-8), 148.8 (C-7), 157.0, (C-14), 169.3,
170.0, 170.1, 170.3 (4 ¥ COCH3); m/z (ESI) 623.17010 (M+ + Na,
C25H32N2O15Na requires 623.16949), 623.2 (100%), 1223.3 (68).
0.60 mmol, 1.5 equiv.) were added at 0 C and the mixture was
stirred for 30 min at 0 ◦C and for 30 min 25 ◦C. Silica gel
(350 mg) was added, solvents were removed and the crude material
was purified by column chromatography (EtOAc–pentane = 2 : 1)
yielding 24 as colourless foam (282 mg, 368 mmol, 92%). Rf 0.63
(EtOAc–pentane = 4 : 1) ; [a]2D3 = +35.0 (c = 0.9, CHCl3) ; lmax
(CH3CN)/nm = 213.0, 264.0 and 431.0 (lg e 1.4111, 1.0752 and
0.0800); nmax/cm-1 = 2964, 1754, 1704, 1619, 1595, 1536, 1492,
˜
1370, 1351, 1219, 1165, 1073, 954, 897, 860, 769, 664, 590, 496;
1H-NMR (599.7 MHz, CDCl3): d = 1.97, 2.02, 2.08, 2.15 (4 ¥ s,
12 H, 4 ¥ COCH3), 3.00 (s, 3 H, NCH3), 3.63 (t, J = 5.2 Hz, 2 H,
H2-15), 4.03 (q, J = 6.6 Hz, 1 H, H-5), 4.12 (mc, 1 H, H-6a), 4.20
(mc, 1 H, H-6b), 4.37 (dt, J = 20.2, 5.3 Hz, 2 H, H2-16), 5.04 (mc, 2
H, H-1, H-3), 5.09 (s, 1 H, H-13), 5.42 (sbr, 1 H, H-4), 5.49 (dd, J =
10.4, 7.9 Hz, 1 H, H-2), 7.29 (mc, 2 H, H-19, H-23), 7.32 (d, J =
9.0 Hz, 1 H, H-12), 7.49 (dd, J = 8.8, 2.3 Hz, 1 H, H-11), 7.77 (dd,
J = 7.6, 1.6 Hz, 1 H, H-9), 8.23 (ddd, J = 10.2, 5.3, 3.3 Hz, 2 H,
H-20, H-22); 13C-NMR (150.8 MHz, CDCl3, internal dynamics):
d = 20.48, 20.55, 20.57 (4 ¥ COCH3), 35.26, 35.62 (NCH3), 47.33,
47.95 (C-15), 61.20 (C-6), 65.37, 65.52 (C-13), 66.44 (C-16), 66.58
(C-4), 67.68 (C-2), 70.40 (C-3), 71.32 (C-5), 100.6 (C-1), 119.5,
119.6 (C-12), 121.6, 121.7 ( C-19, C-23), 124.4, 124.6 (C-9), 125.2
( C-20, C-22), 132.6, 132.8, 133.0, 133.1 ( C-10, C-11), 141.2 (C-8),
145.4 (C-21), 148.9 (C-7), 152.3 (C-18), 155.1, 155.3, 155.5, 156.0
( C-14, C-17), 169.3, 170.0, 170.1, 170.2 (4 ¥ COCH3); C32H35N3O19
(765.63).
n
max/cm-1 = 2943, 1753, 1699, 1623, 1538, 1370, 1235, 1150, 1073,
1
(+)-N,N-Methyl-[4-(2,3,4,6-tetra-O-acetyl-b-D-galactopyra-
nosyl]-3-nitrobenzyl-oxycarbonyl]-O-{{methyl-{(1S,10R)-1-(10-
chloro-ethyl)-3-[(5-(2-(N,N-dimethylamino)-ethoxy)-indol-2-yl)-
carbonyl]-1,2-dihydro-3H-benz[e]indol-5-yl]}}-2-aminoethanol-
carbonate ((1S,10R)-26). To a mixture of the activated alcohol
24 (42.5 mg, 55.5 mmol, 1.0 equiv.) and seco-drug (+)-3 (32.7 mg,
63.6 mmol, 1.15 equiv.) in DMF (6.0 mL) DMAP (16.7 mg,
138 mmol, 2.5 eq.) was added at 0 ◦C in one portion. After 1 h
at 0 ◦C additional DMAP (16.7 mg, 138 mmol, 2.5 eq.) was
added and stirring continued for 2 h at ambient temperature.
The mixture was diluted with CH2Cl2 (4 mL) and sat. LiBr
solution (4 mL). Phases were separated and the aqueous layer
was re-extracted with CH2Cl2 (4 ¥ 4 mL), the combined organic
layers were dried (Na2SO4), the solvents removed and benzol (2 ¥
5 mL) distilled from the residue. After column chromatography
on silica (CH2Cl2–MeOH = 6 : 1) the acetylated spacer prodrug
26 (35.7 mg, 32.3 mmol, 58%) was obtained as slightly yellow solid.
