Ellipticines and 9-acridinylamines as inhibitors of d-alanine:d-alanine ligase

Article abstract of DOI:10.1016/j.bmc.2011.07.020

d-Alanine:d-alanine ligase (Ddl), an intracellular bacterial enzyme essential for cell wall biosynthesis, is an attractive target for development of novel antimicrobial drugs. This study focused on an extensive evaluation of two families of Ddl inhibitors encountered in our previous research. New members of both families were obtained through similarity search and synthesis. Ellipticines and 9-acridinylamines were both found to possess inhibitory activity against Ddl from Escherichia coli and antimicrobial activity against E. coli and Staphylococcus aureus. Ellipticines with a quaternary methylpyridinium moiety were the most potent among all studied compounds, with MIC values as low as 2 mg/L in strains with intact efflux mechanisms. Antimicrobial activity of the studied compounds was connected to membrane damage, making their development as antibacterial drug candidates unlikely unless analogues devoid of this nonspecific effect can be discovered.

Full text of DOI:10.1016/j.bmc.2011.07.020

Bioorganic & Medicinal Chemistry 19 (2011) 5137–5146
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Ellipticines and 9-acridinylamines as inhibitors of D-alanine:D-alanine ligase
a
b
a
c
c
c
ˇ
ˇ b
Blaz Vehar , Martina Hrast , Andreja Kovac , Janez Konc , Katherine Mariner , Ian Chopra , Alex O’Neill ,
b,
ˇ ˇ a
⇑
Dušanka Janezic , Stanislav Gobec
a National Institute of Chemistry, Hajdrihova 19, 1000 Ljubljana, Slovenia
b
ˇ
Faculty of Pharmacy, University of Ljubljana, Aškerceva 7, 1000 Ljubljana, Slovenia
c Institute of Molecular and Cellular Biology and Antimicrobial Research Centre, University of Leeds, Leeds LS2 9JT, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
D-Alanine:D-alanine ligase (Ddl), an intracellular bacterial enzyme essential for cell wall biosynthesis, is
Received 1 June 2011
Revised 11 July 2011
Accepted 12 July 2011
Available online 8 August 2011
an attractive target for development of novel antimicrobial drugs. This study focused on an extensive
evaluation of two families of Ddl inhibitors encountered in our previous research. New members of both
families were obtained through similarity search and synthesis. Ellipticines and 9-acridinylamines were
both found to possess inhibitory activity against Ddl from Escherichia coli and antimicrobial activity
against E. coli and Staphylococcus aureus. Ellipticines with a quaternary methylpyridinium moiety were
the most potent among all studied compounds, with MIC values as low as 2 mg/L in strains with intact
efflux mechanisms. Antimicrobial activity of the studied compounds was connected to membrane dam-
age, making their development as antibacterial drug candidates unlikely unless analogues devoid of this
nonspecific effect can be discovered.
Keywords:
Antimicrobials
Similarity search
Synthesis
Bacterial ligase
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
because of its psychotropic side effects it is only considered a sec-
ond line drug for treatment of tuberculosis.4,8 Several other Ddl
Peptidoglycan is a cross-linked glycopeptide polymer that func-
tions as a vital component of the bacterial cell wall. It provides bac-
teria with the necessary structural integrity to withstand the
osmotic pressure gradient between the cytoplasm and the cell
exterior. Disruption of peptidoglycan assembly is a known and val-
idated mechanism for triggering bacterial cell lysis.1
inhibitors have been discovered, but none are routinely used in
antibacterial therapy.9–20 Most suffer from poor antibacterial activ-
ity, or toxicity. Therefore new inhibitors of Ddl would be desirable.
In our previous research, a structure-based virtual screen was
performed to identify new inhibitors of DdlB using the AutoDock
program with a ligand database of 1990 compounds from the
NCI Diversity Set.21 Several new and structurally diverse com-
pounds were discovered that inhibit DdlB with IC50 values in the
micromolar concentration range. Compound 1, a 9-acridinylamine
(Table 1), and compound 2, a 9-methoxyellipticine derivative (Ta-
ble 2), were also shown to possess antibacterial activity against
both Gram-positive and Gram-negative bacteria. Both were se-
lected as starting compounds for further drug design efforts.
In this study, compounds 1 and 2 were taken as templates for
derivatisation and an exploratory structure–activity relationship
study. Their analogues were obtained through similarity search
and synthetic approaches and were tested for inhibitory activity
against the DdlB enzyme as well as for antimicrobial activity
against several strains of pathogenic bacteria.
D
-Alanine:
essential building block in the intracellular steps of peptidoglycan
assembly. Its product, -alanyl– -alanine, is the substrate for MurF,
D-alanine ligase is a bacterial enzyme that supplies an
D
D
the final enzyme of the Mur ligase pathway which assembles the
intracellular peptidoglycan precursor, UDP-MurNAc-pentapeptide.
The terminal D-Ala–D-Ala dipeptide plays a crucial role in the extra-
cellular steps of peptidoglycan assembly where its peptide bond is
broken and a new bond is formed to cross-link adjacent peptido-
glycan chains.2–5
Two Ddl isoforms, DdlA and DdlB, exist in Escherichia coli and
Salmonella typhimurium. They share 35% amino-acid sequence
identity and express similar kinetic characteristics, substrate spec-
ificity and susceptibility to known inhibitors. Our research was
based on the more extensively studied DdlB isoform.6,7
D
-Cycloserine, a structural analogue of D-Ala, is the only Ddl
2. Results and discussion
2.1. Similarity search
inhibitor that has been used as a therapeutic agent. Nevertheless,
Abbreviations: Ddl, d-alanine:d-alanine ligase; MurNAc, N-acetyl-muramoyl;
NCI, National Cancer Institute; NSC, National Service Center.
To extend the chemical space in our study beyond the NCI
Diversity Set, a similarity search was performed for each of our
⇑
Corresponding author. Tel.: +386 1 476 9585; fax: +386 1 425 8031.
