Synthesis of fluorescence-labelled glycosidic prodrugs based on the cytotoxic antibiotic duocarmycin

Article abstract of DOI:10.1002/ejoc.201000966

The synthesis of the glycosidic prodrugs (1S)-30a, (1S,10R)-30b and (1S,10R)-32 labelled with different fluorescence dyes at different positions at the aromatic A-ring in 2 is described; the compounds are structurally based on the cytotoxic antibiotic duocarmycin SA. For binding, the amino compounds (1S)-3a and (1S,10R)-3b were treated with the commercially available succinimides of the dyes 5-SFX (29) and D10162 (31), respectively. Fluorescence-labelled compounds can be used for verification of interaction with cellular targets on the molecular level by use of confocal laser scanning microscopy for cell cultures and the Explore-Optics instrument (GE) for mice. The described fluorescence-labelled glycosidic prodrugs based on the cytotoxic antibiotic duocarmycine SA have high potential for selective treatment of cancer. Copyright

Full text of DOI:10.1002/ejoc.201000966

FULL PAPER
DOI: 10.1002/ejoc.201000966
Synthesis of Fluorescence-Labelled Glycosidic Prodrugs Based on the
Cytotoxic Antibiotic Duocarmycin
Lutz F. Tietze,*[a] Frank Behrendt,[a] Felix Major,[a] Birgit Krewer,[a] and
J. Marian von Hof[a]
Dedicated to Professor Carmen Najera on the occasion of her 60th birthday
Keywords: Antitumor agents / Cancer therapy / Duocarmycin / Glycosides / Prodrugs / Fluorescence
The synthesis of the glycosidic prodrugs (1S)-30a, (1S,10R)-
30b and (1S,10R)-32 labelled with different fluorescence
dyes at different positions at the aromatic A-ring in 2 is de-
toxic antibiotic duocarmycin SA. For binding, the amino com-
pounds (1S)-3a and (1S,10R)-3b were treated with the com-
mercially available succinimides of the dyes 5-SFX (29) and
scribed; the compounds are structurally based on the cyto- D10162 (31), respectively.
Introduction
Over the last few years we have successfully developed
novel glycosidic prodrugs[1] such as 2a and 2b (Scheme 1),
which can be used for selective treatment of cancer through
antibody-directed enzyme prodrug therapy (ADEPT).[2,3]
The compounds are based on the cytotoxic antibiotic
duocarmycin SA (1, IC50 ≈ 10 pm, L1210)[4] and have as yet
unsurpassed selectivity factors of QIC50 = 3200 and 4800,
respectively (QIC50 = IC50 of prodrug/IC50 of prodrug in
the presence of the cleaving enzyme).[5] Here we describe
the synthesis of the fluorescence-labelled derivatives (1S)-
30a, (1S,10R)-30b and (1S,10R)-32, in order to determine
their cellular uptakes and mode(s) of action. Fluorescence-
labelled compounds can thus be used for verification of
their interaction with cellular targets at the molecular level
either by confocal laser scanning microscopy for cell cul-
tures[6] or with the aid of the Explore-Optics instrument
(GE) for mice.[7]
Scheme 1. Structures of (+)-duocarmycin SA (1) and the glycosidic
For binding to the fluorescence dyes we introduced ami-
nomethyl groups into 2a and 2b at different positions in the
A-ring. This should also allow us to determine the influence
of the binding position on the activities of the labelled com-
prodrugs 2a and 2b, as well as the compounds 3a and 3b, each
containing an aminomethyl group for binding with the fluorescence
dye.
pounds. The synthesis of the corresponding substrates 3a
and 3b started from meta-diiodobenzene and para-diiodo-
benzene, respectively. As dyes, flurorescein (4, Scheme 2)
and dapoxyl (5) were used, through application of the com-
mercially available activated compounds 5-SFX (29,
Scheme 11, below) and D10162 (31, Scheme 13, below).
[a] Georg-August-University Göttingen, Institute of Organic and
Biomolecular Chemistry,
Tammannstraße 2, 37077 Göttingen, Germany
Fax: +49-551-399476
E-mail: ltietze@gwdg.de
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201000966.
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These show absorption maxima of λmax = 494 nm for 4 and case of 10a we obtained a mixture of the desired 11a and
1
λmax = 395 nm for 5 and emission maxima at λmax = 521 nm its regioisomer 11c in a ratio of 2.5:1 (determined by H
for 4 and λmax = 574 nm for 5 with high extinction coeffi- NMR spectroscopy); at this stage it was not possible to
cients.
separate the two compounds. In contrast, when starting
from 10b, compound 11b was formed as the only product.
The acetates 11a/c and 11b were solvolysed with K2CO3
in ethanol to yield the phenols 12a/c and 12b (Scheme 4),
which were protected at their phenolic hydroxy groups by
treatment with BnBr and catalytic amounts of TBAI to give
13a/c and 13b. Exchange of the iodide for CN with CuCN
under microwave conditions led to 14a/c and 14b.[8]
Scheme 2. Structures of fluorescein (4) and dapoxyl (5).
Results and Discussion
Formylation of the diiodobenzenes 6a and 6b (Scheme 3)
by halogen/metal exchange with n-butyllithium at –78 °C in
THF, followed by addition of DMF, led to the iodobenzal-
dehydes 7a and 7b, respectively. The aldehydes were then
converted into the styrene derivatives 8a and 8b through
Wittig–Horner reactions with the phosphonate 9 and NaH
in THF. Acid-catalysed deprotection of 8a and 8b in TFA/
H2O yielded the carboxylic acids 10a and 10b, which were
transformed into the naphthalene derivatives 11a and 11b
through intramolecular Friedel–Crafts reactions by treat-
ment with acetic anhydride and KOAc under reflux. In the
Scheme 4. Synthesis of 16a and 16b starting from the naphthalene
derivatives 11a and 11b, respectively. Reagents and conditions:
(a) K2CO3, EtOH, reflux, 4 h, 84–92%; (b) BnBr, K2CO3, TBAI,
DMF, r.t., 15 h, 82–94%; (c) CuCN, DMF, 140 °C (microwaves),
1 h, 92–94%; (d) LiOH·H2O, THF/MeOH/H2O, r.t., 15 h, 87–98%;
(e) DPPA, NEt3, tBuOH, molecular sieves (4 Å), reflux, 20 h, 95–
100%.
At this stage it was possible to recrystallise the 14a/c mix-
ture from n-pentane/EtOAc to afford pure 14a. Hydrolysis
of the ethyl carboxylates 14a and 14b with lithium hydrox-
ide, followed by formation of the corresponding acyl azides
and Curtius rearrangements with use of diphenylphospho-
ryl azide in tert-butyl alcohol, led to the Boc-protected ami-
nonaphthalenes 16a and 16b (Scheme 4).
Bromination of 16a and 16b with NBS and catalytic
amounts of sulfuric acid gave the compounds 17a and 17b
(Scheme 5), whereas iodination of 16a mediated by NIS and
catalytic amounts of p-toluenesulfonic acid yielded com-
pound 17c.
For the synthesis of the aminomethyl-substituted benzo-
indoles 3a and 3b from 17 we envisaged two different ap-
proaches (Scheme 6): firstly, N-alkylation with the chloroal-
lyl chlorides 18a and 18b, respectively, followed by radical
Scheme 3. Synthesis of the naphthalenes 11a and 11b starting from
the diiodobenzenes 6a and 6b, respectively. Reagents and condi-
tions: (a) nBuLi, THF, –78 °C, 20 min, then DMF, r.t., 2 h; 84–
95%; (b) 9, NaH, THF, 0 °C Ǟ r.t., 19 h; 63–72%; (c) TFA/H2O
(9:1), r.t., 6 h, 70–100%; (d) KOAc, Ac2O, reflux, 1 h, 87–90%.
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Fluorescence-Labelled Glycosidic Prodrugs Based on Duocarmycin
N-Alkylation of 17c and 17b with the allyl halides 18a
and 18b led to 19a and 19b, whereas treatment of 17a and
17b with the epoxy nosylates (2R)-20a and (2R,3R)-20b
gave (2R)-21a and (2R,3R)-21b. Interestingly, compounds
of type 21 each contain an axis of chirality along the
naphthyl–N bond, so they are obtained as mixtures of dia-
stereomers.[9]
Radical cyclisation of 19a and 19b, mediated by tris(tri-
methylsilyl)silane and azobisisobutyronitrile in toluene,
worked well to afford the cyclised products 22a–c
(Scheme 7) in 85–92% yields as racemic mixtures, and in
the case of 19b in a nearly 1:1 ratio of the two possible
Scheme 5. Synthesis of 17a–17c. Reagents and conditions: for 17a/
17b: NBS, cat. H2SO4, THF, –78 °C, 4 h, 81–94%; for 17c: NIS,
cat. TsOH·H2O, THF, 50 °C, 1.5 h, 81%.
cyclisation, and secondly, N-alkylation with the epoxy nosyl- diastereomers 22b/22c.[10] Similarly, metal-induced cyclis-
ates (2R)-20a and (2R,3R)-20b, respectively, followed by ation of (2R,3R)-21b in the presence of ZnCl2, MeLi and
metal-induced cyclisation. The latter approach has the ad- TMS-SCN led to the benzindole derivative (1S,10R)-23b in
vantage of leading to enantiopure – and in the case of 53% yield, in this case as a pure enantiomer (Ͼ99% ee)
(2R,3R)-20b also diastereopure – product(s).
and a pure diastereomer. In comparison, (2R)-21a gave the
Scheme 6. Synthesis of 19 and 21 from 17. Reagents and conditions: (a) NaH, DMF, 18, r.t., 4–14 h, 77–93%; (b) NaH, DMF, 20, r.t.,
21 h, 88–89%.
Scheme 7. Synthesis of rac-22 and enantiopure 23. Reagents and conditions: (a) TTMSS, AIBN, toluene, 80 °C, 4.5 h, 85–92%; (b) (2R)-
20a: nBuLi (3.0 equiv.), –85 °C, 1 h, 12%; (2R,3R)-20b: ZnCl2 (1.5 equiv.), MeLi (6.0 equiv.), TMS-SCN (1.5 equiv.), 55%.
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Scheme 8. Synthesis of the phenols 24. Reagents and conditions: (a) (1S)-24a: Pd/C, H2, EtOAc, r.t., 5 h, 90%; (1S,10R)-24b: Pd/C,
NH4HCO2, THF, 40 °C, 25 min, 86% (transfer hydrogenation).
desired product (1S)-23a in only 12% yield on treatment
with nBuLi at –85 °C. All other attempts to initiate the
cyclisation via a zincate or cuprate failed.
Treatment of (1S)-23a under Appel conditions afforded
enantiopure (1S)-22a,[10] whereas treatment of (1S,10R)-23b
with SOCl2 in the presence of pyridine led to enantiopure
(1S,10R)-22b. Alternatively, the enantiomeric mixture of
rac-22a obtained from 19a could be resolved by preparative
HPLC on a chiral stationary support (Chiralpak IA), with
ee values of 99.7% for (+)-(1R)-22a and 99.2% for (–)-(1S)-
22a. The two diastereomers rac-22b and rac-22c obtained
from 19b were separated by column chromatography on sil-
ica gel, whereas rac-22b was resolved again by preparative
HPLC on chiral support (Chiralpak IA) to obtain the de-
sired (+)-(1S,10R)-22b with an ee value of 99.9%. Neither
the diastereomer 22c nor the enantiomer (–)-(1R,10S)-22b
were included in further investigations, because we have
shown that the corresponding seco drugs – without the cy-
ano group – have much lower cytotoxicities.