Rf 0.33 (CH2Cl2–MeOH = 6 : 1); 1H-NMR (599.8 MHz, DMSO-
d6, 60 ◦C): d = 1.68 (d, J = 6.6 Hz, 3 H, H3-11¢), 1.95, 2.00, 2.14
(3 ¥ s, 12 H, 4 ¥ COCH3), 2.30 (s, 6 H, N(CH3)2), 2.73 (t, J =
5.8 Hz, 2 H, H2-2¢¢¢), 2.95 (sbr, 3 H, NCH3), 3.67 (d, J = 4.7 Hz,
2 H, H2-15), 4.09–4.17 (m, 4 H, H–1¢¢¢, H-6a, H-6b), 4.36 (td, J =
9.4, 2.2 Hz, 1 H, H-1¢), 4.40 (m, 1 H, H-5), 4.45 (t, J = 5.2 Hz,
H2-16), 4.69 (dd, J = 11.0, 2.4 Hz, 1 H, H-2a¢), 4.80 (t, J = 10.2 Hz,
1 H, H-2b¢), 4.85 (dq, J = 6.6, 2.4 Hz, 1 H, H-10¢), 5.14 (sbr, 2 H,
H2-13), 5.20–5.30 (m, 2 H, H-2, H-3), 5.36 (d, J = 0.9 Hz, 1 H,
H-4), 5.49 (d, J = 5.0 Hz, 1 H, H-1), 6.95 (dd, J = 8.9, 2.4 Hz,
1 H, H-6¢¢), 7.19 (d, J = 1.9 Hz, 1 H, H-3¢¢), 7.20 (d, J = 2.2 Hz,
1 H, H-4¢¢), 7.39 (d, J = 8.7 Hz, 1 H, H-12), 7.43 (d, J = 8.8 Hz,
1 H, H-7¢¢), 7.51 (mc, 1 H, H-7¢), 7.62 (t, J = 7.5 Hz, 1 H, H-8¢),
7.70 (dd, J = 8.7, 1.6 Hz, 1 H, H-11), 7.86, 7.88 (2 ¥ sbr, 2 H, H-9,
H-9¢), 8.08 (d, J = 8.4 Hz, 1 H, H-6¢), 8.34 (s, 1 H, H-4¢), 11.47 (s,
N,N-Methyl-[4-(2,3,4,6-tetra-O-acetyl-b-D-galactopyranosyl]-
3-nitrobenzyl-oxycarbonyl]-2-aminoethanol-carbonyl-chloride (22).
To a solution of 19 (120 mg, 0.20 mmol, 1.0 equiv.) in CH2Cl2
(5.0 mL) phosgene (20% in toluene, 0.63 mL, 1.44 mmol, 7.2
equiv.) and Et3N (33.3 mL, 0.24 mmol, 1.2 equiv.) were slowly
added at 0 ◦C. After 1 h at 0 ◦C silica gel (350 mg) was added, all
solvents were removed and the activated alcohol 22 was purified by
column chromatography on silica (EtOAc–pentane = 2 : 1) to give
a colourless sirup (112 mg, 170 mmol, 85%) which was used directly
1
for the following reaction. Rf 0.49 (EtOAc–pentane = 2 : 1); H-
NMR (300.1 MHz, CDCl3, strong internal dynamics): d = 1.98,
2.04, 2.09, 2.16 (4 ¥ s, 12 H, 4 ¥ COCH3), 2.95, 2.97, 3.00 (3 ¥ s,
3 H, NCH3), 3.40–3.79 (m, 4 H, H-15, H-16), 4.02–4.32 (m, 3 H,
H-5, H-6a, H-6b), 5.02–5.12 (m, 4 H, H-1, H-3, H-13), 5.44 (d, J =
3.1 Hz, 1 H, H-4), 5.51 (dd, J = 10.6, 8.1 Hz, 1 H, H-2), 7.31, 7.33
(2 ¥ d, J = 8.3 Hz, 1 H, H-12), 7.50 (d, J = 8.7 Hz, 1 H, H-11), 7.77
(sbr, 1 H, H-9); 13C-NMR (125.7 MHz, CDCl3): d = 20.51, 20.58,
20.61 (4 ¥ COCH3), 31.08, 31.87, 35.28, 35.41, 35.57, 35.67, 35.73
(NCH3), 41.28, 41.54, 46.81, 47.47, 47.53, 48.24, 50.36, 50.85,
51.28, 51.88 ( C-15, C-16), 61.30 (C-6), 65.37, 65.40 (C-13), 66.70
(C-4), 67.80 (C-2), 70.50 (C-3), 71.38 (C-5), 100.6 (C-1), 119.6,
119.7, 120.0 (C-12), 124.5, 124.6, 124.8, 125.2 (C-9), 132.8, 133.1
(C-11), 133.2, 133.5, 133.6 (C-10), 141.1, 141.2 (C-8), 148.9, 149.0,
149.1 (C-7), 154.8, 155.5, 155.9 (C-14), 169.4, 170.1, 170.2, 170.3
(4 ¥ COCH3), 175.3 (C-17); C26H31ClN2O16 (662.98).
