Synthesis of diphenylcarbazoles as cytotoxic DNA binding agents

Article abstract of DOI:10.1039/b401445f

We report the synthesis or a series of novel diphenylcarbazoles designed to interact with DNA. The compounds bearing two or three dimethylaminoalkyloxy side chains were found to bind much more tightly to DNA, preferentially at AT-rich sites, than the corr

Full text of DOI:10.1039/b401445f

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Synthesis of diphenylcarbazoles as cytotoxic DNA binding agents
Ulrich Jacquemard,a Sylvain Routier,*a Arnaud Tatibouët,a Jérôme Kluza,b William Laine,b
Christine Bal,b Christian Bailly*b and Jean-Yves Mérour*a
a Institut de Chimie Organique et Analytique, UMR 6005, Université d’Orléans, B.P. 6759,
45067 Orléans Cedex 2, France. E-mail: sylvain.routier@univ-orleans.fr;
Fax: 33 2 38.41 72.81; Tel: 33 2384 94592
b INSERM U-524 et Laboratoire de Pharmacologie Antitumorale du Centre Oscar Lambret,
IRCL, 59045 Lille, France
Received 30th January 2004, Accepted 23rd March 2004
First published as an Advance Article on the web 19th April 2004
We report the synthesis of a series of novel diphenylcarbazoles designed to interact with DNA. The compounds
bearing two or three dimethylaminoalkyloxy side chains were found to bind much more tightly to DNA,
preferentially at AT-rich sites, than the corresponding hydroxy compounds. The DNA binding compounds
exhibit potent cytotoxic activity toward P388 leukemia cells. The 3,6-diphenylcarbazole thus represent an
interesting scaffold to develop antitumor agents interacting with nucleic acids.
of a new class of symmetric bisbenzimidazole-based minor
Introduction
groove binders showing in vivo antitumor activities.6 The head-
to-head bis-benzimidazole ABA833 (Fig. 1) has revealed strong
cytotoxic activities against various cell lines in vitro, especially
ovarian tumour cells sensitive or resistant to cisplatin and in
vivo activity has been observed using the hollow fiber assay with
ovarian carcinoma cells.6
Small molecules capable of interacting selectively with A–T
base pairs within the minor groove of DNA have been
extensively developed over the past ten years.1 These include (i)
oligopyrrole/imidazole carboxamide, derived from the anti-
biotics netropsin and distamycin, which may serve as molecular
tools to control gene expression in cells,2,3 (ii) diamidine
unfused aromatic cations, typified by the promising anti-
parasitic drug furamidine (DB75, Fig. 1), which is currently
developed in the form of an amidoxime prodrug (DB289) for
the treatment of African trypanosomiasis and opportunistic
infections,4 and (iii) benzimidazole-containing minor groove
binders, derived from the fluorescent dye Hoechst 33258, which
may be useful as anticancer agents.1
More recently, a potent cytotoxic action against breast
cancer cells has also been reported with this compound7 which
exploits water molecules to adapt its extended shape to that
of the minor groove of DNA at AT-rich sequences.8 This
compound thus represents a novel promising model for the
development of DNA-interacting anticancer agents. Bis-
phenylbenzimidazole ABA833 and the diphenylfuran DB75 are
built on the same model. They both contain a heterocycle
“sandwiched” between two phenyl rings substituted with
cationic side chains. This model can be exploited to design
novel potential therapeutic agents. We decided to replace the
benzimidazole system with a carbazole planar chromophore
which may be better adapted to fit to the minor groove surface
of DNA or to stack on DNA base pairs. The carbazole tricycle
is frequently encountered in DNA binding agents, including
intercalating topoisomerase II inhibitors (e.g. pyrrolocarb-
azoles,9 pyridocarbazoles10,11 and benzopyrimidocarbazoles12)
topoisomerase I inhibitors (e.g. indolo carbazole),13 and
most importantly, in DNA minor groove binders such as the
diamidinocarbazoles which have revealed interesting biological
properties as antiparasitic drugs.14
Bisamidinocarbazole dications form tight DNA minor
groove complexes and exhibit a pronounced selectivity for
AT-rich sequences.15–17 Based on these considerations, we there-
fore decided to synthesize a series of bis-phenylcarbazole
derivatives substituted with different side chains, neutral or
cationic, analogous to the dimethylaminoalkyl chains found
in ABA833. Here we report the synthesis of a first series of bis
substituted carbazoles obtained from 2,6-dibromocarbazole
and we present preliminary data about the cytotoxic potential
and DNA interacting capacity (Scheme 1).
Fig. 1 Minor groove binders.
Hoechst 33258 has long been used as a DNA stain in
cytometry experiments and in biology to study the con-
densation/decondensation of nucleic acids. In addition, this
bis-benzimidazole derivative, also known as pibenzimol, has
revealed potent cytotoxic activity and has undergone clinical
trials as an anticancer drug.5 The clinical responses were not
sufficient to warrant a further development but nevertheless
Hoechst 33258 remains an interesting scaffold from which
potential anticancer drugs can be built. A promising develop-
ment has been reported recently with the design by Mann et al.6
Results
To complete the first step, it was reported that the commercially
available 3,6-dibromocarbazole 1, after N-protection, reacted in
the presence of a base to perform a halogen–metal exchange.
Addition of B(OMe)3 or Sn(CH3)3Cl also generates bis-boronic
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 4 7 6 – 1 4 8 3
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 4
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improvement of the synthesis with compound 5 now obtained
in 97% yield using only 10% catalyst.20 This considerable
enhancement illustrates the versatility of cross coupling
reactions. However, it is worth mentioning at this point that
minor variations in the procedure can affect significantly the
yield of the reaction. The protection of the free nitrogen atom
was performed using NaH and benzenesulfonyl chloride in
THF at room temperature to afford 3 in 91% yield. Demethyl-
ation was carried out on 3 and 5, using boron tribromide, to
obtain 4 and 6 in quantitative and 94% yields, respectively.21
The next step consisted of the introduction of various
hydrophilic side chains (Fig. 2, Table 1). Three different side
chains were chosen, in order to introduce hydrophilic functions
spaced by 2 or 3 carbon atoms from the aromatic moieties
(entries 1–6). First non-selective alkylation was realized on
compound 6 with 2-chloroethyldimethylamine hydrochloride 7,
using an excess of caesium carbonate to afford trisubstituted
compound 8, in 49% yield, and disubstituted compound 13
in 34% yield. Decreasing the amounts of base and halogen
derivative, or reaction time did not produce selective O-alkyl-
ation. This can be explained by the poor reactivity of halide 7
in nucleophilic substitutions. Decreasing the reaction time to
a few hours, compound 6, in the presence of more reactive
halogen compounds such as 3-chloropropyldimethylamine
hydrochloride 9 and 3-bromopropoxymethylbenzene 10, leads
to selective O-alkylation, although with limited yields.
Compounds 11 and 12 were also obtained in 67% and 36%
yields, respectively. Several other conditions were tested using
the N-protected carbazole compound 4.
Scheme 1 Retrosynthetic scheme.
acid or bis-stannyl carbazoles18 which were engaged in a Suzuki
or a Stille cross coupling procedure, respectively. In addition,
this synthesis reported a protection of the nitrogen carbazole by
a tosyl group which was cleaved under strong basic conditions
(n-BuLi, 9 eq.).
We first decided to introduce, on the nitrogen of 1, a benzene-
sulfonyl group (Scheme 2), which could be easily removed
under mild basic conditions and was also compatible with the
presence of various reactive groups. Thus, compound 2 was
obtained, using NaH as a base and benzenesulfonylchloride in
dry THF at room temperature, in 86% yield. Halogen exchange,
followed by the addition of B(OMe)3 generated the bis boronic
acid in 79% yield.18 Unfortunately, despite numerous attempts
(variation of temperature, reaction time, palladium catalyst
entity, bases and solvents) the yield of the Suzuki reaction,
between the bis-boronic acid and the 4-bromoanisole remained
limited. Under our best conditions, using 4-bromoanisole
(6 eq.) and an aqueous saturated solution of NaHCO3 as a
base, in a mixture of ethanol and toluene at reflux for 5 h, we
obtained only 38% of the desired product 3. A major drawback
was the low solubility of compound 2.