E-mail address: gobecs@ffa.uni-lj.si (S. Gobec).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.07.020

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B. Vehar et al. / Bioorg. Med. Chem. 19 (2011) 5137–5146
Table 1
9-Acridinylamine derivatives from similarity search and their antimicrobial activity
Compound
NSC no. Similar to
Structurea
IC50
lM
,
MIC, mg/L
E. coli
Membrane integrity in S.
aureus, %
1,%
E. coli
S. aureus
1411
SM1411
SH1000
R
R
R
N
1(reference) 130813 100
162
320
>256
256
64
32b
48.7 9.1
ndc
N
OH
N
3
4
5
10666
31711
12516
95
95
94
128
128
OH
N
161
102
>256
256
>256
256
32
64
47.7 5.3
OH
R
N
ndc
OH
Cl
H
N
N
MeO
6
130106 92
119
>256
>256
>256
ndc
N
OH
Cl
R
ndc
ndc
N
7
8
130109 92
135
32
>256
>256
128
256
OH
O
R
N
28557
92
>256
>256
H
N
R
O
9
192961 92
90
>256
>256
256
ndc
N
HN
N
a
MeO
R =
Cl
b
c
MIC determined for S. aureus 8325-4, the parent strain of SH1000.
not determined because MIC for S. aureus P64 mg/L.
starting compounds to find sufficiently similar structures in the
whole NCI database. Two series of compounds were the result:
13 analogues of 9-acridinylamine similar to compound 1 and 26
ellipticine analogues similar to compound 2. Only 19 of these 39
compounds were available in sufficient quantities (>5 mg) from
the National Cancer Institute and were obtained as solid samples.
Five of the obtained compounds were excluded from further test-
ing due to toxicity concerns, poor solubility or tendency to form
aggregates. All data from this similarity search including structures
of the unavailable and the excluded compounds are gathered in
Supplementary Tables S1 and S2.
The remaining 14 compounds were evaluated for inhibition of
DdlB from E. coli in 250 lM concentrations using the malachite
green assay, followed by determination of IC50 values, and for anti-
microbial activity through determination of minimal inhibitory
concentrations on two strains of E. coli and one strain of S. aureus.
Six of the seven 9-acridinylamine analogues (Table 1) showed
improved enzyme inhibition over their starting compound 1. Com-
pounds 8 and 9, both with a 6-methoxy-8-quinolinyl moiety at-
tached to the general 9-acridinylamine frame, showed promising
IC50 values but lacked any significant microbiological activity.
Compounds 4 and 5, close analogues of 1 with a diethylamine
group and a pyrollidine ring, respectively, replacing the original
piperazine moiety, showed microbiological activity against S. aur-
eus but failed to surpass the starting compound.
Out of seven ellipticine analogues (Table 2), compounds 10–13
exhibited similar or improved IC50 values against DdlB compared
to their starting compound 2. Compounds 10, 12 and 13 showed
promising antimicrobial activity against S. aureus. All three were
also potent antimicrobial agents in the E. coli SM1411 strain de-
leted for the AcrAB efflux pump, with MIC values between 1 and
8 mg/L. Compound 12 was the only similarity search product to

B. Vehar et al. / Bioorg. Med. Chem. 19 (2011) 5137–5146
5139
Table 2
9-Methoxyellipticine derivatives from similarity search and their antimicrobial activity
Compound
NSC no. Similar to
Structure
IC50
lM
,
MIC, mg/L
E. coli
SM1411
Membrane integrity in S.
aureus, %
2,%
E. coli
1411
S. aureus
SH1000
N
O
2(reference) 176327 100
70
32
8
32a
Not determined
N
NH2
N+I-
O
O
10
11
12
155693 96
65
60
43
256
>256
64
8
32
16.0 2.2
N
H
N
69187
95
>256
>256
ndb
N
H
N+I-
155694 95
1
4
17.8 1.2
O
N
N+Ac-
O
O
13
627505 93
89
256
4
64
ndb
N
H
N
Cl
14
15
16
352738 92
317605 92
352737 92
rac 40% 128
32
256
ndb
ndb
ndb
N
H
N
O
N
Cl
Cl
623
330
256
32
256
N
H
N
O
N
>256
>256
>256
N
H
N
2HCl
a
b
c
MIC determined for S. aureus 8325-4, the parent strain of SH1000.
not determined because MIC for S. aureus P64 mg/L.
residual activity at 500 lM; IC50 not measured.
ally, methylation of one or both ellipticine nitrogen atoms was
considered.
possess significant antimicrobial activity against the native E. coli
strain.
The synthetic route for production of ellipticine derivatives is
depicted in Scheme
1 and follows the methods previously
2.2. Synthesis of structural analogues
described.22,23 The ellipticines were obtained from substituted
indoles. First, indoles 17a–e were condensed with acetonylacetone
to obtain corresponding dimethylcarbazoles 18a–e. Vilsmeier–
Haack formylation by N-methylformanilide and POCl3 gave 19a–
e. Schiff bases were formed with aminoacetaldehyde diethyl acetal,
followed immediately by reduction to amine with NaBH4.
Based on promising MIC values demonstrated thus far, ellipt-
icines were selected for further research via chemical synthesis.
Several substitution patterns at positions 7–9 were explored
(see Scheme 1): all-H (series a), 9-methoxy (b), 9-bromo (c), 8-
fluoro (d) and 7-ethyl (e) derivatives were prepared. Addition-

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B. Vehar et al. / Bioorg. Med. Chem. 19 (2011) 5137–5146
O
NH
R1
R2
R1
R2
R1
R2
R1
O
O
1
2
3
N
N
N
N
R2
H
H
H
H
R3
R3
R3
19 a e
R3
17 a e
18 a e
20 a e
–
–
–
–
N
N
N+I-
R1
R2
R1
R2
R1
R2
4
5
6
N
N
N
H
R3
R3
R3
22 a d
–
23 c
–
21 a e
6
R1
R2
R3
N+I-
a
b
c
d
e
-H
-H
-H
-H
-F
-H
-H
-H
-H
-H
-Et
-OMe
-Br
-H
R1
R2
N
H
R3
-H
24 a, c, d
Scheme 1. Synthetic route for production of ellipticine derivatives. Reagents and conditions used: (1) hexane-2,5-dione, pTSA, EtOH, reflux, 1.5 h; (2) POCl3,
N-methylformanilide, 1,2-dichlorobenzene, 105 °C, 4 h; (3) Aminoacetaldehyde diethyl acetal, 115 °C, 3 h; NaBH4, EtOH, 1 h; (4) Na2CO3, TsCl, THF, 2 h; 6 M HCl, dioxane,
reflux, 2 h; (5) NaH, MeI, DMF, 12 h; (6) MeI, acetone, 2 days.