Deprotection of 22a and 22b either with Pd/C under H2
or with ammonium formate, respectively, gave the free
phenols 24a and 24b in good yields (Scheme 8).
Next, the galactose moiety and the DNA binder were
introduced (Scheme 9). For this purpose (1S)-24a and
(1S,10R)-24b were glycosylated with the galactose trichloro-
acetimidate 25 in the presence of BF3·OEt2 at –10 °C. Ad-
dition of an excess of BF3·OEt2 at room temp. led to the
corresponding secondary amine as a result of Boc removal.
This amine was then coupled with the DNA binder DMAI
26 without isolation in the presence of EDC·HCl as the
activating agent.
Scheme 9. Synthesis of the galactosides 27a and 27b. Conditions
and reagents: (a) 26, 0.5 equiv. BF3·OEt2, CH2Cl2, molecular sieves
(4 Å), –10 °C, 3.5 h, then 3 equiv. BF3·OEt2, r.t., 6 h, 27, EDC·HCl,
DMF, r.t., 18 h, 52–53%.
Nitrile reduction in (1S)-27a and (1S,10R)-27b in accord-
ance with the method of Wrobel et al.[11] with PtO2·H2O
under H2 afforded the corresponding primary amines 28a
and 28b (Scheme 10), and these were deprotected under ba- parative HPLC (Kromasil 100 C18), gave the fluorescence-
sic conditions with NaOMe in MeOH to give the desired labelled prodrug (1S)-30a in 51% yield.
aminomethyl-substituted prodrugs (1S)-3a and (1S,10R)-
3b.
In the same manner, coupling of (1S,10R)-3b with 29
(Scheme 12) afforded the fluorescence-labelled prodrug
The final step in the synthesis of the fluorescence-labelled (1S,10R)-30b in 88% yield.
prodrugs was the coupling of (1S)-3a and (1S,10R)-3b with
In addition, the aminomethyl-substituted prodrug
the activated commercially available dyes 5-SFX (29) as a (1S,10R)-3b was also coupled with the dapoxyl derivative
derivative of fluorescein (4) and D10162 (31) as a derivative 31 under the same conditions as described for 29 to give
of dapoxyl (5). Coupling of (1S)-3a with 29 in DMF and the fluorescence-labelled prodrug (1S,10R)-32 in 89% yield
Hünig base (Scheme 11), followed by purification by pre- (Scheme 13).
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Fluorescence-Labelled Glycosidic Prodrugs Based on Duocarmycin
Scheme 10. Synthesis of the aminomethyl-substituted prodrugs 3. Reactions and conditions: (a) HCl/EtOH, PtO2·H2O, H2, r.t., 48 h, 66–
81%; (b) NaOMe/MeOH, MeOH, r.t., 30 min, prep. HPLC (Kromasil 100 C18), 70–95%.
Scheme 11. Coupling of the prodrug (1S)-3a with the NHS ester 29 to give (1S)-30a. Reagents and conditions: (a) iPr2NEt, DMF, r.t.,
18 h, prep. HPLC (Kromasil 100 C18), 51%.
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Scheme 12. Coupling of the prodrug (1S,10R)-3b with the NHS ester 29 to give (1S,10R)-30b. Reagents and conditions: (a) iPr2NEt,
DMF, r.t., 18 h, prep. HPLC (Kromasil 100 C18), 88%.
Scheme 13. Coupling of (1S,10R)-3b with 31 to give (1S,10R)-32. Conditions and reagents: (a) iPr2NEt, DMF, r.t., 9 h, prep. HPLC,
89%.
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Fluorescence-Labelled Glycosidic Prodrugs Based on Duocarmycin
UV (CH3CN): λmax (lg ε) = 221.0 (4.645), 256.0 (4.509), 320.5
(3.828), 354.4 (3.638) nm. ESI-HRMS: m/z = 597.04123 [M + Na]
+; C26H24ClIN2O3 requires 597.04187.
Conclusions
We have prepared the fluorescence-labelled prodrugs
(1S)-30a, (1S,10R)-30b and (1S,10R)-32 to allow investi-
gations of their cellular uptakes and their mode(s) of action
by confocal laser scanning microscopy in the case of cell
cultures and with the Explore Optix (GE) instrument in the
case of mice. The results will be published in due course in
a more biologically orientated journal.
(E/Z)-2-Amino-4-(benzyloxy)-1-bromo-N-(tert-butoxycarbonyl)-N-
(3-chlorobut-2-enyl)-6-cyanonaphthalene (19b): NaH (863 mg, 60%
dispersion in mineral oil, 21.6 mmol, 2.5 equiv.) was added at room
temperature to a solution of 17b (6.52 g, 14.4 mmol, 1.0 equiv.) in
anhydrous DMF (150 mL) and the mixture was stirred for 3 h.
Compound 18b (3.59 g, 3.16 mL, 28.8 mmol, 2.0 equiv.) was then
added dropwise, and stirring was continued for 4 h. After addition
of saturated aqueous NH4Cl (200 mL) and H2O (100 mL), the mix-
ture was extracted with EtOAc (3ϫ 200 mL). The combined or-
ganic layers were washed with H2O (3ϫ 200 mL) and brine (3ϫ
200 mL), dried (MgSO4) and concentrated in vacuo. The residue
was washed with n-pentane (150 mL) and recrystallised from
EtOAc. Workup of the mother liquor by column chromatography
(SiO2; PE/EtOAc, 10:1) afforded another batch of 19b as a white
solid (7.48 g, 13.8 mmol, total 96%). 1H NMR (300 MHz, CDCl3):
δ = 1.26–1.36, 1.52–1.62 [2ϫ m, total 9 H, C(CH3)3], 1.81–1.87,
1.99–2.10 (2ϫ m, total 3 H, 4Ј-H3), 3.90–4.03, 4.23–4.38 (2ϫ m,
total 1 H, 1Ј-Ha), 4.39–4.55 (m, 1 H, 1Ј-Hb), 5.13–5.37 (m, 2 H,
OCH2Ph), 5.61–5.83 (m, 1 H, 2Ј-H), 6.74–6.96 (m, 1 H, 3-H), 7.37–
7.57 (m, 5 H, 5ϫ Ph-H), 7.69–7.81 (m, 1 H, 7-H), 8.33–8.41 (m, 1
H, 8-H), 8.65–8.76 (m, 1 H, 5-H) ppm. 13C NMR (75 MHz,
CDCl3): δ = 20.84, 26.15 (C-4Ј), 28.11, 28.38 [C(CH3)3], 46.19,
47.48 (C-1Ј), 70.83, 70.90 (OCH2Ph), 80.94 [C(CH3)3], 108.8, 109.4,
109.6, 109.8 (C-3, C-6), 114.5, 118.8, 118.9, (C-4a, CN), 121.2,
122.5 (C-2Ј), 124.9 (C-1), 127.4, 127.7, 128.5, 128.7, 128.8, 128.9
(C-5, C-7, C-8, 2ϫ Ph-Co, 2ϫ Ph-Cm, Ph-Cp), 133.3, 134.2, 135.4,
135.5, 142.1, 142.2 (C-2, C-8a, C-3Ј, Ph-Ci), 153.6, 154.1, 154.6 (C-
Experimental Section
General: All reactions were performed in flame-dried glassware un-
der argon. Commercially available reagents were used without fur-
ther purification. Thin layer chromatography (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 flash chromatography. IR spectra were de-
termined with a Bruker Vektor 22 instrument, UV/Vis spectra with
a Perkin–Elmer Lambda 2, and mass spectra with a Finnigan
MAT 95 for EI HRMS and a Bruker Apex IV Fourier transform
ion cyclotron resonance mass spectrometer for ESI HRMS. 1H
NMR spectra were recorded variously with a Varian UNITY
300 MHz, a Varian Inova 500 MHz or a Varian Inova 600 MHz
instrument. 13C NMR spectra were recorded at 75, 125, or
150 MHz. Spectra were taken at room temperature except when
stated otherwise in deuterated solvents as indicated with use of the
solvent peak as internal standard. The purities and stabilities of
the target compounds were checked by HPLC-MS [ESI mass spec-
trometry with an ion-trap mass spectrometer LCQ (Finnigan)]. The
columns used were Phenomenex Synergi Max-RP C12
(150ϫ2 mm, particle size 4 μm). Chromatographic separation by
HPLC was performed with use either of a Kromasil 100 C18 col-
umn (250ϫ20 mm, particle size 7 μm) or of a Chiralpak IA col-
umn (250ϫ20 mm, particle size 5 μm).
4, C=O) ppm. IR (KBr): ν = 3083, 2967, 2223 (CN), 1702 (C=O),
˜
1617, 1592 cm–1. UV (CH3CN): λmax (lgε) = 218.0 (4.655), 256.5
(4.587), 318.0 (3.758), 335.5 (3.762), 350.5 (3.840) nm. ESI-HRMS:
m/z = 541.0888 [M + H]+; C27H26ClIN2O3 requires 541.0888.
(2R)-2-Amino-4-(benzyloxy)-1-bromo-N-(tert-butoxycarbonyl)-7-cy-
ano-N-(2,3-epoxypropyl)naphthalene [(2R)-21a]: NaH (173 mg,
60% dispersion in mineral oil, 4.4 mmol, 4.0 equiv.) was added to a
solution of 17a (500 mg, 1.10 mmol, 1.0 equiv.) in anhydrous DMF
(5 mL), and the mixture was stirred at room temperature for
30 min. The nosylate (2R)-20a (456 mg, 1.76 mmol, 1.6 equiv.) was
added, and stirring was continued for 14 h. The reaction was
quenched by addition of saturated aqueous NaHCO3 (50 mL) and
brine (50 mL). The reaction mixture was extracted with EtOAc (3ϫ
50 mL), and the combined organic layers were dried (MgSO4) and
concentrated in vacuo. Purification of the residue by column
chromatography (SiO2; PE/EtOAc, 5:1) afforded (2R)-21a as a
bright yellow solid (495 mg, 972 μmol, 88%). 1H NMR (300 MHz,
C2D2Cl4): δ = 1.20–1.72 [m, 9-H, C(CH3)3], 2.38–2.50 (m, 1 H, 4Ј-
Ha) 2.67–2.81 (m, 1 H, 4Ј-Hb), 3.08–3.15 (m, 0.5 H, 2Ј-Ha), 3.15–
3.28 (m, 0.5 H, 3Ј-H), 3.30–3.40 (m, 0.5 H, 2Ј-Ha), 3.51–3.73 (m,
0.5 H, 3Ј-H), 3.88–4.21 (m, 1 H, 2Ј-Hb), 5.22–5.35 (m, 2 H,
OCH2Ph), 6.93–7.13 (m, 1 H, 3-H), 7.34–7.54 (m, 5 H, 5ϫ Ph-H),
7.64–7.72 (m, 1 H, 6-H), 8.40–8.49 (m, 1 H, 5-H), 8.64–8.69 (m,
(E/Z)-2-Amino-4-(benzyloxy)-N-(tert-butoxycarbonyl)-N-(3-chloro-
prop-2-enyl)-7-cyano-1-iodonaphthalene (19a): NaH (399 mg, 60%
dispersion in mineral oil, 10.0 mmol, 2.5 equiv.) was added at room
temperature to a solution of 17c (2.00 g, 4.00 mmol, 1.0 equiv.) in
anhydrous DMF (50 mL), and the mixture was stirred for 1.5 h.