For these reasons, we continued to perform the coupling
Suzuki procedure directly between the more soluble carbazole 1
and the 4-methoxyphenylboronic acid.19 The described con-
ditions involving Pd(PPh3)4 (40%), the boronic acid (2.4 eq.)
and sodium hydrogen carbonate as the base, in a homogeneous
mixture of DME/H2O at 80 ЊC for 3 h afforded the desired
compound 5 in 60% yield. Changing the solvent to dioxane and
increasing the reaction time to 18 h gave a lower yield of 51%.
Similar results were obtained by varying the nature of the
base. Disappointed by these modest results, we tested biphasic
conditions to perform this reaction. The use of a mixture of
ethanol and toluene at reflux for 3 h resulted in a significant
Fig. 2 Introduction of hydrophilic side chains (see Table 1).
First, the reaction of compound 4 with compound 7 using
K2CO3 as a base in DMF afforded, after the cleavage of the
protecting group by Bu4NF22 (for an easier purification),
Scheme 2 a) NaH, THF, PhSO2Cl, 0 ЊC to r.t., 12 h, 86%; b) n-BuLi (2.6 eq.), THF, Ϫ70 ЊC, 30 min, B(OMe)3 (3 eq.), 30 min, then r.t., 12 h, 79%; c)
Pd(PPh3)4 (10 mol%.), 4-bromoanisole (6.2 eq.), aq. sat. NaHCO3, ethanol, toluene, reflux, 5 h, 38%; d) BBr3 (2.3 eq.), CH2Cl2, 0 ЊC to r.t., 2 h, 97%;
e) Pd(PPh3)4, 4-methoxyphenylboronic acid, aq. sat. NaHCO3, toluene, reflux, 5 h, 97%; f ) NaH, THF, PhSO2Cl, 0 ЊC to r.t., 2 h, 91%; g) BBr3
(2.3 eq.), CH2Cl2, 0 ЊC to r.t., 3 h, 94%.
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 4 7 6 – 1 4 8 3
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Table 1 Alkylation procedure
Entry
Reactant
Conditions
Product (Yield)
8 (49%)
R1
H
R2
1
2
6
6
7 (6 eq.), Cs2CO3 (10 eq.), DMF, 100 ЊC, 12 h
9 (5 eq.), Cs2CO3 (10 eq.), DMF, 100 ЊC, 1 h
10 (5 eq.), Cs2CO3 (2.5 eq.), DMF, 100 ЊC, 1 h
11 (67%)
3
4
6
4
12 (36%)
13 (70%)
H
H
a) 7 (6 eq.), Cs2CO3 (11 eq.), DMF, 100 ЊC, 12 h
b) Bu4NF (1.2 eq.), THF, reflux, 2 h
5
4
9 (5 eq.), Cs2CO3 (10 eq.), DMF, 100 ЊC, 1 h
14 (81%)
SO2Ph
6
7
4
10 (5 eq.), Cs2CO3 (2.2 eq.), THF/DMF, reflux, 1 h
Bu4NF (1.2 eq.), THF, reflux, 2 h
15 (75%)
11 (80%)
SO2Ph
H
14
8
9
15
12
15
17
Bu4NF (5.0 eq.), THF, reflux, 2 h
12 (quant.)
16 (quant.)
17 (quant.)
16 (32%)
H
H2, Pd(C) 10%, acetic acid/dioxane, r.t. 12 h
H2, Pd(C) 10%, acetic acid/dioxane, r.t. 12 h
Bu4NF (1.2 eq.), THF, reflux, 12 h
H
10
11
SO2Ph
H
Scheme 3 a) Penta-O-acetyl-β--glucopyranose, BF3ؒEt2O, CH2Cl2, 4 Å molecular sieves, r.t, 4 d, 56%; b) MeOH, MeONa, r.t, 2 h, quant.
compound 13 in only 35% yield. Replacing the base by Cs2CO3,
followed by the deprotection increased the yield to 70% for
the two steps. Under these conditions, treatment of 4 with
halides 9 or 10 led to compounds 14 and 15 in 81 and 75%
yields, respectively.
Cleavage of the benzenesulfonyl groups were always
performed using Bu4NF in refluxing THF,22 to obtain
compounds 11 and 12 from 14 and 15 in quantitative yields
(entries 7 and 8). Catalytic hydrogenolysis of 12 and 15 afforded
compounds 16 and 17 in quantitative yields (entries 9 and 10).
Deprotection of benzenesulfonyl group of 17 with Bu4NF led
to 16 in only 32% yield (entry 11). Degradation of the starting
material suggested also an incompatibility between the
hydroxyalkyl group and the reagent.
of the synthetic polynucleotide poly(dAT)2. This alternating
polymer melts at a much lower temperature than calf thymus
DNA (41 ЊC vs 62 ЊC) and thus offers a more sensitive assay to
compare small molecules, especially those targeted to AT-rich
sequences, as it is the case here. Indeed, DNase I footprinting
experiments have indicated that some of these compounds
(8 and 13 in particular) bind preferentially to AT-rich sequences
(data not shown). For each molecule, we measured the differ-
ence in melting temperature for the drug–DNA complexes
and the unbound DNA (∆T m = T mcomplex Ϫ T mDNA). All
compounds were tested at a fixed drug/DNA-nucleotide ratio
of 0.1 with a heating rate of 1 ЊC minϪ1. A typical example of
melting curves obtained with poly(dAT)2 and four compounds
6, 8, 13 and 11 is presented in Fig. 3 and Table 2.
Introduction of the glycosyl moiety was first attempted using
compound 16 (Scheme 3), but no reaction was observed. Con-
sidering the possible implication of the free nitrogen atom of
the carbazole ring in this result, protected carbazole 17 was
used instead. Condensation of 17 and penta-O-acetyl-β--
glucopyranose was undertaken with BF3ؒEt2O as the Lewis
acid, and afforded in 56% yield glycosylated 18 which contains
the expected β--glucoside moiety.23 This was the only anomer
isolated.
It was clear that compound 6 had no effect on the thermal
stability of the DNA whereas the other compounds strongly
Deprotection of the acetates were performed under classical
Zemplen conditions,24 catalytic presence of MeONa in MeOH,
leading to 19 in quantitative yield. Unfortunately, whatever the
experimental conditions used, the deprotection of the benzene-
sulfonyl group of 19 always failed and we could never obtain
the desired unsubstituted carbazole.
complex
Fig. 3 Melting temperature variation ∆T m (T mdrug–DNA
Ϫ
alone
T mDNA
, in ЊC) of poly(dAT)2 after incubation with the
diphenylcarbazoles. The T m measurements were performed at a
constant drug/DNA-phosphate ratio of 10 (2 µM drug, 20 µM DNA-
nucleotide). T m measurements were performed in BPE buffer pH 7.1
(6 mM Na2HPO4, 2 mM NaH2PO4, 1 mM EDTA), in 1 cm quartz
cuvettes at 260 nm with a heating rate of 1 ЊC minϪ1. The T m values
were obtained from first-derivative plots.