Compounds 20a and 20b were then tosylated and cyclised in 6 M
HCl in dioxane to yield ellipticine (21a) and 9-methoxyellipticine
(21b), respectively. To obtain better yields, microwave irradiation
was used for cyclisation of tosylated 20c–e.24 Methylation of indole
nitrogens of 21a–d was performed to afford N-methylated prod-
ucts 22a–d. Finally, 21a, 21c, 21d and 22c were treated with iodo-
methane to give iodide salts 24a, 24c, 24d and 23c. Eleven
ellipticine derivatives obtained through synthesis were evaluated
for activity against DdlB (Table 3).
However, we noted a poor overall correlation between enzyme
inhibitor activity and antimicrobial activity (Tables 1–3), suggest-
ing the possibility that off-target activity25 of the inhibitors might
also contribute to antimicrobial activity. Since non-specific mem-
brane disruption can contribute to the antimicrobial activity of
experimental inhibitors26 we determined whether the compounds
described here could cause membrane damage in S. aureus SH1000
at 4 Â MIC, measured by the BacLight™ assay. We were unable to
test inhibitors displaying high MICs because of solubility problems
with the compounds in this assay. With the exception of 21a, all
other compounds that were tested decreased membrane integrity
by >50% (Tables 1–3).
Some hints about the structure–activity relationship for ellipti-
cines can already be concluded from this study: (i) the presence of
a quaternary methylpyridinium ion at position 2 improves IC50 val-
ues, but exacerbates membrane damage; (ii) -OMe or -Br substitu-
tion at position 9 can be beneficial for inhibitor activity; (iii) -F
substitution at position 8 or -Et substitution at position 7 can cause
diminished antimicrobial activity.
N6-Methylated compounds 22b–d were poorly soluble, causing
difficulties in antimicrobial activity determination. Other synthe-
sized ellipticines did exhibit inhibitory activity against DdlB and
antimicrobial activity against several bacterial strains. Compounds
10–13 were considered in the following analysis as members of the
9-methoxy substituted series b.
N2-Methylated quaternary compounds 24a, 10, 12, 23c, 24c and
24d showed improved MIC values over their analogues, as well as
higher solubility. Notably, to achieve low MIC values against the
native strain of E. coli and IC50 values under 100 lM against DdlB,
a quaternary methylpyridinium moiety was necessary.
Non-methylated ellipticines 21a, 21c and 21d surprisingly
demonstrated improved MIC values compared with their 9-meth-
oxy analogue 11. Moreover, 21a managed to match its quaternary
counterpart 24a and was the only ellipticine without a methyl-
pyridinium moiety in this study to significantly inhibit growth of
the native E. coli strain.
The all-H, 9-methoxy and 9-bromo substitution patterns gener-
ally resulted in active compounds, while 8-fluoro and 7-ethoxy
ellipticines lacked either inhibitor activity or antimicrobial activity,
or both. The exception to this rule was the non-methylated 8-flu-
oroellipticine (21d) with results largely comparable to its bioiso-
stere 21a.
3. Conclusions
A range of ellipticine and 9-acridinylamine derivatives have
been obtained through similarity search and chemical synthesis.
Both classes of compounds exhibit inhibitory activity against DdlB
and antimicrobial activity against E. coli and S. aureus. Some of the
compounds appear to be substrates for the AcrAB efflux pump in
E. coli since their anti-bacterial activities were enhanced in the
AcrAB-deficient mutant E. coli SM1411.
Ellipticines were generally more potent than 9-acridinylamines
in all assays that were performed, but probably achieve part of
their antimicrobial action by damaging the bacterial cytoplasmic
membrane. This makes them unlikely to be developed as antibac-
terial drug candidates until this nonspecific effect can be
diminished through structural changes, without compromising
their inhibitory effect on DdlB.
Best overall activity was achieved by compounds 23c and 24c,
both 9-bromo substituted ellipticines with quaternary methylpy-
ridinium structure. Both demonstrated improved IC50 values
against DdlB compared to top ellipticine derivatives from the sim-
ilarity search. They also maintained similar antibacterial activity.

B. Vehar et al. / Bioorg. Med. Chem. 19 (2011) 5137–5146
5141
Table 3
Antibacterial activity of synthesized ellipticines
Compound
Structure
IC50
,
l
M
MIC, mg/L
Membrane integrity in S. aureus, %
E. coli 1411
E. coli SM1411
S. aureus SH1000
N
21a
192
16
8
4
8
71.9 7.2
N
H
N+I-
24a
46
64
16
37.3 6.2
N
H
N
O
22b
21c
22c
184
86
nda
nda
nda
nda
N
N
Br
>256
16
16
49.6 6.9
N
H
N
Br
Br
Br
297
nda
nda
nda
nda
N
N+I-
23c
24c
36
23
32
16
1
4
2
8
42.4 4.6
N
N+I-
29.8 7.4
N
H
N
21d
22d
103
90
>256
>256
8
4
42.8 5.4
N
F
F
H
N
>256
>256
ndb
N
N+I-
24d
64
>256
4
>256
ndb
N
H
F
(continued on next page)

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B. Vehar et al. / Bioorg. Med. Chem. 19 (2011) 5137–5146
Table 3 (continued)
Compound
Structure
IC50
,
lM
MIC, mg/L
Membrane integrity in S. aureus, %
E. coli 1411
E. coli SM1411
S. aureus SH1000
N
21e
125
>256
>256
8
34.2 7.8
N
H
a
Not determined because of poor solubility.
Not determined because MIC for S. aureus P64 mg/L.
b
4.4. Synthesis
4. Experimental
4.4.1. General procedure for ellipticine synthesis
4.1. Similarity search
Starting compounds indole (17, 37 mmol), hexane-2,5-dione
(4.2 g, 37 mmol) and p-toluenesulfonic acid (3.5 g, 18.5 mmol)
were heated in ethanol (50 mL) at reflux for 1.5 h. The reaction
mixture was cooled to room temperature and solvent volume
was reduced to approximately 25 mL. Water (75 mL) was added
and the aqueous mixture was extracted with ether (3 Â 50 mL).
The organic layer was washed with water (75 mL) and brine
(75 mL), dried (Na2SO4) and concentrated in vacuo to give black
residue. The residue was extracted with hot hexane. The combined
hexane extracts were concentrated in vacuo. Crude residue was
purified by flash chromatography (ethyl acetate:hexane 1:4) which
yielded the product 18 as a white solid.
N-methylformanilide (2.4 mL, 19.2 mmol) and phosphorus oxy-
chloride (1.45 mL, 16 mmol) in 1,2-dichlorobenzene (20 mL) were
stirred for 0.5 h at room temperature. Then 1,4-dimethyl-carbazole
(18, 11.3 mmol) in 1,2-dichlorobenzene (20 mL) was added and the
reaction mixture was heated at 105 °C for 4 h. The reaction mixture
was cooled to room temperature and sodium acetate (5 g) in water
(50 mL) was added. The volume of solvent was reduced to ca.