N,N-Methyl-[4-(2,3,4,6-tetra-O-acetyl-b-D-galactopyranosyl]-
3 - nitrobenzyl - oxycarbonyl ] - 2 - aminoethanol - carbonyl - 4 - nitro-
phenol (24). To a solution of alcohol 19 (241 mg, 0.40 mmol,
1.0 equiv.) in CH2Cl2 (15.0 mL) pyridine (47.6 mg, 48.6 mL,
0.60 mmol, 1.5 equiv.) and 4-nitrophenylchloroformate (121 mg,
1838 | Org. Biomol. Chem., 2010, 8, 1833–1842
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1 H, NH); 13C-NMR (125.7 MHz, DMSO-d6, 35 ◦C): d = 20.15,
20.20, 20.25, 20.31 (4 ¥ COCH3), 23.32 (11¢-CH3), 34.16, 34.76
(NCH3), 45.31 (N(CH3)2), 46.05 (C-1¢), 46.83, 46.93, 47.32 (C-15),
51.85 (C-2¢), 57.61 (C-2¢¢¢), 61.11 (C-6), 61.20 (C-10¢), 64.74, 64.82
(C-13), 66.01 (C-1¢¢¢), 66.16, 66.42 (C-16), 67.00 (C-4), 67.62 (C-2),
69.86 (C-3), 70.71 (C-5), 98.56 (C-1), 103.3 (C-4¢¢), 105.7 (C-3¢¢),
110.1, 110.2 (C-4¢), 113.2 (C-7¢¢), 116.1 (C-5a¢, C-6¢¢), 117.7 (C-12),
122.8, 122.9, 123.1, 123.4, 123.5, 123.6 (C-6¢, C-9, C-9¢), 124.1 (C-
9b¢), 125.2 (C-8¢), 127.5 (C-3a¢¢), 127.6 (C-7¢), 129.5, 130.4, 131.8
(C-2¢¢, C-7a¢¢, C-9a¢), 132.4 (C-10), 133.0, 133.2 (C-11), 140.1 (C-
8), 141.3 (C-3a¢), 146.3, 146.4 (C-17), 147.8 (C-7), 152.9 (C-5¢,
2.02, 2.14 (4 ¥ s, together 12 H, 4 ¥ COCH3), 2.55 (sbr, 3 H,
+
NCH3H2 ), 2.92 (sbr, 3 H, NCH3CO), 3.58 (t, J = 5.2 Hz, 2 H,
H-15), 3.05 (t, J = 5.6 Hz, 2 H, H-16), 4.12 (dd, J = 11.5, 5.3 Hz, 1
H, H-6a), 4.15 (dd, J = 11.4, 5.3 Hz, 1 H, H-6b), 4.48 (t, J = 5.5 Hz,
1 H, H-5), 5.13 (s, 2 H, H-13), 5.24 (dd, J = 10.1, 7.6 Hz, 1 H,
H-2), 5.28 (dd, J = 10.3, 3.2 Hz, 1 H, H-3), 5.37 (dd, J = 2.9 Hz,
1 H, H-4), 5.56 (d, J = 7.6 Hz, 1 H, H-1), 7.45 (d, J = 8.3 Hz, 1 H,
H-12), 7.73 (d, J = 8.1 Hz, 1 H, H-11), 7.88 (sbr, 1 H, H-9), 9.11 (sbr,
2 H, NCH3H2+); 13C-NMR (125.7 MHz, DMSO-d6, 35 ◦C): d =
+
20.16, 20.23, 20.37 (4 ¥ COCH3), 32.43, (NCH3H2 ), 34.09, 34.55
(NCH3CO), 44.42, 44.58, 44.76 (C-16), 45.68, 46.11 (C-15), 61.06
(C-6), 64.80 (C-13), 66.96 (C-4), 67.58 (C-2), 69.77 (C-3), 70.65
(C-5), 98.42 (C-1), 117.7 (C-12), 123.7 (C-9), 132.2 (C-10), 133.2,
133.4 (C-11), 140.0 (C-8), 147.7 (C-7), 154.8, 155.6 (C-14), 168.7,
169.3, 169.7, 169.8 (4 ¥ COCH3); m/z (ESI) 614.21908 (M+ + H,
C26H36N3O14 requires 614.21918), 614.2 (100%).
=
C-5¢¢, 2 Signale), 155.0, 155.1, 155.6 (C-14), 160.1 (NC O), 168.7,
169.4, 169.7, 169.8 (4 ¥ COCH3); m/z (ESI) 1104.34806 (M+ + H,
C53H59N5O19Cl requires 1104.34863), 1104.3 (100%).