DNA interaction
The interaction of the newly synthesized compounds with
DNA was probed by absorption spectroscopy to measure the
capacity of the molecules to stabilize the double helix structure
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 4 7 6 – 1 4 8 3
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Table 2 Binding to DNA and cytotoxicity
Table 3 Drug-induced apoptosis (sub-G1 cells, %)a
Compound
∆T m/ЊCa
IC50/µMb
1
5
Concentration/µM
Time/h
24
48
2.3
23.7
34.3
43.6
26.6
2.6
24
48
6
8
11
13
16
17
19
0
7.23 0.07
4.15 0.05
0.77 0.04
1.53 0.27
1.83 0.27
9.7 0.5
8.3–33.4
13.2
6.0–29.5
2.2
0
6
8
11
13
16
17
19
3
3.5
20
17.8
6.8
2.4
2.5
7.7
14.6
25.2
75.8
57.1
37.1
10.2
4.7
12.6
42.9
30.4
16.6
5.2
0
>50
a T m measurements were performed in BPE buffer pH 7.1 (6 mM
Na2HPO4, 2 mM NaH2PO4, 1 mM EDTA) using 20 µM drug and
20 µM poly(dAT)2 (nucleotide concentration) with a heating rate
2.3
2.8
a Cells (%) in the sub-G1 phase, as determined by flow cytometry.
of 1 ЊC minϪ1 b Drug concentration (µM) that inhibits P388 leukemia
.
cell growth by 50% after incubation in liquid medium for 72 h (n =
2–4).
Cell cycle effects and apoptosis
Treatment of the P388 cells with the different compounds at
1 or 5 µM for 24 h or 48 h led, in most cases, to profound
changes of the cell cycle profiles. The flow cytometry analysis of
propidium iodide-labeled cells indicates that the most cytotoxic
drugs 8, 11 and 13 induce a decrease of the number of cells
in the S phase and a concomitant marked accumulation of cells
in the sub-G1 phase (Fig. 4).
stabilize the duplex structure of the polymer so as to increase
considerably its melting temperature. Monophasic melting
curves were obtained with the dimethylaminopropyloxy
derivative 11 but biphasic curves were observed with the two
ethyloxy analogues 8 and 13, possibly due to a heterogeneous
binding mode. A detailed analysis of the binding mode
and sequence selectivity will be reported separately.25 For each
molecule, the melting temperature was determined from the
first derivative analysis and the ∆T m data are collated in Table
2. In the case of biphasic melting curve, the T m of the two
transitions are indicated. The DNA binding capacity varies
significantly from one drug to another with, as expected, the
cationic dimethylaminoalkyloxy compounds being much more
potent than the uncharged analogues. The hydroxy compounds
6 and 16 show very little interaction with DNA and similarly,
the two carbazoles 19 and 17 (bearing a N-substituted benzene-
sulfonyl group) also failed to stabilize the duplex structure of
DNA. The incorporation of a dimethylaminoalkyloxy chain
thus appears as a profitable scheme to target DNA with these
diphenylcarbazoles.
Cytotoxicity
A tetrazolium-based assay was applied to determine the drug
concentration required to inhibit the growth of murine P388
leukemia cells by 50% after incubation in the culture medium
for 72 h. The IC50 values are collated in Table 2. Interestingly,
the dimethylamino compounds 8, 11 and 13 which bind
strongly to DNA proved to be significantly more cytotoxic than
the uncharged hydroxy derivative 6 and the phenyl sulfonyl
substituted compounds 17 and 19. The benzenesulfonyl pro-
tecting group, which unfortunately could not be removed, is
clearly detrimental to both DNA interaction and cytotoxicity.
This may be an indication that affinity for DNA contributes
to the cytotoxicity and perhaps also that the free NH of the
carbazole ring is necessary for DNA interactions. Although,
there is not a simple correlation between the extent of
DNA binding and the cytotoxic action of the compounds, it
seems clear that DNA binding is necessary to inhibit cell
proliferation. The uncharged molecule 16 is equally toxic to
the cationic derivative 13 despite its poor capacity to stabilize
duplex DNA against heat denaturation. However, the
most active compound in the series is the propyloxy deriv-
ative 11 which is a potent DNA binder. A single parameter,
such as the ∆T m, is often insufficient to account for a complex
biological process. Uptake, distribution, metabolism and
other biological parameters can vary among the different
compounds, especially when comparing compounds with
different size and hyphobic/hydrophilic characters, as is the
case here. Nevertheless, this preliminary screening indicates
that some of the diphenylcarbazole compounds exhibit an
interesting cytotoxic profile, with IC50 values in the low µM
range.
Fig. 4 Cell cycle distribution determined by flow cytometry in P388
cells treated for (a) 24 h or (b) 48 h with the indicated compound at (a) 5
µM or (b) 1 µM. Cells were stained with propidium iodide (PI) and
analysed with a FACScan flow cytometer. In each case, the percentage
of cells in the sub-G1 phase was calculated (Table 3).
The cell cycle perturbations are dose- and time-dependent
(Table 3). The sub-G1 population, with a characteristic
hypo-diploid DNA content, reflects apoptotic cells with
fragmented DNA. Up to 75% of the cells undergo apoptosis
with 11 after 48 h of treatment at 1 µM. Interestingly, there is a
correlation between the extent of drug-induced apoptosis and
the level of cytotoxicity. All these biological data augur well for
a more extended in vitro and in vivo evaluation of compounds
11 and 13 which is currently in progress.
Experimental
1H-NMR and 13C-NMR spectra were recorded on a Bruker
DPX 250 instrument using CDCl3 or DMSO-d6. The chemical
shifts are reported in ppm (δ scale) and all coupling constants
(J) values are in hertz (Hz). The splitting patterns are desig-
nated as follows: s (singlet), d (doublet), t (triplet), q (quartet),
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 4 7 6 – 1 4 8 3
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m (multiplet), and dd (doublet doublet). Melting points are
uncorrected. IR absorption spectra were obtained on a Perkin
acetate 1/1) to afford compound 4 as a white solid (565 mg,
97%). Rf (petroleum ether/ethyl acetate 1/1): 0.53; mp 140 ЊC;
Elmer PARAGON 1000 PC and values were reported in cmϪ1
.
IR (KBr) ν (cmϪ1) 3418, 1610, 1518, 1481, 1447, 1364, 1169,
1
MS spectra (ion spray) were performed on a Perkin Elmer Sciex
PI 300. Monitoring of the reactions was performed using silica
gel TLC plates (silica Merck 60 F254). Spots were visualized by
UV light at 254 nm and 356 nm. Column chromatography was
performed using silica gel 60 (0.063–0.200 mm, Merck).
818; H-NMR (CDCl3, 250 MHz): δ 6.90 (d, 4H, J = 8.6 Hz),
7.49 (d, 2H, J = 8.1 Hz), 7.52 (s, 2H), 7.58–7.66 (m, 5H), 7.81
(dd, 2H, J = 1.7, 8.8 Hz), 7.87 (dd, 2H, J = 1.5, 8.8 Hz), 8.26 (d,
2H, J = 8.6 Hz), 9.60 (s, 2H); 13C-NMR (CDCl3, 62.5 MHz):
δ 114.9 (2 × CH), 115.8 (4 × CH), 118.2 (2 × CH), 125.9
(2 × CH), 126.2 (2 × CH), 126.8 (2 × Cq), 128.0 (4 × CH), 129.8
(2 × CH), 130.3 (2 × Cq), 134.7 (CH), 136.6 (2 × Cq), 136.7 (2 ×
Cq), 139.2 (Cq), 157.2 (2 × Cq); MS (IS): 492 (M ϩ 1)ϩ; Anal.
calcd for C30H21NO4S: C, 73.30; H, 4.31; N, 2.85. Found: C,
73.02; H, 4.48; N, 2.99%.