Compound 18a (986 mg, 823 μL, 8.0 mmol, 2.0 equiv.) was then
added dropwise, and stirring was continued for 14 h. After addition
of saturated aqueous NH4Cl (pH = 7) the mixture was extracted
with EtOAc (3 ϫ 100 mL). The combined organic layers were
washed with H2O (3 ϫ 100 mL) and brine (3 ϫ 100 mL), dried
(MgSO4) and concentrated in vacuo. Purification of the residue by
column chromatography (SiO2; PE/EtOAc, 8:1) afforded 19a as a
yellow solid (1.76 g, 3.06 mmol, 77 %). 1H NMR (300 MHz,
CDCl3): δ = 1.22–1.35, 1.49–1.60 [2ϫ m, total 9 H, C(CH3)3], 3.69–
3.83, 4.17–4.33 (2ϫ m, total 1 H, 1Ј-Ha), 4.42–4.61 (m, 1 H, 1Ј-
Hb), 5.10–5.34 (m, 2 H, OCH2Ph), 5.90–3.12 (m, 1 H, 2Ј-H), 6.74–
6.92 (m, 1 H, 3-H), 7.35–7.69 (m, 5 H, 5ϫ Ph-H), 7.58–7.69 (m, 1
H, 6-H), 8.32–8.44 (m, 1 H, 5-H), 8.57–8.64 (m, 1 H, 8-H) ppm.
13C NMR (150 MHz, CDCl3): δ = 28.12, 28.14 [C(CH3)3], 46.11,
49.19 (C-1Ј), 71.01, 71.10 (OCH2Ph), 81.01, 81.07 [C(CH3)3], 94.57,
94.66 (C-1), 110.41, 110.82 (C-3), 112.19, 112.26 (C-7), 118.31,
1 H, 8-H) ppm. IR (KBr): ν = 2977, 2228, 1703, 1596, 1503, 1370,
˜
1257, 1153 cm–1. UV (CH3CN): λmax (lgε) = 215.5 (4.618), 256.5
(4.596), 316.0 (3.815), 352.0 (3.580) nm. ESI-HRMS: m/z =
509.10705 [M + H]+; C26H25BrN2O4 requires 509.10759.
118.35 (CN), 120.57, 121.72 (C-3Ј), 124.03, 124.07 (C-5), 126.7, (+)-(2R,3R)-2-Amino-4-(benzyloxy)-1-bromo-N-(tert-butoxycarb-
126.8, 127.0, 127.1, 127.2, 128.2, 128.5, 128.6, 129.5, 134.8, 134.9, onyl)-6-cyano-N-(2,3-epoxybutyl)naphthalene [(+)–(2R,3R)-21b]:
135.6, 135.7, 144.8, 145.1 (C-4a, C-8a, C-2, C-2Ј, 6ϫ Ph-C), 138.3, NaH (397 mg, 60 % dispersion in mineral oil, 16.6 mmol,
138.4 (C-8), 152.9, 153.1, 155.2 (C-4, C=O) ppm. IR (KBr): ν =
4.0 equiv.) was added to a solution of 17c (1.88 g, 4.14 mmol,
1.0 equiv.) in anhydrous DMF (30 mL), and the mixture was stirred
˜
2976, 2228, 1703, 1593, 1503, 1410, 1368, 1329, 1257, 1163 cm–1.
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at 60 °C for 60 min. After the system had cooled to room tempera-
ture, the nosylate (2R,3R)-20b (1.70 g, 6.21 mmol, 1.5 equiv.) was
added, and stirring was continued for 2 h. The reaction was
quenched by addition of saturated aqueous NH4Cl (100 mL). The
reaction mixture was extracted with Et2O (3ϫ 100 mL), and the
combined organic layers were dried (Na2SO4) and concentrated in
vacuo. Purification of the residue by column chromatography
(SiO2; PE/EtOAc, 9:1) afforded (+)–(2R,3R)-21b as a white solid
ane/iPrOH, 97:3; flow: 0.8 mLmin–1): (+)-(1R)-22a: 99.7% ee (tR
=
8.4 min), [α]2D0 = +9.5 (c = 0.5 in CHCl3); (–)-(1S)-22a: 99.2% ee
(tR = 6.4 min), [α]2D0 = –11.0 (c = 0.5 in CHCl3).
rac-(1S)-5-(Benzyloxy)-3-(tert-butoxycarbonyl)-1-[(1R)-1-chloroeth-
yl]-7-cyano-1,2-dihydro-3H-benz[e]indole (rac-22b) and rac-(1S)-5-
(Benzyloxy)-3-(tert-butoxycarbonyl)-1-[(1S)-1-chloroethyl]-7-cyano-
1,2-dihydro-3H-benz[e]indole (rac-22c): A solution of 19b (3.20 g,
5.91 mmol, 1.0 equiv.) in anhydrous toluene (150 mL) was flushed
with argon for 10 min. Tris(trimethylsilyl)silane (2.00 mL,
6.48 mmol, 1.1 equiv.) and AIBN (242 mg, 1.47 mmol, 0.25 equiv.)
were added at room temperature, and the solution was stirred at
80 °C with use of a preheated oil bath for 4.5 h. After the system
had cooled to room temperature, the brown solid was washed with
n-pentane. The resulting crude solid was purified by column
chromatography (PE/EtOAc, 30:1 Ǟ 10:1) to afford the syn dia-
stereomer rac-22c as a white solid (560 mg, 1.21 mmol, 20%), a
mixture of the syn diastereomer rac-22c together with the dehalo-
genised cyclisation product as a yellow solid (380 mg) and the de-
sired anti diastereomer rac-22b as a white solid (1.30 g, 2.81 mmol,
48%).
1
(1.93 g, 3.69 mmol, 89%). H NMR (300 MHz, CDCl3): δ = 1.25–
1.41, 1.59–1.68 [2ϫ m, total 12 H, C(CH3)3, 4Ј-H3], 2.60–2.62,
2.76–2.91, 3.04–3.22, 3.27–3.36, 3.52–3.61, 3.71–4.21, 4.30–4.35
(7ϫ m, total 4 H, 1Ј-H2, 2Ј-H, 3Ј-H), 5.20–5.32 (m, 2 H, OCH2Ph),
6.93–7.10 (m, 1 H, 3-H), 7.37–7.54 (m, 5 H, 5ϫ Ph-H), 7.69–7.76
(m, 1 H, 7-H), 8.18–8.21, 8.33–8.38 (2ϫ m, total 1 H, 8-H), 8.66–
8.72 (m, 1 H, 5-H) ppm. 13C NMR (125 MHz, CDCl3): δ = 17.2
(2 signals, C-4Ј), 28.0, 28.1, 28.4 (2 signals, C(CH3)3], 50.5, 52.1,
52.4, 53.0, 53.4, 53.6, 53.7, 54.2, 56.5, 56.8, 57.1, 57.2 (C-1Ј, C-2Ј,
C-3Ј), 68.9, 70.9 (OCH2Ph), 81.0, 81.1, 81.7 (2 signals, C(CH3)3],
109.0, 109.1, 109.3, 109.6, 109.7 (C-3, C-6), 114.0, 114.5, 114.6,
118.9, (2 signals, C-4a, CN), 125.0, 125.1, 125.9 (C-1), 127.5, 127.6,
127.8, 127.9, 128.5 (2 signals), 128.7 (2 signals), 128.8 (2 signals),
128.9 (2 signals, C-5, C-7, C-8, 2ϫ Ph-Co, 2ϫ Ph-Cm, Ph-Cp), 134.1
(2 signals), 134.3, 134.4, 135.4, 135.5, 142.1, 142.2, 142.6 (C-2, C-
8a, Ph-Ci), 153.5, 153.6, 153.7, 154.2 (2 signals), 154.7, 163.5 (C-
1
rac-22c: H NMR (300 MHz, C2D2Cl4, 100 °C): δ = 1.60–1.67 [m,
12 H, 11-H3, C(CH3)3], 3.87 (mc, 1 H, 1-H), 4.13 (dd, J = 11.8,
9.5 Hz, 1 H, 2-Ha), 4.37 (dd, J = 11.8, 2.8 Hz, 1 H, 2-Hb), 4.55
(dq, J = 6.8, 3.7 Hz, 1 H, 10-H), 5.33 (mc, 2 H, OCH2Ph), 7.37–
7.57 (m, 5 H, 5ϫ Ph-H), 7.60 (dd, J = 8.7, 1.6 Hz, 1 H, 8-H), 7.72
(d, J = 8.7 Hz, 1 H, 9-H), 7.87 (br. s, 1 H, 4-H), 8.66 (d, J = 1.6 Hz,
1 H, 6-H) ppm. 13C NMR (75 MHz, C2D2Cl4, 100 °C): δ = 23.04
(C-11), 28.24 [C(CH3)3], 45.54 (C-1), 50.95 (C-2), 59.68 (C-10),
70.79 (OCH2Ph), 81.56 [C(CH3)3], 98.24 (C-4), 105.5 (C-7), 115.5
(C-9b), 119.4, 121.4 (C-5a, CN), 123.0 (C-9), 127.4, 127.6, 128.1,
128.5 (C-8, 2ϫ Ph-Co, 2ϫ Ph-Cm, Ph-Cp), 129.8 (C-6), 131.7 (C-
9a), 135.9 (Ph-Ci), 145.1 (C-3a), 151.7 (C=O), 156.1 (C-5) ppm. IR
4,C=O) ppm. IR (film): ν = 2977, 2228, 1706, 1620, 1591, 1455,
˜
1411, 1369, 1342, 1254, 1151, 1095, 975, 910, 872, 738, 697 cm–1.
UV (CH3CN): λmax (lg ε) = 218.0 (4.630), 256.5 (4.557), 317.0
(3.812), 334.5 (3.765), 350.5 (3.814) nm. ESI-HRMS: m/z =
545.1046 [M + Na]+; C27H27BrN2O4 requires 545.1046.
rac-(1S)-5-(Benzyloxy)-3-(tert-butoxycarbonyl)-1-(chloromethyl)-8-
cyano-1,2-dihydro-3H-benz[e]indole (rac-22a): A solution of 19a
(1.42 g, 2.47 mmol, 1.0 equiv.) in anhydrous toluene (60 mL) was
flushed with argon for 10 min. Tris(trimethylsilyl)silane (836 μL,
2.72 mmol, 1.1 equiv.) and AIBN (102 mg, 618 μmol, 0.25 equiv.)
were added at room temperature, and the solution was stirred at
80 °C with use of a preheated oil bath for 4.5 h. After addition of
SiO2 (3 g), the mixture was concentrated in vacuo, and the residue
was purified by column chromatography (n-hexane/DCM, 9:1) to
afford rac-22a as a white solid (940 mg, 2.10 mmol, 85 %). 1H
NMR (300 MHz, C2D2Cl4): δ = 1.64 [s, 9 H, C(CH3)3], 3.59 (dd,
J = 9.1, 11.2 Hz, 1 H, 2-Ha), 3.89 (dd, J = 3.51, 11.2 Hz, 1 H, 2-
Hb), 4.00 (tt, J = 3.2, 8.8 Hz, 1 H, 1-H), 4.21 (dd, J = 8.8, 11.7 Hz,
1 H, 10-Ha), 4.30 (dd, J = 3.2, 11.8 Hz, 1 H, 10-Hb), 5.32 (s, 2 H,
OCH2Ph), 7.36–7.57 (m, 6 H, 7-H, 5ϫ Ph-H), 7.93 (s, 1 H, 4-H),
8.02 (dd, J = 0.7, 1.5 Hz, 1 H, 9-H), 8.38 (dd, J = 0.5, 8.7 Hz, 1
H, 6-H) ppm. 13C NMR (100 MHz, C2D2Cl4): δ = 28.26 [C(CH3)
3], 41.01 (C-1), 46.37 (C-2), 53.15 (C-10), 70.84 (OCH2Ph), 81.57
[C(CH3)3], 99.71 (C-4), 111.1 (C-8), 114.9 (C-9b), 118.9 (CN), 123.2
(C-7), 123.6 (C-5a), 124.9 (C-6), 127.4 (C-9), 127.4 (2ϫ Ph-Co),
128.0 (Ph-Cp), 128.4 (2 ϫ Ph-Cm), 129.4 (C-9a), 136.1 (Ph-Ci),
(KBr): ν = 2976, 2223 (CN), 1705 (C=O), 1622, 1577, 1478,
˜
1456 cm–1. UV (CH3CN): λmax (lgε) = 214.0 (4.452), 270.5 (4.829),
353.0 (4.198) nm. ESI-HRMS: m/z = 463.1783 [M + H]+;
C26H25ClN2O3 requires 463.1783.