Synthesis of a biscarbamate spacer prodrug
N ,N¢-Dimethyl-[4-(2,3,4,6-tetra-O-acetyl-b-D-galactopyra-
N ,N¢-Dimethyl-[4-(2,3,4,6-tetra-O-acetyl-b-D-galactopyra-
nosyl]-3-nitrobenzyl-oxycarbonyl]-N¢-(tert-butyloxycarbonyl)-ethyl-
endiamine (20a). In analogy to the synthesis of alcohol 19
benzylalcohol 15 (100 mg, 0.20 mmol, 1.0 equiv.) was treated
with pyridine and p-NCF (1.5 equiv. each). After 1 h liquid
amine 18 (63.7 mg, 0.34 mmol, 1.7 equiv.) and DMAP (41.5 mg,
0.34 mmol, 1.7 equiv.) were added and the solution was stirred
for 4.5 h at 25 ◦C. Addition of silica gel (400 mg) followed by
purification by column chromatography (EtOAc–pentane = 2 : 1)
gave 20a as colourless foam (125 mg, 0.18 mmol, 88%). Rf 0.50
(EtOAc–pentane = 4 : 1) ; [a]2D3 = +37.4◦ (c = 0.35, CHCl3) ;
lmax (CH3CN)/nm = 212.5, 254.5 and 309.5 (lg e 1.2757, 0.5179
nosyl]-3-nitrobenzyl-oxycarbonyl]-ethylendiamine
carbamoyl-
chloride (21). The amine salt 20b (54.7 mg, 76.6 mmol, 1.0
equiv.) was suspended in CH2Cl2 (2.10 mL), cooled to 0 ◦C
and phosgene (20% in toluene, 0.24 mL, 0.55 mmol, 7.2 equiv.)
followed by Et3N (12.9 mL, 93.0 mmol, 1.2 equiv.) were added
slowly. After 1 h at this temperature silica gel (200 mg) was
added and the solvents were removed under reduced pressure.
After conventional column chromatography on silica (EtOAc–
pentane = 5 : 1) the activated amine 21 (50.0 mg, 74.0 mmol, 97%)
was obtained as colourless foam. Rf 0.48 (EtOAc–pentane =
1
5 : 1); H-NMR (599.7 MHz, CDCl3): d = 1.98, 2.04, 2.09, 2.16
and 0.2521); nmax/cm-1 = 2978, 1754, 1699, 1623, 1538, 1484,
(4 ¥ s, together 12 H, 4 ¥ COCH3), 2.93, 2.96, 3.04, 3.05, 3.12
(5 ¥ sbr, 6 H, 2 ¥ NCH3), 3.44–3.57 (m, 2 H, H-15), 3.58, 3.62 (2 ¥
t, J = 5.9 Hz, 2 H, H-16), 4.05 (t, J = 6.2 Hz, 1 H, H-5), 4.14
(dd, J = 11.4, 6.2 Hz, 1 H, H-6a), 4.21 (dd, J = 11.2, 7.0 Hz, 1 H,
H-6b), 5.01–5.12 (m, 4 H, H-1, H-3, H-13), 5.40 (dd, J = 3.0 Hz,
1 H, H-4), 5.51 (dd, J = 10.4, 8.0 Hz, 1 H, H-2), 7.32 (dd, J =
8.5, 2.1 Hz, 1 H, H-12), 7.49, 7.55 (2 ¥ d, J = 8.4 Hz, 1 H, H-11),
7.77, 7.78 (2 ¥ sbr, 1 H, H-9); 13C-NMR (125.7 MHz, CDCl3,
25 ◦C, strong internal dynamics): d = 20.53, 20.61, 20.65 (4 ¥
COCH3), 34.58, 35.14, 35.17, 35.41, 36.97, 37.38, 38.74, 39.35
(2 ¥ NCH3), 45.88, 46.13, 46.65, 46.85, 48.27, 49.17, 50.19, 50.57
( C-15, C-16), 61.27 (C-6), 65.32, 65.45, 65.59, 65.75 (C-13), 66.62
(C-4), 67.70 (C-2), 70.46 (C-3), 71.34 (C-5), 100.6 (C-1), 119.7,
119.8 (C-12), 124.5, 124.6, 124.8, 125.0 (C-9), 132.4, 132.6, 132.7,
132.9, 133.1 (C-11), 133.6, 133.7 (C-10), 141.1 (C-8), 148.8, 148.9,
149.0, 149.1 (C-17), 149.8, 150.2 (C-7), 155.4, 155.5, 155.9, 156.1
(C-14), 169.4, 170.1, 170.3 (4 ¥ COCH3); m/z (ESI) 693.20190
(M+ + NH4, C27H38ClN4O15 requires 693.20167), 698.15708
(M+ + Na, C27H34ClN3O15Na requires 698.15750), 698.2 (100%),
714.1 (75), 1373.3 (40), 1389.3 (25).