20 mL and a black solid was collected by filtration. The black solid
was dried over night at 80 °C and extracted with toluene (200 mL)
in a Soxhlet extractor for 24 h. The hot extracts were decolorized
(charcoal), filtered through Celite and concentrated to 30 mL in va-
cuo. The solid was collected by filtration to give the corresponding
aldehyde 19.
A mixture of the aldehyde (19, 4.5 mmol) and aminoacetalde-
hyde diethyl acetal (2.3 mL, 15.7 mmol) was stirred at 115 °C un-
der argon atmosphere for 3 h. The reaction mixture was cooled
to room temperature. Anhydrous ethanol (50 mL) with activated
molecular sieves was added. Sodium borohydride (0.68 g,
18 mmol) was added and the reaction mixture was stirred for 1 h
and concentrated to dryness in vacuo. The residue was dissolved
in ethyl acetate, washed with water, dried and concentrated to give
a brown viscous gum that was purified by flash chromatography
(ethyl acetate:hexane 4:1) to give a yellowish product 20.
To a mixture of amine (20, 3.7 mmol) and Na2CO3 (0.46 g,
4.4 mmol) in tetrahydrofuran (20 mL) and water (10 mL), p-tolu-
enesulfonyl chloride (0.9 g, 4.8 mmol) was added at room temper-
ature. The reaction mixture was stirred for 2 h, diluted with water
(50 mL) and extracted with ethyl acetate (2 Â 50 mL). The com-
bined organic layers were washed with 0.1 M HCl (50 mL), water
(50 mL), saturated solution of NaHCO3 (50 mL), water (50 mL)
and brine (50 mL) and dried (Na2SO4). The organic solvent was
concentrated in vacuo. The crude residue was dissolved in dioxane
(10 mL) and 6 M HCl (10 mL) was slowly added and the reaction
mixture was stirred for 12 h. The separated solid was collected
by filtration and suspended in water (20 mL). The stirred suspen-
sion was cooled to 0 °C and basified to pH 10 with 1 M NaOH.
The National Cancer Institute (NCI) structural database is a li-
brary of over 450,000 compounds. The database has been opti-
mized with use and at the moment over 250,000 compounds are
available for searching at the Enhanced NCI Database Browser. To
date, the main use of the NCI database has been in anticancer re-
search. The compounds are available as solid samples to laborato-
ries engaged in biomedical investigations.27
The search for similar structures within the NCI database was
performed at the Classic Version of the Enhanced NCI Database
Browser server (http://cactus.nci.nih.gov/ncidb2.1). The SMILES
code for each input compound was extracted from the NCI data-
base and copied into the ‘Similarity Search’ field of the server.
The 2D-similarity search was performed using the Tanimoto In-
dex28 with a cut-off value of 92% as the measure of similarity.
4.2. Enzyme inhibition
Residual activity of DdlB ligase in the presence of potential
inhibitors was monitored with the colorimetric malachite green
method in which the orthophosphate generated during the
enzyme reaction is measured. For the initial screening of com-
pounds obtained through similarity search, each compound was
tested in duplicate at a concentration of 250
performed at 37 °C in a mixture (final volume: 50
50 mM Hepes (pH 8.0), 3.25 mM MgCl2, 6.5 mM (NH4)2SO4,
700 -Ala, 500 M ATP, purified His-tagged DdlB (diluted in
l
M. Assays were
lL) containing
lM
D
l
50 mM Hepes (pH 7.2) and 1 mM dithiothreitol), and the test
compound. All compounds were soluble in the assay mixture
containing 5% DMSO. After 20 min of incubation, 100 lL of Bio-
mol reagent was added, and absorbance was read at 650 nm
after 5 min. To exclude possible nonspecific (promiscuous) inhib-
itors, all compounds were also tested in the presence of Triton
X-114 (0.005%).29 IC50 values were determined by measuring
the residual activity at seven different test compound concentra-
tions, and they represent the test compound concentration where
the residual activity was 50%.
4.3. Microbiological evaluation
MIC determinations were performed by broth microdilution
against S. aureus SH1000 and E. coli 1411 (standard laboratory
strains) and E. coli SM1411 (an acrAB deficient derivative of
1411) according to British Society for Antimicrobial Chemotherapy
guidelines.30 The BacLight™ assay (Invitrogen) was used to
measure staphylococcal membrane damage in S. aureus SH1000
induced by agents at 4 Â MIC, compared to a drug free control, as
described previously.26,31

B. Vehar et al. / Bioorg. Med. Chem. 19 (2011) 5137–5146
5143
The yellow suspension was collected by filtration and purified with
flash chromatography to give 21.
(CDCl3, 300 MHz) d 2.54 (s, 3H, CH3–Ar), 2.87 (s, 3H, CH3–Ar),
3.97 (s, 3H, CH3–O), 6.93 (d, J = 7.3 Hz, 1H, Ar–H), 7.10 (dd,
J = 8.8, 2.5 Hz, 1H, Ar–H), 7.14 (d, J = 7.3 Hz, 1H, Ar–H), 7.40 (d,
J = 8.7 Hz, 1H, Ar–H), 7.72 (d, J = 2.5 Hz, 1H, Ar–H), 7.86 (br s, 1H,
NH).
Sodium hydride (0.06 g, 2.5 mmol) was added to an ice cooled
solution of ellipticine derivative (21, 1.9 mmol) in dry N,N-dimeth-
ylformamide (25 mL). After stirring for 30 min at 0 °C, methyl io-
dide (0.13 mL, 2.1 mmol) was added. The reaction mixture was
stirred over night at room temperature. Water was added
(50 mL) and extracted with dichloromethane (3 Â 40 mL). The
combined organic layers were extracted with water (50 mL) and
brine (50 mL) and dried over Na2SO4. The solvent was evaporated
under reduced pressure and the product was purified by flash
chromatography (dichloromethane:methanol 9:1) to obtain 22 as
a yellow solid.
4.4.1.8. 6-Methoxy-1,4-dimethyl-9H-carbazole-3-carbaldehyde
(19b). White crystals. Yield = 42%, mp 205–207 °C (lit.,34
206 °C); 1H NMR (CDCl3, 300 MHz) d 2.58 (s, 3H, CH3–Ar), 3.21 (s,
3H, CH3–Ar), 3.97 (s, 3H, CH3–O), 7.14 (dd, J = 8.77, 2.44 Hz, 1H,
Ar–H), 7.45 (d, J = 8.77 Hz, 1H, Ar–H), 7.76–7.81 (m, 2H, Ar–H),
8.17 (br s, 1H, NH), 10.47 (s, 1H, CHO).