9-Benzenesulfonyl-3,6-dibromo-9H-carbazole (2)
A solution of 3,6-dibromocarbazole 1 (1 g, 3.07 mmol) in dry
THF (25 mL) was cooled to 0 ЊC, under argon, then NaH 60%
dispersion in oil (184 mg, 4.60 mmol) was added portionwise
and the solution was stirred for 1 h. Benzenesulfonylchloride
(0.590 mL, 4.60 mmol) was added, followed after 12 h at room
temperature, by a saturated solution of NH4Cl (30 mL). The
mixture was extracted with CH2Cl2 (3 × 75 mL) and the
combined organic layers were successively washed with a satur-
ated solution of K2CO3 (30 mL), water (30 mL) then dried over
MgSO4. After filtration, the solvents were removed under
reduced pressure to afford compound 2 as a white solid (1.230
g, 86%). Rf (petroleum ether/ethyl acetate 96/4): 0.43; mp >
250 ЊC; IR (KBr) ν (cmϪ1) 1465, 1425, 1372, 1208, 1182, 1125,
3,6-Bis(4-methoxyphenyl)-9H-carbazole (5)
A solution of 3,6-dibromocarbazole 1 (500 mg, 1.53 mmol) and
4-methoxyphenylboronic acid (600 mg, 3.92 mmol) in a mixture
of toluene (31.6 mL), ethanol (19.2 mL) and aqueous saturated
NaHCO3 solution (12.6 mL) was degassed by argon bubbling
for 20 min. Pd(PPh3)4 (10 mol%, 0.153 mmol) was added and
the mixture was immediately transfered to a pre-heated oil bath
and refluxed for 5 h. After hydrolysis (50 mL), the mixture was
extracted with ethyl acetate (2 × 50 mL), washed with brine
(50 mL), dried over MgSO4, then filtered. The solvents were
removed under reduced pressure and the crude material was
purified by flash chromatography (petroleum ether/ethyl acetate
8/2 to ethyl acetate) to afford compound 5 as a white solid
(565 mg, 97%). Rf (petroleum ether/ethyl acetate 8/2): 0.19; mp
198 ЊC; IR (KBr) ν (cmϪ1) 3428, 1607, 2980, 1514, 1482, 1455
1274, 1245, 1036, 820; 1H-NMR (DMSO-d6, 250 MHz): δ 3.81
(s, 6H), 7.05 (d, 4H, J = 8.6 Hz), 7.52 (d, 2H, J = 8.6 Hz), 7.68
(m, 6H), 8.48 (s, 2H), 11.27 (s, NH);13C-NMR (DMSO-d6, 62.5
MHz): δ 55.1 (2 × CH3), 111.3 (2 × CH), 114.3 (4 × CH), 118.0
(2 × CH), 123.3 (2 × Cq), 124.4 (2 × CH), 127.6 (4 × CH), 130.8
(2 × Cq), 133.8 (2 × Cq), 139.4 (2 × Cq), 158.2 (2 × Cq); MS
(IS) : 380 (M ϩ 1)ϩ; Anal. calcd for C26H21NO2: C, 82.3; H,
5.58; N, 3.69. Found: C, 82.57; H, 5.71; N, 3.54%.
1
815, 729, 573; H-NMR (DMSO-d6, 250 MHz): δ 7.52 (t, 2H,
J = 7.5 Hz), 7.67 (t, 1H, J = 7.5 Hz), 7.76 (dd, 2H, J = 2.0,
8.7 Hz), 7.86 (d, 2H, J = 7.5 Hz), 8.19 (d, 2H, J = 8.7 Hz), 8.52
(d, 2H, J = 2 Hz);13C-NMR (DMSO-d6, 62.5 MHz): δ 116.5
(2 × CH), 117.2 (2 × Cq), 124.2 (2 × CH), 126.3 (2 × CH), 126.7
(2 × Cq), 130.0 (2 × CH), 131.2 (2 × CH), 135.2 (CH), 136.1
(Cq), 136.7 (2 × Cq); MS (IS) : 464 (M ϩ 1)ϩ; Anal. calcd for
C18H11Br2NO2S: C, 46.48; H, 2.38; N, 3.01. Found: C, 46.85; H,
2.51; N, 2.83%.
9-Benzenesulfonyl-3,6-bis(4-methoxyphenyl)-9H-carbazole (3)
A solution of compound 5 (670 mg, 1.77 mmol) in dry THF
(15 mL) was cooled to 0 ЊC under argon then NaH (60%
dispersion in oil, 85 mg, 2.12 mmol) was added portionwise and
the solution was stirred for 30 min. Benzenesulfonylchloride
(468 mg, 2.65 mmol) was added dropwise, followed after 1 h at
room temperature, by a saturated solution of NH4Cl (30 mL).
The mixture was extracted with CH2Cl2 (3 × 25 mL). The
combined organic layers were successively washed with a
saturated solution of K2CO3 (20 mL), water (20 mL) then dried
over MgSO4. After filtration, the solvents were removed under
reduced pressure. Purification by flash chromatography (ether
petroleum/ethyl acetate 9/1 to 1/1) afforded compound 3 as a
pale yellow solid (0.84 g, 91%). Rf (ether petroleum/ethyl
acetate 8/2): 0.36; mp 160 ЊC; IR (KBr) ν (cmϪ1) 1608, 1518,
1481, 1449, 1369, 1247, 1209, 1173, 819; 1H-NMR (CDCl3, 250
MHz): δ 3.86 (s, 6H), 7.00 (dd, 4H, J = 2.2, 6.5 Hz), 7.34 (t, 2H,
J = 7.8 Hz), 7.45 (dd, 1H, J = 1.2, 7.3 Hz), 7.60 (dd, 4H, J = 2.2,
6.6 Hz), 7.68 (dd, 2H, J = 1.7, 8.8 Hz), 7.86 (d, 2H, J = 7.4 Hz),
8.08 (d, 2H, J = 1.7 Hz), 8.35 (d, 2H, J = 8.8 Hz); 13C-NMR
(CDCl3, 62.5 MHz): δ 55.5 (2 × CH3), 114.5 (4 × CH), 115.5 (2
× CH), 118.1 (2 × CH), 126.7 (4 × CH), 127.2 (2 × Cq), 128.4
(4 × CH), 129.2 (2 × CH), 133.5 (2 × Cq), 134.0 (CH), 137.3
(2 × Cq), 137.9 (2 × Cq), 138.0 (Cq), 159.3 (2 × Cq); MS (IS) :
520 (M ϩ 1)ϩ; Anal. calcd for C32H25NO4S: C, 73.97; H, 4.85;
N, 2.70. Found: C, 74.38; H, 5.00; N, 2.84%.
3,6-Bis(4-hydroxyphenyl)-9H-carbazole (6)
Same procedure as described for compound 5. To a solution of
compound 4 (519 mg, 1.37 mmol) in CH2Cl2 at 0 ЊC, BBr3 (2.76
mL, 1 M in CH2Cl2, 2.76 mmol) was added dropwise. After 3 h
at room temperature, the reaction mixture was poured into ice
(100 g), extracted with ethyl acetate (2 × 50 mL), dried with
MgSO4, then filtered. The solvents were removed under reduced
pressure and the crude material was purified by flash chromato-
graphy (petroleum ether/ethyl acetate 1/1) to afford compound
6 as a white solid (500 mg, 94%). Rf (ether petroleum/ethyl
acetate 1/1): 0.32; mp > 240 ЊC; IR (KBr) ν (cmϪ1) 3514, 3422,
1
1610, 1517, 1484, 1272, 1205, 819; H-NMR (DMSO-d6, 250
MHz): δ 6.90 (d, 4H, J = 8.6 Hz), 7.49–7.64 (m, 8H), 8.44
(s, 2H), 9.43 (s, 2H), 11.22 (s, NH); 13C-NMR (DMSO-d6, 62.5
MHz): δ 111.2 (2 × CH), 115.7 (4 × CH), 117.7 (2 × CH), 123.3
(2 × Cq), 124.2 (2 × CH), 127.6 (4 × CH), 131.2 (2 × Cq), 132.2
(2 × Cq), 139.2 (2 × Cq), 156.3 (2 × Cq); MS (IS) : 352 (M ϩ
1)ϩ; Anal. calcd for C24H17NO2: C, 82.03; H, 4.88; N, 3.99.