˜
1402, 1368, 1233, 1163, 1127, 1073, 953, 823, 767, 590; 1H-NMR
(599.7 MHz, CDCl3): d = 1.39 (s, 9 H, C(CH3)3), 1.97, 2.03,
2.08, 2.15 (4 ¥ s, together 12 H, 4 ¥ COCH3), 2.78, 2.84 (2 ¥ s, 3
H, NCH3Boc), 2.91 (s, 3 H, NCH3), 3.19–3.53 (m, 4 H, H-15,
H-16), 4.04 (t, J = 6.6 Hz, 1 H, H-5), 4.13 (dd, J = 11.5, 6.2 Hz,
1 H, H-6a), 4.21 (dd, J = 11.2, 7.0 Hz, 1 H, H-6b), 5.03 (d, J =
7.9 Hz, 1 H, H-1), 5.06 (dd, J = 10.5, 3.4 Hz, 1 H, H-3) 5.06
(sbr, 2 H, H-13), 5.43 (dd, J = 3.4, 1.0 Hz, 1 H, H-4), 5.50 (dd,
J = 10.5, 7.9 Hz, 1 H, H-2), 7.30 (d, J = 8.6 Hz, 1 H, H-12),
7.48 (d, J = 8.8 Hz, 1 H, H-11), 7.75 (d, J = 1.9 Hz, 1 H, H-9);
13C-NMR (125.7 MHz, CDCl3): d = 20.50, 20.58, 20.60 (4 ¥
COCH3), 28.31 (C(CH3)3), 34.47, 34.83 (2 ¥ NCH3, 14 signals in
total), 35.27, 35.35 (C(CH3)3), 46.45, 46.70, 46.76, 46.95 ( C-15
,
C-16, 15 signals in total), 61.24 (C-6), 65.16, 65.38 (C-13), 66.62
(C-4), 67.73 (C-2), 70.45 (C-3), 71.34 (C-5), 100.7 (C-1), 119.7 (
C-12, 2 signals), 124.5 (C-9), 133.0, 133.2 ( C-10, C-11), 141.1 (C-8),
148.8, 148.9 (C-7), 155.6, (C-14), 169.3, 170.0, 170.1, 170.2 (4 ¥
COCH3); m/z (ESI) 736.25347 (M+ + H, C31H44N3O16 requires
736.25355), 736.3 (100%), 1449 (20).
N ,N¢-Dimethyl-[4-(2,3,4,6-tetra-O-acetyl-b-D-galactopyra-
nosyl]-3-nitrobenzyl-oxycarbonyl]-ethylendiamine-hydrochloride
(20b). The amine 20a (40 mg, 56 mmol, 1.0 equiv.) was cooled to
0 ◦C and a freshly prepared, pre-cooled solution of HCl (3 M in
EtOAc, 5.0 mL) was added and the mixture was stirred for 30 min
at ambient temperature. The solvent was removed under reduced
pressure and benzol (2 ¥ 5.0 mL) distilled from the residue. The
crude amine salt 20b (37.0 mg) was obtained analytically pure and
used directly in the following reaction. Rf 0.00 (EtOAc–pentane =
4 : 1) ; 1H-NMR (599.7 MHz, DMSO-d6, 85 ◦C): d = 1.94, 2.01,
N ,N¢-Dimethyl-[4-(2,3,4,6-tetra-O-acetyl-b-D-galactopyra-
nosyl]-3-nitrobenzyl-oxycarbonyl]-ethylendiamine
carbamoyl-4-
nitrophenol (23). The crude amine salt 20b (27.2 mg, 41.8 mmol,
1.0 equiv.) in CH2Cl2 (5.0 mL) was treated with pyridine (9.90 mg,
10.1 mL, 126 mmol, 3.0 equiv.) and p-NCF (16.85 mg, 83.6 mmol,
2.0 equiv.) for 30 min at 0 ◦C. After 4 h additional pyridine
(3.0 equiv.) and p-NCF (2.0 equiv.) were added and the reaction
mixture was stirred for 2 h. Silica gel (300 mg) was added, the
solvents were removed and product 23 (32.1 mg, 41.2 mmol,
98% starting from 20b) was obtained as colourless sirup after
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column chromatography (EtOAc–pentane = 4 : 1). Rf 0.39
(EtOAc–pentane = 3 : 1); [a]2D3 = + 18.3◦ (c = 1.31, MeOH) ; lmax