Chromatographic Resolution of rac-22b: A solution of rac-22b
(1.0 g, 2.2 mmol) in a mixture of n-heptane/CH2Cl2 (1:1, 25 mL)
was separated (injection volume: 1.0 mL) by semipreparative
HPLC (Chiralpak IA; 250ϫ20 mm; particle size: 5 μm; n-heptane/
CH2Cl2, 80:20; flow: 18 mLmin–1; UV detector: λ = 250 nm) to
provide (+)-(1S,10R)-22b (tR = 8.2 min) and (–)-(1R,10S)-22b (tR
=
10.8 min). The optical purity was determined by analytical HPLC
(Chiralcel OD; 250 ϫ4.6 mm; particle size: 10 μm; n-hexane/iP-
rOH, 98:2; flow: 0.8 mLmin–1): (+)-(1S,10R)-22b: 99.9% ee (tR
=
37.6 min), [α]2D0 = +24.6 (c = 0.5 in CHCl3); (–)-(1R,10S)-22b:
98.7% ee (tR = 33.9 min), [α]2D0 = –24.4 (c = 0.5 in CHCl3).
(1S)-5-(Benzyloxy)-3-(tert-butoxycarbonyl)-8-cyano-1-(hydroxy-
methyl)-1,2-dihydro-3H-benz[e]indole [(1S)-23a]: A solution of n-
butyllithium in n-hexane (2.5 m, 245 μL, 612 μmol, 3.0 equiv.) was
added slowly at –90 °C to a stirred solution of (2R)-21a (104.0 mg,
20.4 mmol, 1.0 equiv.) in anhydrous THF (10 mL), and stirring was
continued at –78 °C for 1 h. The reaction was quenched by addition
of saturated aqueous NH4Cl (50 mL), and the mixture was ex-
tracted with EtOAc (2ϫ 100 mL). The combined organic layers
were washed with brine (300 mL), dried (MgSO4) and concentrated
in vacuo. Purification of the crude solid by column chromatog-
raphy (SiO2; PE/EtOAc, 4:1) afforded (1S)-23a as a bright brown
143.4 (C-3a), 151.9 (C=O), 155.9 (C-5) ppm. IR (KBr): ν = 2977,
˜
2230, 1693, 1593, 1455, 1409, 1374, 1336, 1257, 1146 cm–1. UV
(CH3CN): λmax (lgε) = 210.0 (4.391), 230.0 (4.469), 268.0 (4.786),
311.5 (3.906), 322.5 (3.977), 381.0 (3.475) nm. ESI-HRMS: m/z =
471.14459 [M + Na]+; C26H25ClN2O3 requires 471.14459.
Chromatographic Resolution of rac-22a: A solution of rac-22a
(500 mg, 1.1 mmol) in a mixture of n-hexane and CH2Cl2 (1:1,
10 mL) was separated (injection volume: 0.15 mL) by semiprepar-
ative HPLC (Chiralpak IA; 250ϫ20 mm; particle size: 5 μm; n-
hexane/CH2Cl2, 85:15; flow: 18 mL min–1; UV detector: λ =
269 nm) to provide (+)-(1R)-22a (tR = 12.5 min) and (–)-(1S)-22a
(tR = 10.8 min). The optical purity was determined by analytical
HPLC (Chiralcel OD; 250ϫ 4.6 mm; particle size: 10 μm; n-hex-
1
solid (10.1 mg, 2.35 mmol, 12%). H NMR (300 MHz, CDCl3): δ
= 1.58 [s, 9 H, C(CH3)3], 3.66–4.26 (m, 5 H, 1-H, 2-H, 10-H), 5.23
(s, 2 H, OCH2Ph), 7.31–7.55 (m, 6 H, 7-H, 5ϫ Ph-H), 7.94–8.05
(m, 1 H, 4-H), 8.05–8.08 (m, 1 H, 9-H), 8.26–8.35 (m, 1 H, 6-
6916
www.eurjoc.org
© 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2010, 6909–6921

Fluorescence-Labelled Glycosidic Prodrugs Based on Duocarmycin
H) ppm. 13C NMR (150 MHz, CDCl3): δ = 28.38 [C(CH3)3], 41.13 EtOAc (800 mL), and the filtrate was concentrated in vacuo. Purifi-
(C-1), 52.48 (C-2), 64.79 (C-10), 70.54 (OCH2Ph), 81.60 [C(CH3) cation of the residue by column chromatography (SiO2; PE/EtOAc,
3], 99.10 (C-4), 110.6 (C-8), 115.0 (C-9b), 119.4 (CN), 123.2 (C-7),
123.5 (C-5a), 124.8 (C-6), 127.4 (C-9), 127.7 (2ϫ Ph-Co), 128.2 90%). H NMR (300 MHz, [D6]DMSO): δ = 1.54 [s, 9 H, C(CH3)
(Ph-Cp), 128.6 (2ϫ Ph-Cm), 129.7 (C-9a), 136.2 (Ph-Ci), 143.7 (C- 3], 3.77–4.31 (m, 5 H, 1-H, 2-H2, 10-H2), 7.50 (dd, J = 1.5, 8.7 Hz,
3a), 152.5 (C=O), 155.4 (C-5) ppm. IR (KBr): ν = 2974, 2227, 1701, 1 H, 7-H), 7.72 (br. s, 1 H, 4-H), 8.18 (d, J = 8.7 Hz, 1 H, 6-H),
5:1) afforded (–)-(1S)-24a as a yellow solid (179 mg, 499 μmol,
1
˜
1623, 1291, 1455, 1369, 1257, 1143, 1092, 1031 cm–1. UV (CH3CN):
8.41 (dd, J = 0.5, 1.5 Hz, 1 H, 9-H), 10.88 (br. s, 1 H, OH) ppm.
λmax (lg ε) = 209.0 (4.393), 229.0 (4.435), 267.5 (4.683), 313.0 13C NMR (75 MHz, [D6]DMSO): δ = 27.98 [C(CH3)3], 47.86 (C-
(3.848), 323.5 (3.878), 375.0 (3.405) nm. ESI-HRMS: m/z = 1), 52.50 (C-2), 61.92 (C-10), 80.8 [C(CH3)3], 101.0 (C-4), 109.7 (C-
431.19653 [M + H]+; C26H26N2O4 requires 431.19653.
8), 114.1 (C-9b), 119.2 (CN), 122.0 (C-5a), 122.5 (C-7), 124.5 (C-
6), 128.8 (C-9), 129.2 (C-9a), 143.2 (C-3a), 151.5 (C=O), 154.6 (C-
(+)-(1S)-5-(Benzyloxy)-3-(tert-butoxycarbonyl)-7-cyano-1-[(1R)-1-
hydroxyethyl]-1,2-dihydro-3H-benz[e]indole [(+)-(1S,10R)-23b]:
Freshly fused ZnCl2 (544 mg, 3.99 mmol, 1.5 equiv.) was suspended
in anhydrous THF (50 mL), and the mixture was cooled to 0 °C.
A solution of methyllithium in Et2O (1.6 m, 9.98 mL, 16.0 mmol,
6.0 equiv.) was slowly added to the stirred mixture, and stirring was
continued at 0 °C for 30 min. The mixture was cooled to –78 °C,
and TMS-NCS (563 μL, 524 mg, 3.99 mmol, 1.5 equiv.) was added
dropwise. The mixture was warmed to 0 °C and stirred for 30 min.
After the mixture had again been cooled to –78 °C, a solution of
5) ppm. IR (KBr): ν = 3350, 2230, 1676, 1626, 1595, 1513, 1422,
˜
1369, 1223, 1144 cm–1. UV (CH3CN): λmax (lgε) = 210.5 (4.221),
229.5 (4.369), 269.0 (4.787), 312.5 (3.826), 323.5 (3.861), 381.0
(3.428) nm. ESI-HRMS: m/z = 381.09764 [M + Na]+
;
C19H19ClN2O3 requires 381.09764.
Chromatographic Resolution of (–)-(1S)-24a: A solution of (–)-(1S)-
24a (250 mg, 697 μmol) in a mixture of n-hexane/iPrOH (1:1,
10 mL) was separated consecutively (injection volume: 0.5 mL) by
semipreparative HPLC (Chiralpak IA; 250ϫ20 mm; particle size:
(+)-(2ЈR,3ЈR)-21b (1.39 g, 2.66 mmol, 1.0 equiv.) in anhydrous 5 μm; n-hexane/iPrOH, 98:2; flow: 18 mLmin–1; UV detector: λ =
THF (25 mL) was added dropwise, and the resulting mixture was
250 nm) to provide (–)-(1S)-24a (tR = 19.4 min), [α]2D0 = –20.0 (c =
stirred at –78 °C for 30 min. After warming to 0 °C, the mixture 0.5 in CHCl3).
was stirred for 1 h, and, after warming to room temperature, it was
(+)-(1S)-3-(tert-Butoxycarbonyl)-1-[(1R)-1-chloroethyl]-7-cyano-5-
stirred for 2.5 h. Saturated aqueous NH4Cl (200 mL) was slowly
added, and the mixture was extracted with CH2Cl2 (4ϫ 200 mL).
The combined organic layers were dried (Na2SO4) and concen-
trated in vacuo. Purification of the residue by column chromatog-
raphy (SiO2; PE/EtOAc, 5:2) afforded (+)-(1S,10R)-23b as a white
solid (840 mg, 1.89 mmol, 71%, 98.8% ee). 1H NMR (300 MHz,
C2D2Cl4, 100 °C): δ = 1.27 (d, J = 6.4 Hz, 3 H, 10-CH3), 1.63 [br.
s, 9 H, C(CH3)3], 3.71 (dt, J = 9.9, 3.1 Hz, 1 H, 1-H), 4.09 (dd, J
= 11.6, 9.9 Hz, 1 H, 2-Ha), 4.26 (dd, J = 11.6, 3.4 Hz, 1 H, 2-Hb),
4.33 (mc, 1 H, 10-H), 5.32 (s, 2 H, OCH2Ph), 7.38–7.54 (m, 5 H,
5ϫ Ph-H), 7.58 (dd, J = 8.8, 1.7 Hz, 1 H, 8-H), 7.83 (d, J = 8.8 Hz,
1 H, 9-H), 7.89 (br. s, 1 H, 4-H), 8.64 (d, J = 1.6 Hz, 1 H, 6-
H) ppm. 13C NMR (150 MHz, C2D2Cl4, 100 °C): δ = 20.2 (C-11),
28.3 [C(CH3)3], 45.0 (C-1), 49.9 (C-2), 68.7 (C-10), 70.8 (OCH2Ph),
81.4 [C(CH3)3], 98.3 (C-4), 105.5 (C-7), 115.7 (C-9b), 119.5, 121.4
(C-5a, CN), 123.7 (C-9), 127.4 (2 signals), 128.1, 128.5 (C-8, 5ϫ
Ph-CH), 129.7 (C-6), 132.0 (C-9a), 136.0 (Ph-Ci), 145.2 (C-3a),
hydroxy-1,2-dihydro-3H-benz[e]indole [(+)-(1S,10R)-24b]: A solu-
tion of (+)-(1S,10R)-22b (130 mg, 281 μmol, 1.0 equiv.) in anhy-
drous THF (9 mL) was warmed to 40 °C and Pd/C (10%, 30 mg,
28 μmol, 0.1 equiv. based on Pd) was added. A solution of
NH4CHO2 in H2O (25%, 0.30 mL) was added dropwise, and the
mixture was stirred at 40 °C for 25 min. The mixture was filtered
through Celite, the filter cake was washed with acetone (300 mL),
and the filtrate was concentrated in vacuo. Purification of the resi-
due by column chromatography (SiO2; PE/EtOAc, 10:1Ǟ5:1Ǟ3:1)
afforded (+)-(1S,10R)-24b as a white solid (92 mg, 247 μmol, 88%).