The ellipticine derivative (21 or 22, 0.31 mmol) was dissolved in
acetone (200 mL) and methyl iodide (1 mL) was added. The reaction
mixture was stirred in the dark for 2 days. The yellow solid was col-
lected by filtration and washed with ether to obtain 23 or 24.
4.4.1.9. 2,2-Diethoxy-N-((6-methoxy-1,4-dimethyl-9H-carbazol-
3-yl)methyl)ethanamine (20b). Brown oil. Yield = 76%; 1H NMR
(CDCl3, 300 MHz) d 1.23 (t, J = 7.0 Hz, 6H, CH3–CH2), 1.68 (br s,
1H, NH), 2.49 (s, 3H, CH3–Ar), 2.86 (s, 4H, CH3–Ar + NH–CH2–CH),
2.88 (s, 1H, NH–CH2–CH), 3.51–3.61 (m, 2H, CH2–CH3), 3.6–3.77
(m, 2H, CH2–CH3), 3.95 (s, 3H, CH3–O), 3.98 (s, 2H, NH–CH2–Ar),
4.68 (t, J = 5.6 Hz, 1H, CH), 7.08 (dd, J = 8.7, 2.5 Hz, 1H, Ar–H),
7.17 (s, 1H, Ar–H), 7.37 (d, J = 8.7 Hz, 1H, Ar–H), 7.77 (d,
J = 2.4 Hz, 1H, Ar–H), 7.95 (br s, 1H, NH).35
4.4.1.1. 1,4-Dimethyl-9H-carbazole (18a). White crystals.
Yield = 54%, mp 93–94 °C (lit.,22 97–98 °C); 1H NMR (CDCl3,
300 MHz) d 2.47 (s, 3H, CH3), 2.78 (s, 3H, CH3), 6.87 (d, J = 7.3 Hz,
1H, Ar–H), 7.06 (d, J = 7.3 Hz, 1H, Ar–H), 7.17 (m, 1H, Ar–H), 7.34
(m, 1H, Ar–H), 7.41 (d, J = 8.0 Hz, 1H, Ar–H), 7.91 (br s, 1H, NH),
8.10 (d, J = 7.9 Hz, 1H, Ar–H).
4.4.1.10. 9-Methoxy-5,11-dimethyl-6H-pyrido[4,3-b]carbazole
(21b). Yellow solid. Yield = 43%, mp: 288–289 °C (lit.,36 276.3–
278.5 °C); 1H NMR (CDCl3, 300 MHz) d 2.77 (s, 3H, CH3), 3.26 (s,
3H, CH3), 3.91 (s, 3H, CH3–O), 7.20 (dd, J = 8.71, 2.38 Hz, 1H, Ar–
H), 7.49 (d, J = 8.71 Hz, 1H, Ar–H), 7.87–7.91 (m, 2H, Ar–H), 8.40
(d, J = 6.03 Hz, 1H, Ar–H), 9.68 (s, 1H, Ar–H), 11.19 (s, 1H, NH).
4.4.1.2. 1,4-Dimethyl-9H-carbazole-3-carbaldehyde (19a). Yel-
low crystals. Yield = 38%, mp 204–206 °C (lit.,32 216–218 °C); 1H
NMR (CDCl3, 300 MHz) d 2.56 (s, 3H, CH3), 3.18 (s, 3H, CH3), 7.31
(t, J = 7.3 Hz, 1H, Ar–H), 7.43–7.53 (m, 2H, Ar–H), 7.75 (s, 1H, Ar–
H), 8.26 (d, J = 8.4 Hz, 1H, Ar–H), 8.31 (br s, 1H, NH), 10.44 (s, 1H,
CHO).
4.4.1.11.
9-Methoxy-5,6,11-trimethyl-6H-pyrido[4,3-b]carba-
zole (22b). Yellow powder. Yield = 55%, mp 278–281 °C (lit.,34
158 °C); 1H NMR (DMSO-d6, 300 MHz) d 2.91 (s, 3H, CH3–Ar),
3.11 (s, 3H, CH3–Ar), 3.89 (s, 3H, CH3), 3.97 (s, 3H, CH3), 7.12 (dd,
J = 8.8, 2.3 Hz, 1H, Ar–H), 7.19 (d, J = 1.7 Hz, 1H, Ar–H), 7.76–7.78
(m, 2H, Ar–H), 8.40 (d, J = 6.1 Hz, 1H, Ar–H), 9.58 (s, 1H, Ar–H).
4.4.1.3. N-((1,4-Dimethyl-9H-carbazol-3-yl)methyl)-2,2-dieth-
oxyethanamine (20a). Yellow solid. Yield = 77 %, mp 87–88 °C
(lit.,24 105–106 °C); 1H NMR (CDCl3, 300 MHz)
d 1.23 (t,
J = 7.1 Hz, 6H, CH3–CH2), 1.64 (br s, 1H, CH2–NH–CH2), 2.51 (s,
3H, CH3–Ar), 2.87–2.89 (m, 5H, CH3–Ar + NH–CH2–CH), 3.51–3.61
(m, 2H, CH2–CH3), 3.66–3.76 (m, 2H, CH2–CH3), 4.00 (s, 2H, NH–
CH2–Ar), 4.69 (t, J = 5.7 Hz, 1H, CH), 7.19 (s, 1H, Ar–H), 7.23–7.26
(m, 1H, Ar–H), 7.42–7,45 (m, 1H, Ar–H), 7.48 (d, J = 7.5 Hz, 1H,
Ar–H), 8.14 (br s, 1H, NH), 8.26 (d, J = 7.9 Hz, 1H, Ar–H).
4.4.1.12. 6-Bromo-1,4-dimethyl-9H-carbazole (18c). White
crystals. Yield = 55%, mp 137–138 °C (lit.,37 137–138 °C); 1H NMR
(CDCl3, 300 MHz) d 2.55 (s, 3H, CH3), 2.84 (s, 3H, CH3), 6.97 (d,
J = 7.3 Hz, 1H, Ar–H), 7.18 (d, J = 7.3 Hz, 1H, Ar–H), 7.37 (d,
J = 8.6 Hz, 1H, Ar–H), 7.52 (dd, J = 8.6, 1.9 Hz, 1H, Ar–H), 8.01 (br
s, 1H, NH), 8.29 (d, J = 1.7 Hz, 1H, Ar–H).