H-4¢), 11.40 (s, 1 H, NH); 13C-NMR (125.7 MHz, DMSO-d6,
35 C): d = 20.17, 20.20, 20.25, 20.30 (4 ¥ COCH3), 23.35 (11¢-
◦
(CH3CN)/nm = 272.0 (lg e 1.0096) ; nmax/cm-1 = 2961, 1754,
CH3), 33.76, 34.23, 34.48, 34.62, 34.75, 35.67 (2 ¥ NCH3), 45.36
(N(CH3)2), 46.04 (C-1¢), 45.94, 46.23, 46.33, 46.64, 46.77 ( C-15, C-
16), 51.89 (C-2¢), 57.66 (C-2¢¢¢), 61.08 (C-6), 61.25 (C-10¢), 64.60,
64.71, 64.81, 64.87 (C-13), 66.09 (C-1¢¢¢), 66.99 (C-4), 67.63 (C-2),
69.86 (C-3), 70.71 (C-5), 98.58 (C-1), 103.3 (C-4¢¢), 105.6 (C-3¢¢),
110.4, 110.5, 110.7 (C-4¢), 113.1 (C-7¢¢), 116.0 (C-6¢¢), 117.7 (C-
5a¢, C-12), 123.0 (C-6¢, C-9b¢), 123.4 (C-9¢), 123.6, 123.7, 123.9,
124.0 (C-9), 124.7 (C-7¢), 127.2 (C-8¢), 127.5 (C-3a¢¢), 129.4, 130.5,
131.7 (C-2¢¢, C-7a¢¢, C-9a¢), 132.5, 132.6 (C-10), 133.1, 133.3 (C-
11), 140.0, 140.1 (C-8), 141.3 (C-3a¢), 147.2, 147.3, 147.8 (C-7),
˜
1704, 1616, 1595, 1537, 1404, 1348, 1220, 1162, 1073, 864, 821,
749, 685, 590; 1H-NMR (599.7 MHz, CDCl3, strong internal
dynamics): d = 1.99, 2.04, 2.10, 2.17 (4 ¥ s, together 12 H, 4 ¥
COCH3), 2.95, 2.97, 3.03, 3.07, 3.12 (5 ¥ sbr, 6 H, 2 ¥ NCH3),
3.43–3.64 (m, 4 H, H-15, H-16), 3.97–4.09 (m, 1 H, H-5), 4.13
(dd, J = 11.2, 6.3 Hz, 1 H, H-6a), 4.18–4.23 (m, 1 H, H-6b),
5.00–5.11 (m, 4 H, H-1, H-3, H-13), 5.44 (d, J = 2.4 Hz, 1 H,
H-4), 5.51 (dd, J = 10.1, 8.7 Hz, 1 H, H-2), 7.12, 7.21 (2 ¥ d,
J = 8.9 Hz, 1 H, H-19, H-23), 7.26–7.31 (m, 1 H, H-12), 7.46 (t,
J = 10.0 Hz, 1 H, H-11), 7.77 (m, 1 H, H-9), 8.15–8.24 (m, 2 H,
=
152.9, 153.7 (C-5¢, C-5¢¢), 155.5 (C-14), 160.0 (NC O), 162.2 (C-
◦
H-20, H-22); 13C-NMR (125.7 MHz, CDCl3, 27 C, very strong
17), 168.7, 169.4, 169.7, 169.8 (4 ¥ COCH3); m/z (ESI) 1117.38056
internal dynamics): d = 20.53, 20.60, 20.62 (4 ¥ COCH3), 34.57,
34.91, 35.23, 35.35, 35.44, 35.48 (2 ¥ NCH3), 46.12, 46.50, 46.75,
46.91, 47.30, 47.39, 47.49 ( C-15, C-16), 61.25 (C-6), 65.26, 65.42,
65.52, 65.80 (C-13), 66.64 (C-4), 67.78 (C-2), 70.49 (C-3), 71.41
(C-5), 100.7 (C-1), 119.5, 119.6, 119.7, 119.8 (C-12), 122.0, 122.1,
122.4 ( C-19, C-23), 124.5, 124.6 (C-9), 125.0, 125.1 ( C-20, C-22),
132.3, 132.7, 132.9, 133.2, 133.3, 133.7 ( C-10, C-11), 141.1, 141.3
(C-8), 144.8, 145.1 (C-21), 148.9, 149.0, 149.1 (C-7), 153.1 153.3,
153.6 (C-18), 155.6, 155.7, 155.9, 156.0, 156.1, 156.2 ( C-14, C-17),
169.3, 170.1, 170.2, 170.3 (4 ¥ COCH3); C33H38N4O18 (778.67).
(M+ + H, C54H62N6O18Cl requires 1117.38036), 1117.4 (100%).
(+)-N ,N¢-Dimethyl-[4-(b-D-galactopyranosyl]-3-nitrobenzyl-
oxycarbonyl]-O-{{methyl-{(1S,10R)-1-(10-chloro-ethyl)-3-[(5-(2-
(N,N-dimethylamino)-ethoxy)-indol-2-yl)carbonyl]-1,2-dihydro-
3H-benz[e]indol-5-yl]}}-ethylendiamine carbamate ((1S,10R)-11).