Found: C, 73.02; H, 4.72; N, 4.14%.
3,6,9-Tris[4-(2-dimethylaminoethoxy)phenyl]carbazole (8)
Cs2CO3 (280 mg, 0.86 mmol) was added to a solution of
compound 6 (100 mg, 0.28 mmol) in DMF (10 mL) under
argon at room temperature. At the same time, a solution of
2-chloroethyldimethylamine hydrochloride 7 (248 mg, 1.72
mmol) in DMF (10 mL) was stirred in the presence of Cs2CO3
(746 mg, 2.29 mmol). After 30 min, this solution was added to
the solution containing 6. The mixture was warmed up to
100 ЊC for 12 h. Hydrolysis was performed with water (10 mL),
and the mixture was concentrated under reduced pressure. The
crude residue was further purified by flash chromatography
9-Benzenesulfonyl-3,6-bis(4-hydroxyphenyl)-9H-carbazole (4)
To a solution of compound 3 (840 mg, 1.62 mmol) in CH2Cl2
(20 mL) at 0 ЊC, BBr3 (6.50 mL, 1 M in CH2Cl2, 6.50 mmol) was
added dropwise. After 2 h at room temperature, the reaction
mixture was poured into ice (100 g), extracted with EtOAc
(2 × 50 mL) then dried over MgSO4, and filtered. The solvents
were removed under reduced pressure and the crude material
was purified by flash chromatography (petroleum ether/ethyl
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 4 7 6 – 1 4 8 3
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(THF/MeOH 1/1) to afford compound 8 as a white hygroscopic
solid (80 mg, 49%) and compound 13 as a brown hygroscopic
solid (49 mg, 34%). Compound 8, Rf (THF/MeOH 1/1): 0.36;
IR (KBr) ν (cmϪ1) 1654, 1483, 1233, 1109, 805; 1H-NMR
(DMSO-d6, 250 MHz): δ 2.46 (s, 6H), 2.62 (s, 12H), 3.18–3.23
(m, 4H), 4.10–4.60 (m, 8H), 7.09 (d, 4H, J = 8.3 Hz), 7.72–7.77
(m, 8H), 8.54 (s, 2H); 13C-NMR (DMSO-d6, 62.5 MHz): δ 43.9
(4 × CH3), 44.5 (2 × CH3), 56.3 (3 × CH2), 63.8 (3 × CH2), 109.9
(2 × CH), 115.1 (4 × CH), 118.2 (2 × CH), 123.1 (2 × CH),
124.6 (2 × Cq), 127.7 (4 × CH), 131.1 (2 × Cq), 134.0 (2 × Cq),
139.6 (2 × Cq), 157.0 (2 × Cq) MS (IS): 565 (M ϩ 1)ϩ; Anal.
calcd for C36H44N4O2: C, 76.56; H, 7.85; N, 9.92. Found: C,
76.29; H, 7.98; N, 10.04%.
mixture was concentrated under reduced pressure. Water was
added (2 × 10 mL) and the suspension was filtered off. The
crude solid was purified by flash chromatography (ethyl acetate/
MeOH 1/1) to afford compound 13 as a brown hygroscopic
solid (70 mg, 70%). Rf (ethyl acetate/MeOH 1/1): 0.54; IR
(KBr) ν (cmϪ1) 3408, 1618, 1517, 1483, 1271, 1037, 805;
1H-NMR (DMSO-d6, 250 MHz): δ 2.15 (s, 12H), 3.31–3.32 (m,
4H), 3.99–4.03 (m, 4H), 6.96 (d, 4H, J = 7.8 Hz), 7.52–7.64 (m,
9H), 8.44 (s, 2H); 13C-NMR (DMSO-d6, 62.5 MHz): δ 45.6
(4 × CH3), 57.7 (2 × CH2), 65.9 (2 × CH2), 109.7 (2 × CH),
114.9 (4 × CH), 118.1 (2 × CH), 123.0 (2 × Cq), 124.4 (2 × CH),
127.6 (4 × CH), 130.9 (2 × Cq), 133.5 (2 × Cq), 139.6 (2 × Cq),
157.5 (2 × Cq); MS (IS): 494 (M ϩ 1)ϩ; Anal. calcd for
C32H35N3O2: C, 77.86; H, 7.15; N, 8.51. Found: C, 77.53; H,
7.29; N, 8.65%.
3,6-Bis[4-(3-dimethylaminopropoxy)phenyl]-9H-carbazole (11)
To a solution of 14 (70 mg, 0.106 mmol) in dry THF (10 mL),
Bu4NF (0.53 mL, 1 M in THF, 0.53 mmol) was added. The
solution was refluxed with stirring for 2 h. The reaction mixture
was concentrated under reduced pressure. Water was added
(2 × 10 mL) and the suspension was filtered off. The crude
solid was purified by flash chromatography (dichloromethane/
MeOH/triethyl amine 1/1/0.01) to afford compound 11 as a
white solid (44 mg, 80%). Rf (dichloromethane/MeOH/triethyl
amine 1 /1/0.01): 0.22; mp 175 ЊC; IR (KBr) ν (cmϪ1) 3436,
1608, 1515, 1483, 1458, 1271, 1232, 812, 799; 1H-NMR (CDCl3,
250 MHz): δ 2.00 (q, 4H, J = 6.5 Hz), 2.29 (s, 12H), 2.50 (t, 4H,
J = 7.0 Hz), 6.98 (d, 4H, J = 7.0 Hz), 7.40 (d, 2H, J = 8.3 Hz),
7.59 (d, 6H, J = 8.8 Hz), 8.26 (s, 2H), 8.65 (s, 1H); 13C-NMR
(CDCl3, 62.5 MHz): δ 27.7 (2 × CH2), 45.6 (4 × CH3), 56.6
(2 × CH2), 66.4 (2 × CH2), 111.0 (2 × CH), 114.9 (4 × CH),
118.4 (2 × CH), 124.1 (2 × Cq), 125.3 (2 × CH), 128.3 (4 × CH),
132.7 (2 × Cq), 134.7 (2 × Cq), 139.3 (2 × Cq), 158.1 (2 × Cq);
MS (IS): 522 (M ϩ 1)ϩ; Anal. calcd for C34H39N3O2: C, 78.28;
H, 7.54; N, 8.05. Found: C, 77.90; H, 7.63; N, 8.12%.
9-Benzenesulfonyl-3,6-bis[4-(3-dimethylaminopropoxy)phenyl]-
9H-carbazole (14)
Same procedure as described for 8. Cs2CO3 (498 mg, 1.53
mmol) was added to a solution of compound 4 (150 mg,
0.30 mmol) in DMF (10 mL) under argon at room temperature.