4.4.1.4. 5,11-Dimethyl-6H-pyrido[4,3-b]carbazole (21a). Yel-
low powder. Yield = 42%, mp 311–313 °C (lit.,24 309–310 °C); 1H
NMR (DMSO-d6, 300 MHz) d 2.80 (s, 3H, CH3), 3.27 (s, 3H, CH3),
7.27 (t, J = 7.4 Hz, 1H, Ar–H), 7.53–7.59 (m, 2H, Ar–H), 7.92 (d,
J = 6.0 Hz, 1H, Ar–H), 8.37–8.43 (m, 2H, Ar–H), 9.70 (s, 1H, Ar–H),
11.37 (s, 1H, NH).
4.4.1.13. 6-Bromo-1,4-dimethyl-9H-carbazole-3-carbaldehyde
(19c). Yellow solid. Yield = 39%, mp 265–267 °C (lit.,34 267–
268 °C); 1H NMR (CDCl3, 300 MHz) d 2.60 (s, 3H, CH3), 3.19 (s,
3H, CH3), 7.43 (d, J = 8.5 Hz, 1H, Ar–H), 7.59 (dd, J = 8.5, 1.5 Hz,
1H, Ar–H), 7.81 (s, 1H, Ar–H), 8.31 (br s, 1H, NH), 8.41 (s, 1H, Ar–
H), 10.49 (s, 1H, CHO).
4.4.1.5. 5,6,11-Trimethyl-6H-pyrido[4,3-b]carbazole (22a). Yel-
low powder. Yield = 49%, mp 208–210 °C (lit.,33 207–208 °C); 1H
NMR (DMSO-d6, 300 MHz) d 3.06 (s, 3H, CH3), 3.27 (s, 3H, CH3),
4.14 (s, 3H, CH3), 7.31–7.36 (m, 1H, Ar–H), 7.42 (d, J = 8.2 Hz, 1H,
Ar–H), 7.57–7.63 (m, 1H, Ar–H), 7.91 (d, J = 6.1 Hz, 1H, Ar–H),
8.37 (d, J = 7.9 Hz, 1H, Ar–H), 8.53 (d, J = 5.3 Hz, 1H, Ar–H), 9.72
(s, 1H, Ar–H).
4.4.1.14. N-((6-Bromo-1,4-dimethyl-9H-carbazol-3-yl)methyl)-
2,2-diethoxyethanamine (20c). Pale yellow solid. Yield = 62%,
mp 99–100 °C; 1H NMR (CDCl3, 300 MHz) d 1.23 (t, J = 6.3 Hz, 6H,
CH3-CH2), 1.51 (br s, 1H, NH), 2.49 (s, 3H, CH3–Ar), 2.82 (s, 3H,
CH3–Ar), 2.86 (d, J = 5.6 Hz, 2H, CH–CH2–NH), 3.53–3.59 (m, 2H,
CH2–CH3), 3.69–3.76 (m, 2H, CH2–CH3), 3.97 (s, 2H, CH2–Ar), 4.68
(t, J = 5.6 Hz, 1H, CH), 7.20 (s, 1H, Ar–H), 7.34 (d, J = 8.6 Hz, 1H,
Ar–H), 7.49 (dd, J = 8.6, 1.9 Hz, 1H, Ar–H), 8.20 (br s, 1H, NH),
8.32 (d, J = 1.7 Hz, 1H, Ar–H); 13C NMR (DMSO-d6, 300 Hz) d
15.37, 15.72, 16.37, 51.49, 51.79, 62.37, 102.08, 117.77, 111.88,
116.85, 121.09, 126.16, 126.16, 127.42, 129.06, 129.20, 138.34,
138.57; IR (KBr) mmax 3528, 3318, 3153, 2973, 2931, 2853, 2366,
2345, 1610, 1458, 1438, 1406, 1374, 1362, 1335, 1278, 1242,
1226, 1147, 1124, 1112, 1082, 1057, 1010, 961, 930 cmÀ1; ESI-
HRMS m/z calcd. for [M+(H)]+ 419.1334, found 419.1346. Anal.
4.4.1.6. 2,5,11-Trimethyl-6H-pyrido[4,3-b]carbazol-2-ium io-
dide (24a). Yellow powder. Yield = 73%, mp 316–319 °C; 1H
NMR (DMSO-d6, 300 MHz) d 2.84 (s, 3H, CH3), 3.29 (s, 3H, CH3),
4.45 (s, 3H, CH3-N), 7.37–7.42 (m, 1H, Ar–H), 7.67 (d, J = 3.32 Hz,
2H, Ar–H), 8.39–8.46 (m, 3H, Ar–H), 10.00 (s, 1H, Ar–H). 12.11
(br s, 1H, NH).
4.4.1.7. 6-Methoxy-1,4-dimethyl-9H-carbazole (18b). White
crystals. Yield = 44%, mp 136–137 °C (lit.,34 136.5 °C); 1H NMR

5144
B. Vehar et al. / Bioorg. Med. Chem. 19 (2011) 5137–5146
Calcd. for C21H27N2O2Br  0.6 H2O: C, 58.63; H, 6.61; N, 6.51.
Ar–H), 8.21 (dd, J = 8.8, 5.3 Hz, 1H, Ar–H), 8.33 (br s, 1H, NH),
10.47 (s, 1H, CHO); 13C NMR (CDCl3, 300 Hz) d 14.79, 16.14,
Found C, 58.30; H, 6.62; N, 6.53.
2
2
97.73 (d, JF,C = 26.26 Hz), 108.54 (d, JF,C = 23.64 Hz), 117.34,
3
4.4.1.15.
9-Bromo-5,11-dimethyl-6H-pyrido[4,3-b]carbazole
122.42, 123.98 (d, JF,C = 10.22 Hz), 126.92, 127.11, 128.80,
(21c). Yellow powder. Yield = 72%, mp 329–331 °C (lit.,24 330–
332 °C); 1H NMR (DMSO-d6, 300 MHz) d 2.79 (s, 3H, CH3), 3.25 (s,
3H, CH3), 7.53 (d, J = 8.6 Hz, 1H, Ar–H), 7.67 (dd, J = 8.6, 1.9 Hz,
1H, Ar–H), 7.94 (d, J = 6.1 Hz, 1H, Ar–H), 8.43–8.47 (m, 2H, Ar–H),
9.72 (s, 1H, Ar–H), 11.54 (s, 1H, NH).