[α]2D0 = +22.8 (c = 0.5 in CHCl3). 1H NMR (300 MHz, [D6]DMSO):
δ = 1.54 [s, 9 H, C(CH3)3], 1.58 (d, J = 6.8 Hz, 3 H, 11-H3), 3.98–
4.16 (m, 3 H, 1-H, 2-H2), 4.71 (mc, 1 H, 10-H), 7.65 (mc, 2 H, 4-
H, 8-H), 7.93 (d, J = 8.8 Hz, 1 H, 9-H), 8.46 (d, J = 1.2 Hz, 1 H, 6-
H), 10.89 (br. s, 1 H, OH) ppm. 13C NMR (75 MHz, [D6]DMSO): δ
= 22.92 (C-11), 27.96 [C(CH3)3], 44.07 (C-1), 49.46 (C-2), 61.24 (C-
10), 80.93 [C(CH3)3], 99.61 (C-4), 103.8 (C-7), 114.7 (C-9b), 119.6,
119.9 (C-5a, CN), 123.9 (C-9), 127.2 (C-8), 129.5 (C-6), 131.3 (C-
9a), 144.7 (C-3a), 151.3 (C=O), 155.1 (C-5) ppm. UV (CH3CN):
λmax (lg ε) = 214.5 (4.347), 271.5 (4.820), 355.5 (4.148) nm. IR
151.8 (C=O), 155.9 (C-5) ppm. IR (KBr): ν = 2973, 2222, 1702,
˜
1620, 1577, 1479, 1455, 1413, 1373, 1325, 1255, 1144, 909, 822,
736, 696 cm–1. UV (CH3CN): λmax (lg ε) = 215.0 (4.462), 271.5
(4.835), 355.5 (4.211) nm. ESI-HRMS: m/z = 445.2121 [M + H]+;
C27H28N2O4 requires 445.2122.
(KBr): ν = 3361 (C=O), 2978, 2221 (CN), 1687 (C=O), 1623,
˜
1577 cm–1. ESI-HRMS: m/z = 373.1314 [M + H]+; C20H21ClN2O3
(+)-(1S)-5-(Benzyloxy)-3-(tert-butoxycarbonyl)-1-[(1R)-1-chloroeth-
yl]-7-cyano-1,2-dihydro-3H-benz[e]indole [(+)-(1S,10R)-22b]: SOCl2
(206 μL, 337 mg, 2.83 mmol, 10.0 equiv.) and pyridine (572 μL,
560 mg, 7.09 mmol, 25.0 equiv.) were added at 0 °C to a solution
of (+)-(1S,10R)-23b (126 mg, 283 μmol, 1.0 equiv.) in anhydrous
CH2Cl2 (6 mL). The reaction mixture was stirred at room tempera-
ture for 4 h. After concentration, the crude product was purified
by column chromatography (PE/EtOAc, 5:1) to afford (+)-
(1S,10R)-22b as a white solid (57.8 mg, 164 μmol, 58%, 99.0% ee).
requires 373.1314.
(1S)-1-(Chloromethyl)-8-cyano-3-({5-[2-(dimethylamino)ethoxy]indol-
2-yl}carbonyl)-1,2-dihydro-3H-benz[e]indol-5-yl 2,3,4,6-Tetra-O-
acetyl-β-D-galactopyranoside [(+)-(1S)-27a]: A mixture of the phe-
nol (–)-(1S)-24a (283 mg, 663 μmol, 1.00 equiv.) in dry CH2Cl2
(20 mL) and freshly activated molecular sieves (4 Å, 2.00 g) was
stirred at room temperature for 30 min. After addition of the tri-
chloroacetimidate 25 (427 mg, 870 μmol, 1.3 equiv.) and cooling to
–10 °C, the promoter BF3·OEt2 (50 μL) in dry CH2Cl2 (4 mL) was
(–)-(1S)-3-(tert-Butoxycarbonyl)-1-(chloromethyl)-8-cyano-5-hydroxy- added dropwise, and the mixture was stirred for 3.5 h. Additional
1,2-dihydro-3H-benz[e]indole [(–)-(1S)-24a]: Pd/C (10 %, 140 mg, BF3·OEt2 (250 μL) in CH2Cl2 (4 mL) was then added, and the mix-
129 μmol, 0.1 equiv. based on Pd) was added to a solution of (–)-
(1S)-22a (250 mg, 557 μmol, 1.0 equiv.) in anhydrous EtOAc
(17 mL). The mixture was flushed with argon and H2, and the mix-
ture was stirred at room temperature under H2 for 5 h. The reaction
mixture was filtered through Celite, the filter cake was washed with
ture was allowed to warm to room temperature and stirred for 6 h.
The reaction mixture was transferred by cannula into a new flask
for separation of the molecular sieves, which were washed with
CH2Cl2 (30 mL). The obtained organic solution was concentrated
in vacuo, and the residue was dissolved in DMF (38 mL) and co-
Eur. J. Org. Chem. 2010, 6909–6921
© 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
6917

L. F. Tietze et al.
FULL PAPER
oled to 0 °C. EDC·HCl (450 mg, 2.36 mmol, 3.5 equiv.) and
6 H, NMe2), 2.81 (t, J = 5.6 Hz, 2 H, 2ЈЈ-H2), 4.02–4.07 (m, 2 H,
DMAI·HCl (26, 336 mg, 1.35 mmol, 2.0 equiv.) were added, and 1-H, 5ЈЈЈ-H), 4.11–4.23 (m, 4 H, 1ЈЈ-H2, 6ЈЈЈ-H2), 4.52 (mc, 1 H,
the reaction mixture was stirred at room temperature for 18 h. Af-
ter dilution with EtOAc (70 mL), H2O (70 mL) and saturated aque-
ous NaHCO3 (70 mL) were added, and the aqueous phase was ex-
tracted with EtOAc (4ϫ 150 mL). The combined organic layers
were washed with brine (4ϫ 200 mL), dried (MgSO4) and concen-
trated in vacuo. Purification of the residue by column chromatog-
10-H), 4.63 (mc, 1 H, 2-Ha), 4.86 (dd, J = 10.6, 2.1 Hz, 1 H, 2-Hb),
5.10 (dd, J = 10.5, 3.5 Hz, 1 H, 3ЈЈЈ-H), 5.30 (d, J = 8.0 Hz, 1 H,
1ЈЈЈ-H), 5.44 (mc, 1 H, 4ЈЈЈ-H), 5.66 (dd, J = 10.5, 8.0 Hz, 1 H,
2ЈЈЈ-H), 7.01–7.06 (m, 2 H, 3Ј-H, 6Ј-H), 7.13 (d, J = 2.3 Hz, 1 H,
4Ј-H), 7.36 (d, J = 8.9 Hz, 1 H, 7Ј-H), 7.62 (dd, J = 8.7, 1.5 Hz, 1
H, 8-H), 7.75 (d, J = 8.7 Hz, 1 H, 9-H), 8.43 (br. s, 1 H, 4-H), 8.51
raphy (SiO2; CH2Cl2/MeOH, 6:1) afforded (+)-(1S)-27a as a white (d, J = 1.5 Hz, 1 H, 6-H), 9.62 (br. s, 1 H, NH) ppm. 13C NMR
solid (340 mg, 416 μmol, 53%). [α]2D0 = +6.0 (c = 0.3 in CHCl3). 1H
(150 MHz, CDCl3): δ = 20.6 (3 signals), 20.7 [4ϫ C(O)CH3], 23.69
NMR (600 MHz, CDCl3): δ = 2.01 [s, 6 H, 2ϫ C(O)CH3], 2.03 [s, (C-11), 45.74 (NMe2), 47.15 (C-1), 53.06 (C-2), 58.25 (C-2ЈЈ), 59.32
3 H, C(O)CH3], 2.16 [s, 3 H, C(O)CH3], 2.37 (s, 6 H, NMe2), 2.79
(t, J = 5.7 Hz, 2 H, 2ЈЈ-H2), 3.55 (dd, J = 11.5, 10.0 Hz, 1 H, 10-
Ha), 3.78–3.85 (m, 1 H, 1-H), 3.90 (dd, J = 11.5, 3.2 Hz, 1 H, 10-
Hb), 4.05–4.23 (m, 5 H, 1ЈЈ-H2, 5ЈЈЈ-H, 6ЈЈЈ-H2), 4.69–4.75 (m, 1 H,
2-Ha), 4.84 (dd, J = 10.6, 2.0 Hz, 1 H, 2-Hb), 4.99 (dd, J = 10.5,
3.2 Hz, 1 H, 3ЈЈЈ-H), 5.27 (d, J = 7.9 Hz, 1 H, 1ЈЈЈ-H), 5.38 (d, J =
3.3 Hz, 1 H, 4ЈЈЈ-H), 5.63 (dd, J = 10.5, 7.9 Hz, 1 H, 2ЈЈЈ-H), 7.03–
(C-10), 61.09 (C-6ЈЈЈ), 66.26 (C-1ЈЈ), 66.74 (C-4ЈЈЈ), 68.46 (C-2ЈЈЈ),
70.58 (C-3ЈЈЈ), 71.19 (C-5ЈЈЈ), 99.44 (C-1ЈЈЈ), 103.3, 103.5 (C-4, C-
4Ј), 106.3 (C-3Ј), 107.4 (C-7), 112.8 (C-7Ј), 117.7 (C-6Ј), 119.2,
119.4, 122.6 (C-5a, C-9b, CN), 123.6 (C-9), 128.1, 128.2 (C-8, C-
3aЈ), 129.8, 130.1, 131.1, 131.6 (C-6, C-9a, C-2Ј, C-7aЈ), 145.3 (C-
3a), 153.7, 153.9 (C-5, C-5Ј), 160.7 (C=O), 169.4, 170.0, 170.2,
170.5 [4ϫ C(O)CH ] ppm. IR (KBr): ν = 3406 (NH), 2940, 2226
˜
3
7.06 (m, 2 H, 3Ј-H, 6Ј-H), 7.14 (d, J = 2.2 Hz, 1 H, 4Ј-H), 7.37 (d, (CN), 1752 (C=O), 1624, 1521, 1457 cm–1. UV (CH3CN): λmax (lgε)
J = 8.9 Hz, 1 H, 7Ј-H), 7.53 (dd, J = 8.7, 1.5 Hz, 1 H, 7-H), 8.08 = 218.0 (4.656), 266.5 (4.415), 309.0 (4.487), 359.0 (4.540) nm. ESI-
(d, J = 0.8 Hz, 1 H, 9-H), 8.21 (d, J = 8.7 Hz, 1 H, 6-H), 8.51 (s, HRMS: m/z = 833.2795 [M + H]+; C42H45ClN4O12 requires
1 H, 4-H), 10.05 (s, 1 H, NH) ppm. 13C NMR (125 MHz, CDCl3):
δ = 20.63, 20.69, 20.72, 20.84 [4ϫ C(O)CH3], 45.84 (NMe2), 45.96
(C-10), 54.99 (C-2), 58.35 (C-2ЈЈ), 61.16 (C-6ЈЈЈ), 66.33 (C-1ЈЈ),
66.78 (C-4ЈЈЈ), 68.43 (C-2ЈЈЈ), 70.51 (C-3ЈЈЈ), 71.20 (C-1), 99.02 (C-
1ЈЈЈ), 103.52 (C-4Ј), 103.9 (C-4), 106.4 (C-6Ј*), 111.6 (C-5a), 112.7
(C-7Ј), 117.8 (C-3Ј*), 118.6 (C-8), 118.8, 124.4 (C-5a, C-9b), 124.8
(C-6), 125.0 (C-7), 128.0 (C-9), 128.2, 128.7, 130.0, 131.5 (CN, C-
2Ј, C-3aЈ, C-7aЈ), 143.7 (C-3a), 153.3 (C-5), 153.9 (C-5Ј), 160.8
(C=O), 169.5, 169.7, 170.0, 170.3 [4ϫ C(O)CH3] ppm. IR (KBr):
833.2796.