The acetylated prodrug 25 (14.6 mg, 13.1 mmol, 1.0 equiv.)
was dissolved in MeOH (3.00 mL) and treated at 0 ◦C with
NaOMe (0.065 M in MeOH, 100 ml, 6.50 mmol, 0.5 equiv.)
for 30 min under stirring. Neutralisation was realised using a
solution of HCl (0.10 M in MeOH, 65.0 ml, 0.5 equiv.) and
the solvents removed. Conventional column chromatography on
silica (CH2Cl2–MeOH = 3 : 1), removal of the solvents to 1 mL
and filtration through a membrane filter following by solvent
evaporation under reduced pressure gave the spacer prodrug
(1S,10R)-11 (9.80 mg, 10.3 mmol, 79%) as colourless, amorphous
solid. Rf 0.12 (CH2Cl2–MeOH = 3 : 1); lmax (CH3CN)/nm = 253.0,
˜
(+)-N,N¢-Dimethyl-[4-(2,3,4,6-tetra-O-acetyl-b-D-galactopyra-
nosyl]-3-nitrobenzyl-oxycarbonyl]-O-{{methyl-{(1S,10R)-1-(10-
chloro-ethyl)-3-[(5-(2-(N,N-dimethylamino)-ethoxy)-indol-2-yl)-
carbonyl]-1,2-dihydro-3H-benz[e]indol-5-yl]}}-ethylendiamine car-
bamate ((1S,10R)-25). To a cooled solution (0 ◦C) of the
activated amine 21 (30.0 mg, 44.5 mmol, 1.0 equiv.) and seco-
drug (+)-3 (22.8 mg, 63.6 mmol, 1.0 equiv.) in DMF (6.0 mL)
DMAP (53.7 mg, 443 mmol, 10 equiv.) was slowly added (10 min)
followed by dropwise addition of Et3N (9.25 ml, 66.8 mmol, 1.5
equiv.) and stirring was continued at 0 ◦C for 2 h. The mixture
was diluted with CH2Cl2 (4.0 mL) and sat. LiBr solution (2.5 mL).
Phases were separated and the aqueous layer was re-extracted
with CH2Cl2 (4 ¥ 5 mL), the combined organic layers were
dried (Na2SO4), the solvents removed and benzol (2 ¥ 5 mL)
was destilled from the residue. After column chromatography on
silica (CH2Cl2–MeOH = 6 : 1) the acetylated spacer prodrug 25
(18.9 mg, 16.9 mmol, 38%) was obtained as colourless solid. Rf
297.0 and 330.5 (lg e 1.1978, 1.2604 and 1.2348), nmax/cm-1
=
1
3386, 2925, 1715, 1623, 1533, 1409, 1212, 1072, 759; H-NMR
(599.8 MHz, DMSO-d6, 70 ◦C): d = 1.67 (d, J = 6.6 Hz, 3 H,
H3-11¢), 2.29 (s, 6 H, N(CH3)2), 2.72 (t, J = 5.8 Hz, 2 H, H2-2¢¢¢),
2.88–3.39 (m, 6 H, 2 ¥ NCH3, underneath H2O signal), 3.42 (dd,
J = 9.4, 3.1 Hz, 1 H, H-3), 3.45–3.64 (m, 6 H, H-2, H-5, H2-6,
H-15, H-16), 3.75 (d, J = 2.9 Hz, 1 H, H-4), 4.11 (t, J = 5.8 Hz,
2 H, H2-1¢¢), 4.34 (mc, 1 H, H-1¢), 4.39–4.62 (mbr, 4 H, 4 ¥ OH),
4.67 (dd, J = 11.2, 2.3 Hz, 1 H, H-2a¢), 4.77 (t, J = 10.4 Hz, 1
H, H-2b¢), 4.84 (mc, 1 H, H-10¢), 5.47 (d, J = 7.1 Hz, 1 H, H-1),
5.11 (sbr, 2 H, H2-13), 6.94 (dd, J = 8.9, 2.4 Hz, 1 H, H-6¢¢), 7.16
(sbr, 1 H, H-3¢¢), 7.19 (d, J = 2.1 Hz, 1 H, H-4¢¢), 7.34 (sbr, 1 H,
H-12), 7.42 (d, J = 9.0 Hz, 1 H, H-7¢¢), 7.46 (mc, 1 H, H-7¢), 7.59
(mc, 2 H, H-11, H-8¢), 7.79, 7.87 (2 ¥ sbr, 2 H, H-9, H-9¢), 8.03 (d,
J = 8.4 Hz, 1 H, H-6¢), 8.20 (s, 1 H, H-4¢), 11.41 (s, 1 H, NH);
0.36 (CH2Cl2–MeOH = 5 : 1); [a]23 = +24.0◦ (c = 0.4, MeOH);
D
lmax (CH3CN)/nm = 204.0, 250.0, 296.5 and 332.0 (lg e 1.8025,
1.3737, 1.5361 and 1.4955); nmax/cm-1 = 2934, 1754, 1626, 1537,
˜
1
1461, 1408, 1370, 1232, 1123, 1072, 758; H-NMR (599.8 MHz,
DMSO-d6, 70 C): d = 1.68 (d, J = 6.6 Hz, 3 H, H3-11¢), 1.95,
◦
◦
13C-NMR (125.7 MHz, DMSO-d6, 35 C): d = 23.38 (11¢-CH3),
1.99, 2.00, 2.14 (4 ¥ s, together 12 H, 4 ¥ COCH3), 2.29 (s, 6 H,
N(CH3)2), 2.72 (t, J = 5.8 Hz, 2 H, H2-2¢¢¢), 2.90–3.81 (m, 10 H,
2 ¥ NCH3, H2-15, H2-16), 4.08–4.14 (m, 4 H, H2-1¢¢¢, H2-6), 4.33
(td, J = 9.5, 2.4 Hz, 1 H, H-1¢), 4.39 (t, J = 6.3 Hz, 1 H, H-5),