135.59, 140.21, 142.38, 161.50 (d, JF,C = 243.43 Hz), 191.43; IR
1
(KBr) mmax 3482, 3311, 3098, 2920, 2862, 2767, 2373, 2346, 1734,
1718, 1664, 1648, 1636, 1624, 1594, 1577, 1458, 1438, 1371,
1339, 1330, 1282, 1224, 1202, 1160, 1140, 1112, 1044, 960 cmÀ1
;
ESI-HRMS m/z calcd. for [M+(H)]+ 242.0981, found 242.0981. Anal.
Calcd. for C15H12NOF: C, 74.67; H, 5.01; N, 5.81. Found C, 74.44; H,
4.76; N, 5.74.
4.4.1.16. 9-Bromo-5,6,11-trimethyl-6H-pyrido[4,3-b]carbazole
(22c). Yellow powder. Yield = 35%, mp 208–210 °C; 1H NMR
(DMSO-d6, 300 MHz) d 2.97 (s, 3H, CH3–Ar), 3.10 (s, 3H, CH3–Ar),
4.02 (s, 3H, CH3–N), 7.21 (d, J = 8.66 Hz, 1H, Ar–H), 7.64 (dd,
J = 8.64, 1.89 Hz, 1H, Ar–H), 7.85 (d, J = 6.17 Hz, 1H, Ar–H), 8.34
(d, J = 1.83 Hz, 1H, Ar–H), 8.51 (d, J = 6.18 Hz, 1H, Ar–H), 9.64 (s,
1H, Ar–H); 13C NMR (DMSO-d6, 300 Hz) d 13.42, 14.31, 33.70,
109.45, 110.98, 111.42, 116.12, 121.12, 122.72, 124.13, 125.58,
129.36, 129.53, 133.88, 140.17, 141.12, 143.27, 149.25; IR (KBr)
mmax 3448, 3067, 2926, 2365, 2345, 1588, 1508, 1498, 1474,
1458, 1439, 1388, 1379, 1355, 1340, 1299, 1287, 1241, 1200,
1160, 1144, 1107, 1024 cmÀ1; ESI-HRMS m/z calcd. for [M+(H)]+
339.0497, found 339.0492. Anal. Calcd. for C18H15N2Br  0.5 H2O:
C, 62.08; H, 4.63; N, 8.04. Found C, 62.06; H, 4.60; N, 7.88.
4.4.1.21. 2,2-Diethoxy-N-((7-fluoro-1,4-dimethyl-9H-carbazol-
3-yl)methyl)ethanamine (20d). Yellow solid. Yield = 51%, mp
65–67 °C; 1H NMR (CDCl3, 300 MHz) d 1.24 (t, J = 7.05 Hz, 6H,
CH3–CH2), 1.78 (br s, 1H, NH), 2.41 (s, 3H, CH3–Ar), 2.81 (s, 3H,
CH3–Ar), 2.92 (d, J = 5.59 Hz, 2H, CH–CH2–NH), 3.55–3.60 (m, 2H,
CH2–CH3), 3.71–3.77 (m, 2H, CH2–CH3), 3.99 (s, 2H, CH2-Ar), 4.71
(t, J = 5.56 Hz, 1H, CH), 6.92–6.99 (m, 1H, Ar–H), 7.08 (dd,
J = 10.06, 2.78 Hz, 2H, Ar–H), 8.09 (dd, J = 8.73, 5.43 Hz, 1H, Ar–
H), 8.55 (br s, 1H, NH); 13C NMR (CDCl3, 300 Hz) d 15.37, 15.73,
2
16.39, 37.03, 51.52, 51.64, 62.43, 97.17 (d, JF,C = 26.09 Hz),
2
101.96, 107.26 (d, JF,C = 23.63 Hz), 116.71, 121.01, 121.66, 123.57
3
(d, JF,C = 10.25 Hz), 128.20, 128.68, 128.87, 138.64, 140.42 (d,
1
3JF,C = 12.32 Hz), 161.30 (d, JF,C = 241.17 Hz); IR (KBr) mmax 3316,
4.4.1.17.
9-Bromo-2,5,6,11-tetramethyl-6H-pyrido[4,3-b]car-
3123, 3023, 2974, 2910, 2885, 2827, 1734, 1605, 1590, 1534,
1522, 1502, 1490, 1472, 1438, 1406, 1394, 1372, 1339, 1324,
1294, 1273, 1225, 1202, 1139, 1126, 1095, 1080, 1060, 1024,
973, 940, 928 cmÀ1; ESI-HRMS m/z calcd. for [M+(H)]+ 359.2135,
bazol-2-ium iodide (23c). Yellow powder. Yield = 57%, mp 299–
301 °C; 1H NMR (DMSO-d6, 300 MHz) d 3.11 (s, 3H, CH3–Ar), 3.25
(s, 3H, CH3–Ar), 4.24 (s, 3H, CH3–N), 4.46 (s, 3H, CH3–N), 7.77 (d,
J = 8.81 Hz, 1H, Ar–H), 7.85 (dd, J = 8.78, 1.69 Hz, 1H, Ar–H), 8.47
(d, J = 7.20 Hz, 1H, Ar–H), 8.52 (d, J = 1.54 Hz, 1H, Ar–H), 8.58 (d,
J = 7.29 Hz, 1H, Ar–H), 10.08 (s, 1H, Ar–H); 13C NMR (DMSO-d6,
300 Hz) d 13.56, 15.11, 33.82, 46.61, 111.50, 111.95, 113.08,
120.12, 120.44, 123.04, 125.08, 126.23, 130.97, 132.36, 133.62,
133.80, 143.17, 144.07, 147.38; IR (KBr) mmax 3448, 3039, 2368,
2346, 1636, 1609, 1586, 1566, 1458, 1392, 1364, 1334, 1300,
1288, 1243, 1189, 1142, 1104, 1078, 1053 cmÀ1; ESI-HRMS m/z
calcd. for [M+(H)]+ 353.0653, found 353.0642.
found 359.2140. Anal. Calcd. for
67.32; H, 7.75; N, 7.48. Found C, 67.05; H, 7.59; N, 7.85.
C
21H27N2O2F Â 0.9 H2O: C,
4.4.1.22.