(+)-(1S)-8-(Aminomethyl)-1-(chloromethyl)-3-({5-[2-(dimethylamino)-
ethoxy]indol-2-yl}carbonyl)-1,2-dihydro-3H-benz[e]indol-5-yl
2,3,4,6-Tetra-O-acetyl-β-D-galactopyranoside [(+)-(1S)-28a]: A
solution of (+)-(1S)-27a (50 mg, 61 μmol, 1.0 equiv.) in anhydrous
EtOH (3 mL) was flushed with argon, and a solution of HCl in
EtOH (1.25 m, 98 μL, 122 μmol, 2.0 equiv.), H2O (0.15 mL) and
PtO2·H2O (3.0 mg, 12.2 μmol, 0.2 equiv.) were added. The mixture
was stirred at room temperature under H2 for 48 h and then filtered
through Celite, with washing with MeOH (200 mL), and concen-
trated in vacuo. Purification of the residue by column chromatog-
raphy (SiO2; CH2Cl2/MeOH, 5:1) afforded (+)-(1S)-28a as a white
ν = 3406 (NH), 2942, 2229 (CN), 1754 (C=O), 1626, 1521, 1457,
˜
1396, 1226 cm–1. UV (CH3CN): λmax (lgε) = 212.5 (4.712), 261.5
(4.431), 302.0 (4.546), 399.0 (4.519) nm. ESI-HRMS: m/z =
819.2651 [M + H]+; C41H43ClN4O12 requires 819.2639.
1
solid (33 mg, 40 μmol, 66%). [α]2D0 = +2.0 (c = 0.3 in DMSO). H
NMR (600 MHz, [D6]DMSO): δ = 1.98 [s, 3 H, C(O)CH3], 2.01 [s,
3 H, C(O)CH3], 2.04 [s, 3 H, C(O)CH3], 2.18 [s, 3 H, C(O)CH3],
2.24 (s, 6 H, NMe2), 2.65 (t, J = 5.8 Hz, 2 H, 2ЈЈ-H2), 3.89–3.97
(m, 3 H, CH2NH2, 10-Ha), 4.07 (t, J = 5.9 Hz, 2 H, 1ЈЈ-H2), 4.14
(m, 3 H, 6ЈЈЈ-H2, 10-Hb), 4.26 (m, 1 H, 1-H), 4.49 (m, 1 H, 5ЈЈЈ-
H), 4.57–4.62 (m, 1 H, 2-Ha), 4.83 (m, 1 H, 2-Hb), 5.38–5.46 (m, 3
H, 2ЈЈЈ-H, 3ЈЈЈ-H, 4ЈЈЈ-H), 5.56 (m, 1 H, 1ЈЈЈ-H), 6.92 (dd, J = 8.9,
2.3 Hz, 1 H, 6Ј-H), 7.10 (s, 1 H, 3Ј-H), 7.17 (d, J = 2.1 Hz, 1 H,
4Ј-H), 7.41 (d, J = 8.9 Hz, 1 H, 7Ј-H), 7.44 (d, J = 8.7 Hz, 1 H, 7-
H), 7.86 (s, 1 H, 9-H), 7.90 (d, J = 8.7 Hz, 1 H, 6-H), 8.18 (s, 1 H,
4-H), 11.55 (s, 1 H, NH) ppm. 13C NMR (125 MHz): δ = 20.29,
20.34, 20.50 [4 ϫ C(O)CH3], 40.09 (C-1), 41.14 (C-10), 45.51
(CH2NH2), 47.42 (NMe2), 55.01 (C-2), 57.76 (C-2ЈЈ), 60.94 (C-1ЈЈ),
66.22 (C-6ЈЈЈ), 67.03, 68.49, 69.66, 70.29 (C-2ЈЈЈ, C-3ЈЈЈ, C-4ЈЈЈ, C-
5ЈЈЈ), 98.90 (C-1ЈЈЈ), 101.6 (C-4), 103.1 (C-4Ј), 105.2 (C-3Ј), 113.1
(C-7Ј), 115.7 (C-6Ј), 118.8, 121.2, 127.3, 129.5, 130.7, 131.5 (C-5a,
C-9a, C-9b, C-8, C-2Ј, C-3aЈ, C-7aЈ), 121.6 (C-6), 124.5 (C-7), 141.8
(C-3a), 152.6, 152.8 (C-5, C-5Ј), 160.1 (C=O), 169.2, 169.3, 169.7,
(+)-(1S)-1-[(1R)-1-Chloroethyl]-7-cyano-3-({5-[2-(dimethylamino)eth-
oxy]indol-2-yl}carbonyl)-1,2-dihydro-3H-benz[e]indol-5-yl 2,3,4,6-
Tetra-O-acetyl-β-D-galactopyranoside [(+)-(1S,10R)-27b]: A mix-
ture of the phenol (+)-(1S,10R)-24b (218 mg, 585 μmol, 1.00 equiv.)
in dry CH2Cl2 (29 mL) and freshly activated molecular sieves (4 Å,
1.50 g) was stirred at room temperature for 30 min. After addition
of the trichloroacetimidate 25 (317 mg, 643 μmol, 1.1 equiv.) and
cooling to –10 °C, the promoter BF3·OEt2 (37 μL, 292 μmol,
0.5 equiv.) in dry CH2Cl2 (2.9 mL) was added dropwise, and the
mixture was stirred for 3.5 h. Additional BF3·OEt2 (222 μL,
1.75 mmol, 3.0 equiv.) in CH2Cl2 (2.8 mL) was then added, and the
mixture was allowed to warm to room temperature and stirred for
6 h. The reaction mixture was transferred by cannula into a new
flask for separation of the molecular sieves, which were washed
with CH2Cl2 (2ϫ 20 mL). The obtained organic solution was con-
centrated in vacuo, and the residue was dissolved in DMF (29 mL)
and cooled to 0 °C. EDC·HCl (336 mg, 1.75 mmol, 3.0 equiv.) and
DMAI·HCl (26, 250 mg, 878 μmol, 1.5 equiv.) were added, and the
reaction mixture was stirred at room temperature for 18 h. After
dilution with EtOAc (50 mL), H2O (50 mL) and saturated aqueous
NaHCO3 (50 mL) were added, and the aqueous phase was ex-
tracted with EtOAc (4ϫ 100 mL). The combined organic layers
were washed with brine (4ϫ 200 mL) and dried (MgSO4) and con-
centrated in vacuo. Purification of the residue by column
169.8 [4ϫ C(O)CH ] ppm. IR (KBr): ν = 3421 (NH), 1749 (C=O),
˜
3
1628, 1519, 1404 cm–1. UV (CH3CN): λmax (lgε) = 206.0 (4.461),
300.0 (4.312), 336.0 (4.249) nm. ESI-HRMS: m/z = 823.2941 [M +
H]+; C41H47ClN4O12 requires 823.2952.
(+)-(1S)-7-(Aminomethyl)-1-[(1R)-1-chloroethyl]-3-({5-[2-(dimethyl-
amino)ethoxy]indol-2-yl}carbonyl)-1,2-dihydro-3H-benz[e]indol-5-yl
chromatography (SiO2; CH2Cl2/MeOH, 10:1) afforded (+)- 2,3,4,6-Tetra-O-acetyl-β-D-galactopyranoside [(+)-(1S,10R)-28b]: A
(1S,10R)-27b as a white solid (254 mg, 305 μmol, 52%). [α]2D0
+6.7 (c = 0.3 in CHCl3). 1H NMR (600 MHz, CDCl3): δ = 1.63
=
solution of (+)-(1S,10R)-27b (50 mg, 60 μmol, 1.0 equiv.) in anhy-
drous EtOH (3 mL) was flushed with argon, and a solution of HCl
(d, J = 6.7 Hz, 3 H, 11-H3), 2.00 [s, 3 H, C(O)CH3], 2.02 [s, 3 H, in EtOH (1.25 m, 96 μL, 120 μmol, 2.0 equiv.), H2O (0.15 mL) and
C(O)CH3], 2.06 [s, 3 H, C(O)CH3], 2.19 [s, 3 H, C(O)CH3], 2.39 (s, PtO2·H2O (3.0 mg, 12.0 μmol, 0.2 equiv.) were added. The mixture
6918
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Eur. J. Org. Chem. 2010, 6909–6921

Fluorescence-Labelled Glycosidic Prodrugs Based on Duocarmycin
was stirred at room temperature under H2 for 48 h and was then
filtered through Celite, with washing with MeOH (100 mL), and
concentrated in vacuo. Purification of the residue by column
= 7). The reaction mixture was filtered, the filter cake was washed
with MeOH (120 mL), and the filtrate was concentrated in vacuo.