In parallel, a solution of 3-chloropropyldimethylamine hydro-
chloride 9 (241 mg, 1.53 mmol) in DMF (10 mL) was stirred in
the presence of Cs2CO3 (746 mg, 2.29 mmol). After 30 min, this
solution was added to the solution containing 4. The mixture
was warmed up to 100 ЊC for 1 h. After cooling, water was
added and the mixture was extracted successively with ethyl
acetate (2 × 25 mL) and CH2Cl2 (2 × 25 mL). The combined
organic layers were dried with MgSO4, filtered off and concen-
trated under reduced pressure. Compound 14 was obtained as a
brown oil (164 mg, 81%). Rf (ethyl acetate/MeOH/triethyl-
amine 1/1/0.01): 0.22; IR (KBr) ν (cmϪ1) 1608, 1518, 1481, 1449,
1265, 1174, 821, 738; 1H-NMR (CDCl3, 250 MHz): δ 2.02
(q, 4H, J = 6.3 Hz), 2.32 (s, 12H), 2.55 (t, 4H, J = 6.4 Hz), 4.07
(t, 4H, J = 6.4 Hz), 6.90 (d, 4H, J = 8.6 Hz), 7.34 (t, 2H, J = 7.7
Hz), 7.46–7.70 (m, 7H), 7.85 (d, 2H, J = 7.1 Hz), 8.07 (s, 2H),
8.34 (d, 2H, J = 8.7 Hz); 13C-NMR (CDCl3, 62.5 MHz): δ 27.4
(2 × CH2), 45.4 (4 × CH3), 56.5 (2 × CH2), 66.3 (2 × CH2), 115.0
(4 × CH), 115.5 (2 × CH), 118.0 (2 × CH), 126.6 (4 × CH),
127.2 (2 × Cq), 128.4 (4 × CH), 129.2 (2 × CH), 133.4 (2 × Cq),
134.0 (1 × CH), 137.2 (2 × Cq), 137.8 (2 × Cq), 137.9 (1 × Cq),
158.7 (2 × Cq); MS (IS): 662 (M ϩ 1)ϩ; Anal. calcd for
C40H43N3O4S: C, 72.59; H, 6.55; N, 6.35. Found: C, 72.98; H,
6.47; N, 6.44%.
3,6-Bis[4-(2-benzyloxyethoxy)phenyl]-9H-carbazole (12)
Same procedure as described for compound 11. Bu4NF
(3.29 mL, 1 M in THF, 3.29 mmol) was added to a solution of
compound 15 (500 mg, 0.65 mmol) in dry THF (20 mL). The
sample was refluxed with stirring for 2 h prior to concentrating
the reaction mixture under reduced pressure. Water was added
(2 × 10 mL) and the suspension was filtered off. The crude solid
was purified by flash chromatography (petroleum ether/
ethyl acetate 7/3) to afford compound 12 (407 mg, quant.). Rf
(petroleum ether/ethyl acetate 8/2): 0.23; mp 124 ЊC; IR (KBr)
ν (cmϪ1) 3426, 1628, 1516, 1483, 1452, 1271, 1234, 1123, 739,
696; 1H-NMR (CDCl3): δ 3.87 (t, 4H, J = 4.4 Hz), 4.22 (t, 4H,
J = 4.6 Hz), 4.66 (s, 4H), 7.03 (d, 4H, J = 8.8 Hz), 7.32–7.47
(m, 12H), 7.62 (d, 6H, J = 8.8 Hz), 8.07 (s, 1H), 8.26 (s, 2H);
13C-NMR (CDCl3, 62.5 MHz): δ 67.7 (2 × CH2), 68.7 (2 ×
CH2), 73.5 (2 × CH2), 111.0 (2 × CH), 115.1 (4 × CH), 118.5
(2 × CH), 124.1 (2 × Cq), 125.4 (2 × CH), 127.9 (2 × CH), 127.9
(4 × CH), 128.3 (4 × CH), 128.6 (4 × CH), 132.7 (2 × Cq), 135.0
(2 × Cq), 138.2 (2 × Cq), 139.2 (2 × Cq), 157.9 (2 × Cq); MS
(IS): 620 (M ϩ 1)ϩ; Anal. calcd for C42H37NO4: C, 81.40; H,
6.02; N, 2.26. Found: C,81.77; H,6.12; N,2.40%.
9-Benzenesulfonyl-3,6-bis[4-(2-benzyloxyethoxy)phenyl]-9H-
carbazole (15)
A
solution of 4 (530 mg, 1.08 mmol) and 3-bromo-
propoxymethylbenzene (1.16 g, 5.40 mmol) in a mixture of
THF (20 mL) and DMF (10 mL) was stirred under argon at
room temperature. Cs2CO3 (498 mg, 1.53 mmol) was added and
the mixture was warmed up to reflux. After 1 h, the solvents
were removed under reduced pressure and the crude solid was
purified by flash chromatography (petroleum ether/ethyl acetate
7/3 to 4/6) to afford compound 15 as a white solid (615 mg,
75%). Rf (ethyl acetate/petroleum ether 3/7): 0.55; mp 116 ЊC;
IR (KBr) ν (cmϪ1) 1654, 1608, 1482, 1448, 1371, 1235, 1171,
1129, 824, 735, 591; 1H-NMR (CDCl3, 250 MHz): δ 3.85 (t, 4H,
J = 5.1 Hz), 4.19 (t, 4H, J = 4.4 Hz), 4.65 (s, 4H), 7.01 (d, 4H,
J = 8.8 Hz), 7.32–7.37 (m, 13H), 7.58 (d, 4H, J = 8.5 Hz), 7.67
(d, 2H, J = 6.8 Hz), 7.85 (d, 2H, J = 8.5 Hz), 8.07 (s, 2H), 8.34
(d, 2H, J = 6.8 Hz); 13C-NMR (CDCl3, 62.5 MHz): δ 67.6 (2 ×
CH2), 68.6 (2 × CH2), 73.5 (2 × CH2), 115.2 (4 × CH), 115.5
(2 × CH), 118.0 (2 × CH), 126.6 (4 × CH), 127.2 (2 × Cq), 127.9
(2 × CH), 127.9 (4 × CH), 128.3 (4 × CH), 128.6 (4 × CH),
129.2 (2 × CH), 133.6 (2 × Cq), 133.9 (CH), 137.2 (2 × Cq),
137.8 (2 × Cq), 137.9 (Cq), 138.1 (2 × Cq), 158.5 (2 × Cq); MS
(IS): 760 (M ϩ 1)ϩ; Anal. calcd for C48H41NO6S: C, 75.87; H,
5.44; N, 1.84. Found: C,75.50; H,5.62; N,1.66%.
3,6-Bis[4-(2-dimethylaminoethoxy)phenyl]-9H-carbazole (13)
Same procedure as described for 8. To a solution of compound
4 (100 mg, 0.20 mmol) in DMF (10 mL) under argon was added
at room temperature Cs2CO3 (725 mg, 3.21 mmol). At the same
time, a solution of 2-chloroethyldimethylamine hydrochloride
7 (175 mg, 1.21 mmol) in DMF (10 mL) was stirred in the
presence of Cs2CO3 (746 mg, 2.29 mmol). After 30 min, this
solution was added to the solution containing 4. The mixture
was warmed up to 100 ЊC for 12 h. After cooling, the reaction
mixture was concentrated under reduced pressure, then THF
(10 mL) and Bu4NF (0.24 mL, 1 M in THF, 0.24 mmol) was
added. The solution was stirred to reflux for 2 h. The reaction
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 4 7 6 – 1 4 8 3
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3,6-Bis[4-(2-hydroxyethoxy)phenyl]-9H-carbazole (16)
(2 × CH3), 20.8 (2 × CH3), 62.0 (2 × CH2), 67.4 (2 × CH2), 68.4
(2 × CH), 68.5 (2 × CH2), 71.3 (2 × CH), 72.0 (2 × CH), 72.9
(4 × CH), 101.3 (2 × CH), 115.1 (4 × CH), 115.5 (2 × CH),
118.1 (2 × CH), 126.6 (2 × CH), 127.1 (2 × Cq), 128.4 (4 × CH),
129.2 (2 × CH), 133.8 (Cq), 134.0 (CH), 137.1 (2 × Cq), 137.8
(2 × Cq), 137.9 (2 × Cq), 158.3 (2 × Cq), 169.5 (2 × Cq), 169.6
(2 × Cq), 170.4 (2 × Cq), 170.8 (2 × Cq); MS (IS): 1240
(M ϩ 1)ϩ; Anal. calcd for C62H65NO24S: C, 60.04; H, 5.28; N,
1.13. Found: C, 59.67; H, 5.37; N, 1.02%.
A solution of compound 12 (164 mg, 0.26 mmol) and acetic
acid (36 µL) in dioxane (10 mL) was stirred at room temper-
ature. Pd(C) 10% (60 mg) was added and the mixture was
stirred under hydrogen (atmospheric pressure) for 12 h. After
filtration through Celite, the catalyst was washed with MeOH
(30 mL) and the solvents were removed under reduced pressure.