4.68 (dd, J = 11.0, 1.7 Hz, 1 H, H-2a¢), 4.77 (t, J = 10.2 Hz, 1
H, H-2b¢), 4.84 (dq, J = 6.6, 2.4 Hz, 1 H, H-10¢), 5.14 (sbr, 2 H,
H2-13), 5.21–5.27 (m, 2 H, H-2, H-3), 5.36 (d, J = 2.1 Hz, 1 H,
H-4), 5.47 (d, J = 6.3 Hz, 1 H, H-1), 6.94 (dd, J = 8.9, 2.2 Hz, 1
H, H-6¢¢), 7.16 (sbr, 1 H, H-3¢¢), 7.20 (d, J = 2.0 Hz, 1 H, H-4¢¢),
7.36 (sbr, 1 H, H-12), 7.43 (d, J = 8.9 Hz, 1 H, H-7¢¢), 7.45 (mc, 1
H, H-7¢), 7.58 (t, J = 7.6 Hz, 1 H, H-8¢), 7.66 (sbr, 1 H, H-11), 7.85
(sbr, 2 H, H-9, H-9¢), 8.03 (d, J = 8.4 Hz, 1 H, H-6¢), 8.20 (s, 1 H,
31.19, 31.50, 34.67, 34.80, 34.84, 38.52, 38.61 (2 ¥ NCH3), 45.33
(N(CH3)2), 46.03 (C-1¢), 40.41, 40.49, 46.40 ( C-15, C-16), 51.90 (C-
2¢), 57.62 (C-2¢¢¢), 60.11 (C-6), 61.29 (C-10¢), 64.81, 64.83, 64.95
(C-13), 66.03 (C-1¢¢¢), 67.82 (C-4), 70.00 (C-5), 73.30 (C-3), 75.68
(C-2), 101.1 (C-1), 103.3 (C-4¢¢), 105.6 (C-3¢¢), 110.5, 110.7 (C-
4¢), 113.2 (C-7¢¢), 116.0 (C-6¢¢), 117.0 (C-5a¢, C-12), 122.2, 123.1,
123.5, 123.6 (C-6¢, C-9, C-9¢, C-9b¢), 124.4 (C-7¢), 127.3 (C-8¢),
127.5 (C-3a¢¢), 129.4, 130.6, 131.7 (C-2¢¢, C-7a¢¢, C-9a¢), 131.7 (C-
10), 133.3 (C-11), 139.8 (C-8), 141.3 (C-3a¢), 147.2 (C-7), 150.6 (C-
=
5¢), 152.9 (C-5¢¢), 155.5 (C-14), 160.0 (NC O), 162.7 (C-17); m/z
(ESI) 949.33796 (M+ + H, C46H54ClN6O14 requires 949.33810),
949.3 (100%), 971.2 (41).
1840 | Org. Biomol. Chem., 2010, 8, 1833–1842
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In vitro cytotoxicity assay
MS/MS measurements were carried out by fragmentation of ions
isolated in the ICR cell using Argon as collision gas.
Adherent cells of line A549 were sown in triplicate in 6 multiwell
plates at concentrations of 102, 103, and 104 cells per cavity. Culture
medium was removed using suction after 24 h and cells were
washed in the incubation medium UltraCultureTM (UC, serum-
free special medium, purchased from Lonza). Incubation with
compound 11 was then performed in UltraCultureTM medium at
6–8 various concentrations for 24 h. All substances were used
as freshly prepared solutions in DMSO (Merck, Darmstadt,
Germany) diluted withincubation mediumto afinalconcentration
of DMSO of 1% in the wells. After 24 h of exposure the test
substance was removed and the cells were washed with fresh
medium. Cultivation was accomplished at 37 ◦C and 7.5% CO2
in air for 9–10 days. The medium was removed and the clones
were dried and stained with Lo¨ffler’s methylene blue (Merck,
Darmstadt, Germany). They were then counted macroscopically.
The IC50 values are based on the relative clone forming rate,
which was determined according to the following formula: relative
clone forming rate [%] = 100 ¥ (number of clones counted
after exposure)/(number of clones counted in the control). The
obtained data points and graph can be found in the ESI.‡
Liberation of the drug from its glycosidic prodrug was achieved
by addition of 4 U mL-1 b-galactosidase (E.C. 3.2.1.23) from
Escherichia coli G 5635 (Sigma), to the cells during incubation
with the substances.
Acknowledgements
We are grateful to R. Machinek and team for his assistance with
the high temperature NMR measurements. Financial support was
provided by the Deutsche Forschungsgemeinschaft (DFG), the
Friedrich-Ebert-Stiftung (Ph.D. scholarship for H. J. S.) and the
Deutsche Telekom Stiftung (Ph.D. scholarship for B. K.).
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©