The residue was dissolved in H2O + 0.1% CF3CO2H/CH3CN (4:1)
chromatography (SiO2; CH2Cl2/MeOH, 5:1) afforded (+)-(1S,10R)- (12 mL) and purified by prep. HPLC to afford (–)-(1S,10R)-3b as
28b as a white solid (41 mg, 49 μmol, 82%). [α]2D0 = +4.4 (c = 0.25
a white solid (75 mg, 84 μmol, 70%). [α]2D0 = –9.0 (c = 0.3 in
1
in DMSO). 1H NMR (600 MHz, [D6]DMSO): δ = 1.64 (d, J = DMSO). H NMR (600 MHz, [D6]DMSO, 100 °C): δ = 1.65 (d, J
6.3 Hz, 3 H, 11-H3), 1.98, 1.99, 2.06, 2.18 [4ϫ s, 12 H, 4ϫ C(O) = 6.5 Hz, 3 H, 11-H3), 2.90 (s, 6 H, NMe2), 3.48–3.55, 3.56–3.64
CH3], 2.25 (mc, 6 H, NMe2), 2.67 (mc, 2 H, 2ЈЈ-H2), 3.93–4.19 (m,
(2ϫ m, 5 H, 2ЈЈ-H2, 3ЈЈЈ-H, 5ЈЈЈ-H, 6ЈЈЈ-Ha), 3.67–3.73 (m, 1 H,
6 H, 1ЈЈ-H2, 6ЈЈЈ-H2, CH2NH2), 4.27 (mc, 1 H, 1-H), 4.45–4.53 (m, 6ЈЈЈ-Hb), 3.82–3.89 (m, 2 H, 2ЈЈЈ-H, 4ЈЈЈ-H), 4.18–4.29 (m, 3 H, 1-
1 H, 5ЈЈЈ-H), 4.58–4.65 (m, 1 H, 2-Ha), 4.73–4.84 (m, 2 H, 2-Hb, 10-
H), 5.35–5.48 (m, 3 H, 2ЈЈЈ-H, 3ЈЈЈ-H, 4ЈЈЈ-H), 5.59 (d, J = 7.5 Hz, 1
H, CH2NH3+), 4.36 (mc, 2 H, 1ЈЈ-H2), 4.62–4.67 (m, 1 H, 2-Ha),
4.74 (mc, 1 H, 2-Hb), 4.82 (mc, 1 H, 10-H), 4.97 (d, J = 7.6 Hz, 1
H, 1ЈЈЈ-H), 6.92 (mc, 1 H, 6Ј-H), 7.14–7.20 (m, 2 H, 3Ј-H, 4Ј-H), H, 1ЈЈЈ-H), 7.02 (mc, 1 H, 6Ј-H), 7.15 (d, J = 1.8 Hz, 1 H, 3Ј-H),
7.40–7.44 (m, 1 H, 7Ј-H), 7.72–7.79 (m, 1 H, 6-H), 7.93 (br. s, 1 H,
9-H*), 8.03 (d, J = 8.6 Hz, 1 H, 8-H*), 8.24 (br. s, 1 H, 4-H), 11.57
7.28 (br. s, 1 H, 4Ј-H), 7.47 (d, J = 8.9 Hz, 1 H, 7Ј-H), 7.64 (d, J
= 8.8 Hz, 1 H, 8-H), 8.03 (d, J = 8.6 Hz, 1 H, 9-H), 8.24 (br. s, 1
(mc, 1 H, NH) ppm. 13C NMR (125 MHz, [D6]DMSO): δ = 20.30 H, 4-H), 8.41 (br. s, 1 H, 6-H), 11.52 (s, 1 H, NH) ppm. 13C NMR
(3 signals), 20.60 [4ϫ C(O)CH3], 23.29 (C-11), 44.29 (CH2NH2), (125 MHz, [D6]DMSO): δ = 23.28 (C-11), 42.65 (CH2NH3+), 42.84
45.45 (NMe2), 45.92 (C-1), 52.07 (C-2), 57.74 (C-2ЈЈ), 61.03, 61.25
(NMe2), 45.89 (C-1), 52.03 (C-2), 55.60 (C-2ЈЈ), 59.60 (C-6ЈЈЈ),
(C-10, C-6ЈЈЈ), 66.24 (C-1ЈЈ), 67.14, 68.66, 69.89 (C-2ЈЈЈ, C-3ЈЈЈ, C- 61.27 (C-10), 62.81 (C-1ЈЈ), 67.48, 70.56 (C-2ЈЈЈ, C-4ЈЈЈ), 73.01,
4ЈЈЈ), 70.43 (C-5ЈЈЈ), 99.16 (C-1ЈЈЈ), 102.8 (C-4), 103.3 (C-4Ј), 105.6
(C-3Ј), 113.2 (C-7Ј), 115.9 (C-6Ј), 120.2, 121.4, 122.4, 123.5 (C-7,
75.20 (C-3ЈЈЈ, C-5ЈЈЈ), 101.9 (C-4), 102.2 (C-1ЈЈЈ), 104.1 (C-4Ј),
105.5 (C-3Ј), 113.3 (C-7Ј), 115.7 (C-6Ј), 118.8, 122.5 (C-7, C-9b),
C-8, C-9, C-9b*), 127.5, 127.9, 128.8, 130.7, 131.8, 134.5 (C-6, C- 123.6 (C-9), 123.8 (C-6), 127.4, 127.7, 129.1, 129.2, 131.1, 132.1 (C-
5a*, C-9a, C-2Ј, C-3aЈ, C-7aЈ), 141.9 (C-3a), 152.6, 153.0 (C-5, C- 5a, C-8, C-9a, C-2Ј, C-3aЈ, C-7aЈ), 142.6 (C-3a), 152.0, 153.7 (C-5,
2
5Ј), 160.2 (C=O), 169.4, 169.6, 169.9, 170.0 [4ϫ C(O)CH3] ppm. C-5Ј), 158.1 [q, JC,F = 32 Hz, CF3C(O)], 160.1 (C=O) ppm. IR
IR (KBr): ν = 3417 (NH), 1750 (C=O), 1624, 1520, 1471, (KBr): ν = 3406 (NH), 1679, 1624, 1521, 1472, 1412 cm–1. UV
˜
˜
1409 cm–1. UV (CH3CN): λmax (lgε) = 202.5 (4.555), 249.0 (4.248), (CH3CN): λmax (lgε) = 207.5 (4.416), 250.5 (4.158), 299.0 (4.247),
298.0 (4.343), 337.5 (4.331) nm. ESI-HRMS: m/z = 837.3108 [M +
337.5 nm (4.230). ESI-HRMS: m/z = 669.2686 [M – 2ϫ CF3CO2 –
H]+; C42H49ClN4O12 requires 837.3109.
H]+; C38H43ClF6N4O12 requires 669.2685.
(1S)-8-(Aminomethyl)-1-(chloromethyl)-3-({5-[2-(dimethylamino)-
ethoxy]indol-2-yl}carbonyl)-1,2-dihydro-3H-benz[e]indol-5-yl β-D-
Synthesis of the Fluorescein-Labelled Compound (1S)-30a: A solu-
tion of 3a (11.9 mg, 18.2 μmol, 1.3 equiv.) and iPr2NEt (11 μL,
14.5 μg, 66 μmol, 4.5 equiv.) in anhydrous DMF (150 μL) was
added to a solution of 29 (8.90 mg, 15.2 μmol, 1.0 equiv.) in anhy-
drous DMF (150 μL), and the mixture was stirred at room tem-
perature for 18 h; then H2O + 0.1% CF3CO2H (3.0 mL) and
CH3CN (2.5 mL) were added, and the compound was purified by
prep. HPLC to afford (1S)-30a as a yellow solid (7.8 mg, 6.9 μmol,
46%). 1H NMR (600 MHz, [D6]DMSO): δ = 1.08–1.70 (m, 6 H,
3S-H2, 4S-H2, 5S-H2), 2.20 (m, 2 H, 2S-H), 2.87 (s, 6 H, NMe2),
3.18–4.26 (m, 13 H, 2ЈЈЈ-H, 3ЈЈЈ-H, 4ЈЈЈ-H, 5ЈЈЈ-H, 6ЈЈЈ-H2, 10-H2,
1-H, 6S-H2, 1ЈЈ-H2), 4.37 (m, 2 H, 2ЈЈ-H), 4.47 (m, 2 H, Ar-CH2),
4.59 (m, 1 H, 2-Ha), 4.85 (m, 1 H, 2-Hb), 4.90 (m, 1 H, 1ЈЈЈ-H),
6.53–6.60 (m, 4 H, Ar-H), 6.69 (d, J = 2.1 Hz, 1-H, Ar-H), 7.01
(dd, J = 8.9, 2.4 Hz, 1 H, 6Ј-H), 7.18 (m, 1 H, 3Ј-H), 7.27 (d, J =
2.1 Hz, 1 H, 4Ј-H), 7.30–7.38 (m, 2 H, 7Ј-H, Ar-H), 7.45 (d, J =
8.9 Hz, 1 H, 7-H), 7.72 (m, 1 H, Ar-H), 8.24 (m, 1 H, Ar-H), 8.30
(d, J = 8.8 Hz, 1 H, 6-H), 8.41 (t, J = 6.0 Hz, 1 H, Ar-H), 8.45 (d,
J = 9.6 Hz, 1 H, Ar-H), 8.79 (t, J = 5.4 Hz, 1 H, Ar-H), 9.75, 10.05
(s, 2 H, OH, CO2 H), 11.70 (s, 1 H, indole-NH) ppm. ESI-HRMS:
m/z = 1126.3832 [M + H]+; C60H60ClN5O15 requires 1126.3847.
Galactopyranoside [(1S)-3a]: A solution of NaOMe in MeOH
(5.4 m, 8.5 μL, 46 μmol, 2.0 equiv.) was added at 0 °C to a solution
of (+)-(1S)-28a (19 mg, 23 μmol, 1.0 equiv.) in MeOH (3 mL). The
mixture was allowed to warm to room temperature and stirred for
30 min. MeOH (1 mL), H2O (1 mL) and Amberlite IR-120 were
then added (pH = 7). The mixture was filtered, the filter cake was
washed with MeOH (25 mL), and the filtrate was concentrated in
vacuo. The residue was dissolved in H2O + 0.1 % CF3CO2H/
CH3CN (4:1) (4 mL) and purified by prep. HPLC to afford 3a as
a white solid (12.7 mg, 19.4 μmol, 84%). 1H NMR (600 MHz, [D6]-
DMSO): δ = 2.90 (s, 6 H, NMe2), 3.44–4.21 (m, 7 H, 1ЈЈ-H, 2ЈЈЈ-
H, 3ЈЈЈ-H, 4ЈЈЈ-H, 5ЈЈЈ-H, 6ЈЈЈ-H), 4.20–4.31 (m, 3 H, CH2NH2, 1-
H), 4.31–4.40 (m, 2 H, 2ЈЈ-H2), 4.57–4.66 (m, 2 H, 10-H2), 4.82–
4.93 (m, 3 H, 2-H2, 1ЈЈЈ-H), 7.01 (dd, J = 8.9, 2.4 Hz, 1 H, 6Ј-H),
7.14 (d, J = 1.6 Hz, 1 H, 3Ј-H), 7.26 (d, J = 2.3 Hz, 1 H, 4Ј-H),
7.45 (d, J = 8.9 Hz, 1 H, 7Ј-H), 7.49 (dd, J = 8.8, 1.3 Hz, 1 H, 7-
H), 8.01 (s, 1 H, 9-H), 8.37 (d, J = 8.7 Hz, 1 H, 6-H), 8.42 (s, 1 H,
4-H), 11.74 (s, 1 H, NH) ppm. 13C NMR (125 MHz, [D6]DMSO):
δ = 41.29 (C-1), 42.30 (CH2NH2), 42.80 (NMe2), 47.27 (C-10),
54.89 (C-2), 55.55 (C-1ЈЈ), 59.45 (C-6ЈЈЈ), 62.72 (C-2ЈЈ), 67.40,
70.38, 73.02, 75.05 (C-2ЈЈЈ, C-3ЈЈЈ, C-4ЈЈЈ, C-5ЈЈЈ), 102.1 (C-1ЈЈЈ),
103.9 (C-4Ј), 105.2 (C-3Ј), 113.2 (C-7Ј), 115.6 (C-6Ј), 117.8 (C-4*),
122.3 (C-9b), 122.5 (C-9), 123.7 (C-6), 124.1 (C-7), 127.2, 129.1,
131.0, 131.9, 133.1 (C-5a, C-9a, C-2Ј, C-3aЈ, C-7aЈ, C-8), 142.5 (C-
3a), 151.9, 153.6 (C-5, C-5Ј), 160.1 (C=O) ppm. ESI-HRMS: m/z
= 655.2526 [M + H]+; C33H39ClN4O8 requires 655.2529.