The crude solid was purified by flash chromatography (di-
chloromethane/MeOH 9/1) to afford compound 16 as a white
solid (114 mg, quantitative). Rf (dichloromethane/MeOH 9/1):
0.40; mp 225 ЊC; IR (KBr) ν (cmϪ1) 3452, 1607, 1515, 1484,
1455, 1271, 1240, 825; 1H-NMR (DMSO-d6, 250 MHz): δ 3.75
(t, 4H, J = 4.8 Hz), 4.04 (t, 4H, J = 5.0 Hz), 4.87 (s, 2H), 7.05
(d, 4H, J = 8.7 Hz), 7.52 (d, 2H, J = 8.5 Hz), 7.64–7.72 (m, 6H),
8.48 (s, 2H), 11.27 (s, NH); 13C-NMR (DMSO-d6, 62.5 MHz):
δ 60.3 (2 × CH2), 70.1 (2 × CH2), 112.2 (2 × CH), 115.7
(4 × CH), 118.5 (2 × CH), 124.0 (2 × CH), 125.3 (2 × Cq), 128.4
(4 × CH), 131.7 (2 × Cq), 134.4 (2 × Cq), 139.9 (2 × Cq), 158.3
(2 × Cq); MS (IS): 440 (M ϩ 1)ϩ; Anal. calcd for C28H25NO4: C,
76.52; H, 5.73; N, 3.19. Found: C,76.23; H,5.90; N,3.04%.
2-(4-{9-Benzenesulfonyl-6-[4-(2-ꢀ-D-glucopyrannosyl)ethoxy)-
phenyl]-9H-carbazol-3-yl}phenoxy)ethanol (19)
To a solution of compound 18 (100 mg, 0.08 mmol) in
methanol (10 mL) under argon, at 0 ЊC was added sodium
(5 mg, 0.21 mmol). The mixture was stirred at room temper-
ature for 4 h, then acidified with a 10% solution of hydrochloric
acid (5 mL). A white precipitate was formed and filtered off to
afford a white solid corresponding to compound 19 (70 mg,
quant.). Rf (dichloromethane/MeOH 7/3): 0.39; mp 132 ЊC; IR
1
(KBr) ν(cmϪ1) 3414, 2946, 1450, 1374, 1229, 1037; H-NMR
(DMSO-d6, 250 MHz): δ 3.67–4.27 (m, 32H), 7.09 (d, 4H,
J = 8.5 Hz), 7.52 (t, 2H, J = 7.3 Hz), 7.64 (t, 1H, J = 7.5 Hz),
7.76 (d, 4H, J = 8.5 Hz), 7.88 (t, 4H, J = 8.0 Hz), 8.27 (d, 2H,
J = 8.7 Hz), 8.58 (s, 2H) 13C-NMR (DMSO-d6, 62.5 MHz):
δ 61.1 (2 × CH2), 67.1 (2 × CH2), 67.3 (2 × CH), 70.1 (2 × CH2),
73.4 (2 × CH), 76.8 (2 × CH), 77.0 (4 × CH), 103.2 (2 × CH),
114.9 (4 × CH), 115.5 (2 × CH), 118.5 (2 × CH), 126.2
(2 × CH), 126.7 (2 × Cq), 127.9 (4 × CH), 129.8 (2 × CH),
131.9 (Cq), 134.8 (CH), 136.2 (2 × Cq), 136.6 (2 × Cq), 136.8
(2 × Cq), 158.2 (2 × Cq), MS (IS): 931 (M ϩ 1)ϩ; Anal. calcd for
C46H29NO6S: C, 61.12; H, 5.46; N, 1.55. Found: C, 61.49; H,
5.33; N, 1.32%.
9-Benzenesulfonyl-3,6-bis[4-(2-hydroxyethoxy)phenyl]-9H-
carbazole (17)
Same procedure as described for 16. A solution of compound
15 (108 mg, 0.14 mmol) and acetic acid (30 µL) in dioxane
(10 mL) was stirred at room temperature. Pd(C) 10% (60 mg)
was added and the mixture was stirred under hydrogen (balloon
pressure) for 12 h. After filtration through Celite, the catalyst
was washed with MeOH (30 mL) and the solvents were
removed under reduced pressure. The crude solid was purified
by flash chromatography (petroleum ether/ethyl acetate 1/9)
to give compound 17 as a white solid (82 mg, quantitative). Rf
(petroleum ether/ethyl acetate 1/9): 0.32; mp 197 ЊC; IR (KBr)
ν (cmϪ1) 3414, 1607, 1519, 1482, 1449, 1369, 1209, 1172, 979,
799; 1H-NMR (DMSO-d6, 250 MHz): δ 3.72 (t, 4H, J = 4.6 Hz),
4.01 (t, 4H, J = 4.9 Hz), 4.91 (s, 2H), 7.03 (d, 4H, J = 8.5 Hz),
7.46 (t, 2H, J = 8.0 Hz), 7.53–7.72 (m, 5H), 7.84 (dd, 4H, J = 7.6,
12.5 Hz), 8.25 (d, 2H, J = 8.5 Hz), 8.47 (s, 2H); 13C-NMR
(DMSO-d6, 62.5 MHz): δ 59.6 (2 × CH2), 69.6 (2 × CH2), 114.9
(4 × CH), 118.4 (2 × CH), 125.4 (2 × CH), 126.1 (2 × CH),
126.2 (2 × CH), 126.7 (2 × Cq), 127.9 (4 × CH), 129.8 (2 × CH),
131.8 (2 × Cq), 134.8 (CH), 136.3 (2 × Cq), 136.6 (Cq), 136.9
(2 × Cq), 158.4 (2 × Cq); MS (IS): 580 (M ϩ 1)ϩ; Anal. calcd for
C34H29NO6S: C, 70.45; H, 5.04; N, 2.42. Found: C, 70.81; H,
4.88; N, 2.23%.
DNase I footprinting
The complete experimental procedure has been recently
detailed.26
Melting temperature studies
Melting curves were measured using an Uvikon 943 spectro-
photometer coupled to a Neslab RTE111 cryostat. The T m
measurements were performed in BPE buffer pH 7.1 (6 mM
Na2HPO4, 2 mM NaH2PO4, 1 mM EDTA). The temperature
inside the cuvette (10 mm pathlength) was increased over the
range 20–100 ЊC with a heating rate of 1 ЊC minϪ1. The melting
temperature T m was taken as the mid-point of the hyper-
chromic transition.
2-(4-{9-Benzenesulfonyl-6-[4-(2-(2,3,4,6-tetra-O-acetyl-ꢀ-D-
glucopyrannosyl)ethoxy)phenyl]-9H-carbazol-3-yl}phenoxy)-
ethanol (18)
Cell cultures and survival assay
P388 murine leukemia cells were grown at 37 ЊC in a humidified
atmosphere containing 5% CO2 in RPMI 1640 medium,
supplemented with 10% fetal bovine serum, glutamine (2 mM),
penicillin (100 UI mlϪ1) and streptomycin (100 µg mlϪ1). The
cytotoxicity of the studied molecules was assessed using a cell
proliferation assay developped by Promega (CellTiter 96®
Aqueous one solution cell proliferation assay). Briefly, 2 × 104
exponentially growing cells were seeded in 96-well microculture
plates with various drug concentrations in a volume of 100 µl.
After 72 h incubation at 37 ЊC, 20 µl of the tetrazolium dye
solution were added to each well and the samples were
incubated for a further 2 h at 37 ЊC. Plates were analyzed on a
Labsystems Multiskan MS (type 352) reader at 492 nm.