Chromatographic Resolution of (1S)-30a: Compound (1S)-30a was
separated consecutively (injection volume: 2.0 mL) by semiprepar-
ative HPLC [Kromasil 100 C18; 250ϫ20 mm; particle size: 7 μm;
gradient: H2O (0.1% CF3CO2H)/CH3CN, 83:17 for 60 min, then
H2O (0.1% CF3CO2H)/CH3CN, 50:50 for 10 min, then H2O (0.1%
CF3CO2H)/CH3CN, 83:17 for 10 min; flow: 12 mLmin–1; UV de-
tector: λ = 250 nm] to provide (1S)-30a (tR = 33.1 min).
(–)-(1S)-7-(Aminomethyl)-1-[(1R)-1-chloroethyl]-3-({5-[2-(dimethyl-
amino)ethoxy]indol-2-yl}carbonyl)-1,2-dihydro-3H-benz[e]indol-5-yl Synthesis of the Fluorescein-Labelled Compound (1S,10R)-30b: A
β-
D
-Galactopyranoside Bis(trifluoroacetate) [(–)-(1S,10R)-3b]:
A
solution of (–)-(1S,10R)-3b (11.95 mg, 13.3 μmol, 1.0 equiv.) and
iPr2NEt (11 μL, 14.5 μg, 66 μmol, 5.0 equiv.) in anhydrous DMF
(150 μL) was added to a solution of 29 (8.28 mg, 14.1 μmol,
1.0 equiv.) in anhydrous DMF (150 μL), and the mixture was
stirred at room temperature for 19 h; then H2O + 0.1% CF3CO2H
(3.0 mL) and CH3CN (2.7 mL) were added, and the compound was
solution of NaOMe in MeOH (5.4 m, 44 μL, 239 μmol, 2.0 equiv.)
was added at 0 °C to a solution of (+)-(1S,10R)-28b (100 mg,
119 μmol, 1.0 equiv.) in MeOH (12 mL). The reaction mixture was
allowed to warm to room temperature and stirred for 30 min.
MeOH (4 mL), H2O (4 mL) and Amberlite IR-120 were added (pH
Eur. J. Org. Chem. 2010, 6909–6921
© 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
6919

L. F. Tietze et al.
FULL PAPER
purified by prep. HPLC to afford (1S,10R)-30b as a yellow solid
(14.7 mg, 11.7 μmol, 88%). 1H NMR (600 MHz, [D6]DMSO,
Chromatographic Purification of (–)-(1S,10R)-32: Semipreparative
HPLC [Kromasil 100 C18; 250ϫ20 mm; particle size: 7 μm; gradi-
80 °C): δ = 1.37–1.45 (m, 2 H, 4S-H2), 1.57–1.69 (m, 7 H, 11-H3, ent: H2O (0.1% CF3CO2H)/CH3CN, 83:17 for 60 min, then H2O
3S-H2, 5S-H2), 2.22 (mc, 2 H, 2S-H2), 2.92 (s, 6 H, NMe2), 3.34 (mc,
2 H, 6S-H2), 3.47–3.63 (m, 5 H, 2ЈЈ-H2, 3ЈЈЈ-H, 5ЈЈЈ-H, 6ЈЈЈ-Ha), CF3CO2H)/CH3CN, 83:17 for 10 min; flow: 12 mLmin–1; UV de-
(0.1% CF3CO2H)/CH3CN, 50:50 for 10 min, then H2O (0.1%
3.67–3.72 (m, 1 H, 6ЈЈЈ-Hb), 3.81–3.87 (m, 2 H, 2ЈЈЈ-H, 4ЈЈЈ-H), tector: λ = 250 nm; injection volume: 2.0 mL]. (1S,10R)-32 (tR
4.22 (mc, 1 H, 1-H), 4.36 (mc, 2 H, 1ЈЈ-H2), 4.45 (mc, 2 H, ArCH2), 38.5 min). [α]2D0 = –14.0 (c = 0.15, MeOH).
4.63 (mc, 1 H, 2-Ha), 4.71 (mc, 1 H, 2-Hb), 4.81 (mc, 1 H, 10-H),
=
Supporting Information (see footnote on the first page of this arti-
cle): Experimental Section for the synthesis of molecules 7a–17c.
4.93 (d, J = 7.5 Hz, 1 H, 1ЈЈЈ-H), 6.56 (mc, 4 H, 4ϫ Ar-H), 6.70
(br. s, 2 H, 2ϫ Ar-H), 7.02 (mc, 1 H, 6Ј-H), 7.13 (d, J = 1.5 Hz, 1
H, 3Ј-H), 7.25–7.35, 7.44–7.51 (2ϫ m, 4 H, 8-H, 4Ј-H, 7Ј-H, Ar-
H), 7.91 (d, J = 8.7 Hz, 1 H, 9-H), 8.07–8.27 (m, 4 H, 4-H, 2ϫ
Ar-H, NH), 8.44 (br. s, 1 H, 6-H), 8.58 (br. s, 1 H, NH), 9.89, 9.59
(2ϫ br. s, 2 H, OH, CO2 H), 11.51 (br. s, 1 H, indole-NH) ppm.
ESI-HRMS: m/z = 1140.4004 [M – 2ϫ CF3CO2]+; C63H63ClF3-
N5O17 requires 1140.4004.
Acknowledgments
The work was supported by the Deutsche Forschungsgemeinschaft
and the Fonds der Chemischen Industrie. F. M. thanks the Studien-
stiftung des Deutschen Volkes (German National Academic Foun-
dation) for a Ph.D. scholarship. B. K. is grateful to the Deutsche
Telekom Foundation for a Ph.D. scholarship.
Chromatographic Purification of (1S,10R)-30b: Semipreparative
HPLC [Kromasil 100 C18; 250ϫ20 mm; particle size: 7 μm; gradi-
ent: H2O (0.1% CF3CO2H)/CH3CN, 83:17 for 60 min, then H2O
(0.1% CF3CO2H)/CH3CN, 50:50 for 10 min, then H2O (0.1%
CF3CO2H)/CH3CN, 83:17 for 10 min; flow: 12 mLmin–1; UV de-
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tector: λ = 250 nm; injection volume: 2.0 mL]. (1S)-30a (tR
36.6 min).
=
Synthesis of the Dapoxyl-Labelled Compound (–)-(1S,10R)-32: A
solution of (–)-(1S,10R)-3b (25.0 mg, 27.9 μmol, 1.0 equiv.) and
iPr2NEt (23 μL, 30.3 μg, 139 μmol, 5.8 equiv.) in anhydrous DMF
(150 μL) was added to a solution of 31 (12.3 mg, 24.0 μmol,
1.0 equiv.) in anhydrous DMF (200 μL), and the mixture was
stirred at room temperature for 9 h. H2O (+ 0.1% CF3CO2H)
(3.0 mL) and CH3CN (2.5 mL) were added, and the compound was
purified by prep. HPLC to afford (–)-(1S,10R)-32 as a yellow solid
(27.8 mg, 21.5 μmol, 89%). 1H NMR (600 MHz, [D6]DMSO,
80 °C): δ = 1.63 (d, J = 6.6 Hz, 3 H, 11-H3), 2.40 (t, J = 7.4 Hz, 2
H, 2S-H2), 2.93 (s, 6 H, 2ЈЈ-NMe2), 2.98 (s, 6 H, 12#-NMe2), 3.14
(t, J = 7.4 Hz, 2 H, 3S-H2), 3.48 (dd, J = 9.6, 3.2 Hz, 1 H, 3ЈЈЈ-H),
3.52–3.57 (m, 3 H, 5ЈЈЈ-H, 2ЈЈ-H2), 3.60 (dd, J = 10.6, 5.5 Hz, 1 H,
6ЈЈЈ-Ha), 3.69 (dd, J = 10.6, 7.2 Hz, 1 H, 6ЈЈЈ-Hb), 3.81–3.86 (m, 2
H, 2ЈЈЈ-H, 4ЈЈЈ-H), 4.20 (dt, J = 9.4, 2.6 Hz, 1 H, 1-H), 4.36 (t, J
= 5.0 Hz, 2 H, 1ЈЈ-H2), 4.42 (mc, 2 H, 12-H2), 4.62 (dd, J = 11.0,
2.6 Hz, 1 H, 2-Ha), 4.70 (dd, J = 11.0, 9.4 Hz, 1 H, 2-Hb), 4.79
(dq, J = 6.6, 3.0 Hz, 1 H, 10-H), 4.92 (d, J = 7.8 Hz, 1 H, 1ЈЈЈ-H),
6.83 (d, J = 9.0 Hz, 2 H, 2ϫ Ar-H), 7.01 (dd, J = 9.0, 2.2 Hz, 1
H, 6Ј-H), 7.12 (d, J = 2.0 Hz, 1 H, 3Ј-H), 7.28 (d, J = 2.2 Hz, 1 H,
4Ј-H), 7.45–7.48 (m, 2 H, 7Ј-H, 8-H), 7.54–7.56 (m, 2 H, 3S-NH,
8#-H), 7.65 (d, J = 9.0 Hz, 2 H, 2ϫ Ar-H), 7.89 (d, J = 8.5 Hz, 1
H, 9-H), 7.96 (d, J = 8.4 Hz, 2 H, 2ϫ Ar-H), 8.18 (s, 1 H, 12-NH),
8.20–8.23 (m, 4 H, 4-H, 6-H, 2ϫ Ar-H), 9.64 (br. s, 1 H, NH+),
11.5 (s, 1 H, 1Ј-NH) ppm. 13C NMR (125 MHz, [D6]DMSO): δ =
23.4 (C-11), 35.5 (C-2S), 38.7 (C-3S), 40.1 (12#-NMe2), 42.5 (C-12),
42.9 (2ЈЈ-NMe2), 46.0 (C-1), 52.0 (C-2), 55.7 (C-2ЈЈ), 59.5 (C-6ЈЈЈ),
61.4 (C-10), 62.7 (C-1ЈЈ), 67.5 (C-4ЈЈЈ), 70.5 (C-2ЈЈЈ), 73.1 (C-3ЈЈЈ),
75.2 (C-5ЈЈЈ), 101.8 (C-9b), 102.1 (C-1ЈЈЈ), 104.0 (C-4Ј), 105.4 (C-
3Ј), 112.1 (2ϫ Ar-C), 113.3 (C-7Ј), 114.6 (CF3CO2), 115.6 (C-6Ј),
118.9 (C-5a), 121.4 (C-8#), 121.7 (C-4, C-6), 122.7 (C-7), 123.3 (C-
9), 125.5 (2ϫ Ar-C), 126.1 (2ϫ Ar-C), 127.4 (C-8), 127.4 (2ϫ Ar-
C), 128.5 (C-4#), 130.2 (C-9#), 131.2 (C-9a), 132.0 (C-3aЈ), 134.7
(C-7aЈ), 141.1 (C-2Ј, C-1#), 141.6 (C-3a), 150.1 (C-7#), 152.0 (C-
12#), 152.7 (C-5Ј), 153.4 (C-5), 157.5 (C-5#, 1Ј-C=O), 160.0
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(CF CO ), 169.6 (2S-C=O) ppm. IR (KBr): ν = 3406, 2924, 2819,
˜
3
2
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202.5 (4.821), 252.0 (4.514), 289.0 (4.546), 299.5 (4.582), 349.0 nm
(4.627). ESI-HRMS: m/z
C56H61ClF3N7O14S requires 1066.3782.
= 1066.3778 [M –
2ϫ CF3CO2]+;
6920
www.eurjoc.org
© 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2010, 6909–6921

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Received: July 8, 2010
Published Online: November 2, 2010
Eur. J. Org. Chem. 2010, 6909–6921
© 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
6921