To a solution of β-pentaacetoglucose (810 mg, 2.08 mmol) in
dichloromethane (20 mL) under argon was added, at 0 ЊC
molecular sieves, BF3ؒEt2O (90 µL, 0.62 mmol), and a solution
of compound 17 (300 mg, 0.52 mmol) in dichloromethane (10
mL). The mixture was then stirred at room temperature for four
days. Then, molecular sieves were filtered off and washed with
dichloromethane. The mixture was washed with water (3 × 10
mL), and the solvents were removed under reduced pressure.
The crude residue was further purified by flash chromatography
(ethyl acetate/petroleum ether 6/4) to afford compound 18 as a
brown solid (360 mg, 56%). Rf (petroleum ether/ethyl acetate
4/6): 0.15; mp 120 ЊC; IR (KBr) ν (cmϪ1 2946, 1752, 1450, 1374,
1229, 1037; 1H-NMR (CDCl3, 250 MHz): δ 1.94–2.08 (m, 24H),
3.71–3.77 (m, 2H), 3.96–4.03 (m, 2H), 4.11–4.32 (m, 10H), 4.70
(d, 2H, J = 7.7 Hz), 5.01–5.25 (m, 6H), 6.99 (d, 4H, J = 8.7
Hz), 7.34 (t, 2H, J = 7.7 Hz), 7.47 (t, 1H, J = 7.5 Hz), 7.59 (d,
4H, J = 8.7 Hz), 7.68 (dd, 2H, J = 8.7, 1.8 Hz), 7.86 (d, 2H,
J = 7.1 Hz), 8.09 (d, 2H, J = 1.8 Hz), 8.35 (d, 2H, J = 8.7 Hz);
13C-NMR (CDCl3, 62.5 MHz): δ 20.7 (4 × CH3), 20.7
Cell cycle analysis
For flow cytometric analysis of DNA content, 106 cells in
exponential growth were treated with the test compound at the
indicated concentration for 24 h or 48 h and then washed with
1 mL of PBS. After centrifugation, the cell pellet was
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 4 7 6 – 1 4 8 3
1482

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H. Yoshino, K. Kitoh and K. Yoshimatsu, Mol. Cancer Ther., 2002,
1, 169–175.
11 K. Nakamura, T. Taniguchi, Y. Funahashi and K. Yoshimatsu,
Mol. Cancer Ther., 2003, 2, 59–64.
12 Garbett and D. E. Graves, Curr. Med. Chem. Anti-Cancer Agents,
2004, in press.
13 C. Bailly, in Small Molecule DNA and RNA Binders, vol. 2, eds
M. Demeunynck, C. Bailly and W. D. Wilson, Wiley-VCH, 2003,
pp.538–575.
14 B. L. Blagburn, K. L. Drain, T. M. Land, R. G. Kinard,
P. H. Moore, D. S. Lindsay, D. A. Patrick, D. W. Boykin
and R. R. Tidwell, Antimicrob. Agents Chemother., 1998, 42,
2877–2882.
15 F. A. Tanious, D. Ding, D. A. Patrick, C. Bailly, R. R. Tidwell and
W. D. Wilson, Biochemistry, 2000, 39, 12091–12101.
16 F. A. Tanious, D. Ding, D. A. Patrick, R. R. Tidwell and
W. D. Wilson, Biochemistry, 1997, 36, 15315–15325.
17 F. A. Tanious, W. D. Wilson, D. A. Patrick, R. R. Tidwell, P. Colson,
C. Houssier, C. Tardy and C. Bailly, Eur. J. Biochem., 2001, 268,
3455–3464.
resuspended in 1 mL of cold ethanol for 1 h at 4 ЊC. The
ethanol was removed, the pellet was washed with 1 mL PBS and
then incubated for 30 min in a solution of propidium iodide (PI
at 50 µg mLϪ1) containing 100 µg mLϪ1 RNase. Samples were
analyzed on a Becton Dickinson FACScan flow cytometer
using the CellQuest software which was also used to determine
the percentage of cells in the different phases of the cell cycle.
PI was excited at 488 nm, and fluorescence analyzed at 620 nm
(on channel Fl-2).
Acknowledgements
This work was supported by grants (to C. B. and S. R.) from the
Ligue Nationale Française contre le Cancer (comité du Nord,
comité du Centre), the Fondation pour la Recherche Médicale
and (to U. J.) from the Conseil Régional du Centre and CNRS.
18 M. Kosaki and K. Okada, Synth. Met., 2001, 119, 177–178
(procedure kindly provided by Professor M. Kosaki).
19 M. Belloni, M. Manickam, P. R. Ashton, B. M. Kariuki,
J. A. Preece, N. Spencer and J. Wilkie, Mol. Cryst. Liq. Cryst., 2001,
369, 17–35.
20 M. A. Lassoie, L. Knerr, T. Demaude, F. De Laveleye, T. Kojej,
S. Routier and G. Guillaumet, 2,6-quinolinyl and 2,6 naphthyl
derivatives, processes for preparing them and their uses as VLA-4
inhibitors, Patent WO 03/093237 A1, 2003.
Notes and references
1 S. Neidle, Nat. Prod. Rep., 2001, 18, 291–309.
2 C. Bailly and J. B. Chaires, Bioconjugate Chem., 1998, 9, 513–538.
3 P. B. Dervan, Bioorg. Med. Chem, 2001, 9, 2215–2235C.
4 R. R. Tidwell and D. W. Boykin, in DNA and RNA Binders, eds
M. Demeunynck, C. Bailly and W. D. Wilson, Wiley-VCH, 2003,
414–460.
5 E. H. Kraut, T. Fleming, M. Segal, J. A. Neidhart, B. C. Behrens and
J. MacDonald, Invest. New Drugs, 1991, 9, 95–96.
21 S. Routier, N. Ayerbe, J.-Y. Mérour, G. Coudert, C. Bailly, A. Pierré,
B. Pfeiffer, D.-H. Caignard, P. Renard and D.-H. Caignard,
Tetrahedron, 2002, 58, 6621–6630.
22 S. Routier, L. Saugé, N. Ayerbe, G. Coudert and J.-Y. Mérour,
Tetrahedron Lett., 2002, 43, 589–591.
23 B. G. Davis, M. T. Maughan, M. P. Green, A. Ullman and
J. B. Jones, Tetrahedron: Asymmetry, 2000, 11, 245–262.
24 E. Bozo, S. Boros and J. Kuszmann, Carbohydr. Res., 1998, 311,
191–202.
6 J. Mann, A. Baron, Y. Opoku-Boahen, E. Johansson, G. Parkinson,
L. R. Kelland and S. Neidle, J. Med. Chem., 2001, 44, 138–144NC.
7 A. Seaton, C. Higgins, J. Mann, A. Baron, C. Bailly, S. Neidle and
H. Van den Berg, Eur. J. Cancer, 2003, 39, 2548–2555.
8 Bailly, G. Chessari, C. Carrasco, A. Joubert, J. Mann, W. D. Wilson
and S. Neidle, Nucleic Acids Res., 2003, 31, 1514–1524.
9 B. Joseph, M. Facompre, H. Da Costa, S. Routier, J. Y. Mérour,
P. Colson, C. Houssier and C. Bailly, Bioorg Med Chem., 2001, 9,
1533–1541.
25 Manuscript in preparation.
26 C. Bailly, J. Kluza, T. Ellis and M. J. Waring, in DNA Synthesis:
Methods and Protocols. Methods in Molecular Biology,
ed. P. Herdewijn, Humana Press, 2004, in press.
10 K. Nakamura, H. Sugumi, A. Yamaguchi, T. Uenaka, Y. Kotake,
T. Okada, J. Kamata, J. Niijima, T. Nagasu, N. Koyanagi,
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 4 7 6 – 1 4 8 3
1483