8-Fluoro-5,11-dimethyl-6H-pyrido[4,3-b]carbazole
(21d). Yield = 52%, mp 283–286 °C; 1H NMR (DMSO-d6,
300 MHz) d 2.82 (s, 3H, CH3), 3.24 (s, 3H, CH3), 7.15 (td, J = 9.33,
2.38 Hz, 1H, Ar–H), 7.35 (dd, J = 9.50, 2.32 Hz, 1H, Ar–H), 8.28 (d,
J = 6.70 Hz, 1H, Ar–H), 8.37–8.45 (m, 2H, Ar–H), 9.87 (s, 1H, Ar–
H), 12.14 (s, 1H, NH); 13C NMR (DMSO-d6, 300 Hz) d 11.82, 14.14,
2
2
97.15 (d, JF,C = 26.00 Hz), 106.59 (d, JF,C = 23.46 Hz), 108.25,
115.86, 119.78, 122.07, 122.72, 125.09 (d, JF,C = 10.58 Hz),
127.47, 132.29, 140.52, 140.89, 143.68 (d, JF,C = 12.79 Hz),
149.54, 161.73 (d, JF,C = 241.58 Hz); IR (KBr) mmax 3904, 3854,
3
4.4.1.18. 9-Bromo-2,5,11-trimethyl-6H-pyrido[4,3-b]carbazol-
2-ium iodide (24c). Yellow powder. Yield = 88%, mp 328–330 °C;
1H NMR (DMSO-d6, 400 MHz) d 2.84 (s, 3H, CH3–Ar), 3.29 (s, 3H,
CH3–Ar), 4.47 (s, 3H, CH3–N), 7.61 (d, J = 8.4 Hz, 1H, Ar–H), 7.79
(d, J = 8.4 Hz, 1H, Ar–H), 8.43–8.56 (m, 3H, Ar–H), 10.09 (s, 1H,
Ar–H), 12.26 (s, 1H, NH); IR (KBr) mmax 3429, 3144, 2360, 1599,
1449, 1410, 1284, 1243, 1184, 1127, 1066 cmÀ1; ESI-HRMS m/z
calcd. for [M+(H)]+ 339.0497, found 339.0506.
3
1
3839, 3690, 3368, 3163, 3004, 2924, 2375, 2345, 1605, 1591,
1490, 1458, 1449, 1420, 1398, 1388, 1333, 1299, 1283, 1170,
1150, 1111, 1034, 1010, 963 cmÀ1; ESI-HRMS m/z calcd. for
[M+(H)]+ 265.1141, found 265.1140.
4.4.1.23. 8-Fluoro-5,6,11-trimethyl-6H-pyrido[4,3-b]carbazole
(22d). Yellow powder. Yield = 42%, mp 232–234 °C; 1H NMR
(DMSO-d6, 300 MHz) d 3.05 (s, 3H, CH3–Ar), 3.21 (s, 3H, CH3–Ar),
4.15 (s, 3H, CH3–N), 7.11 (td, J = 9.33, 2.38 Hz, 1H, Ar–H), 7.55
(dd, J = 10.61, 2.35 Hz, 1H, Ar–H), 8.02 (d, J = 6.00 Hz, 1H, Ar–H),
8.38 (dd, J = 8.67, 5.60 Hz, 1H, Ar–H), 8.46 (d, J = 6.12 Hz, 1H, Ar–
H), 9.71 (s, 1H, Ar–H); 13C NMR (DMSO-d6, 300 Hz) d 13.25,
4.4.1.19. 7-Fluoro-1,4-dimethyl-9H-carbazole (18d). White
crystals. Yield = 46%, mp 114–115 °C; 1H NMR (CDCl3, 300 MHz) d
2.55 (s, 3H, CH3), 2.84 (s, 3H, CH3), 6.96–7.04 (m, 2H, Ar–H),
7.13–7.19 (m, 2H, Ar–H), 8.01 (br s, 1H, NH), 8.09 (dd, J = 8.7,
5.4 Hz, 1H, Ar–H); 13C NMR (CDCl3, 300 Hz) d 16.49, 20.36, 97.20
2
2
(d, JF,C = 26.3 Hz), 107.47 (d, JF,C = 23.8 Hz), 117.02, 120.99,
3
2
2
121.31, 123.34 (d, JF,C = 10.3 Hz), 125.91, 130.30, 139.10, 139.86,
14.27, 33.90, 96.38 (d, JF,C = 27.20 Hz), 106.82 (d, JF,C = 23.39 Hz),
1
3
140.02, 161.42 (d, JF,C = 241.1 Hz); IR (KBr) mmax 3423, 3080,
109.31, 116.10, 118.97, 122.14, 123.41, 125.01 (d, JF,C = 10.57 Hz),
2975, 2944, 2920, 2865, 2371, 1870, 1617, 1593, 1522, 1492,
1458, 1447, 1387, 1375, 1335, 1315, 1296, 1274, 1218, 1141,
1121, 1099, 1040, 1007, 958 cmÀ1; ESI-HRMS m/z calcd. for
[M+(H)]+ 214.1032, found 214.1022. Anal. Calcd. for C14H12NF: C,
78.85; H, 5,67; N, 6.57. Found C, 79.12; H, 5.57; N, 6.48.
127.91, 133.47, 140.34, 141.60, 145.96 (d, 3JF,C = 12.56 Hz), 149.19,
1
162.12 (d, JF,C = 243.73 Hz); IR (KBr)
m
max 3480, 3020, 2927, 2374,
2346, 1617, 1590, 1560, 1458, 1438, 1420, 1412, 1382, 1362, 1340,
1310, 1276, 1242, 1212, 1198, 1159, 1097, 1086, 1025, 998, 952,
896 cmÀ1; ESI-HRMS m/z calcd. for [M+(H)]+ 279.1298, found
279.1291. Anal. Calcd. for C18H15N2F Â 0.3 H2O: C, 76.20; H, 5.54;
N, 9.87. Found C, 76.67; H, 5.34; N, 9.76.
4.4.1.20. 7-Fluoro-1,4-dimethyl-9H-carbazole-3-carbaldehyde
(19d). Grey powder. Yield = 42%, mp 197–200 °C; 1H NMR (CDCl3,
300 MHz) d 2.60 (s, 3H, CH3), 3.19 (s, 3H, CH3), 7.08 (td, J = 9.1,
2.4 Hz, 1H, Ar–H), 7.22 (dd, J = 9.2, 2.3 Hz, 1H, Ar–H), 7.77 (s, 1H,
4.4.1.24. 8-Fluoro-2,5,11-trimethyl-6H-pyrido[4,3-b]carbazol-
2-ium iodide (24d). Yellow powder. Yield = 54%, mp 323–

B. Vehar et al. / Bioorg. Med. Chem. 19 (2011) 5137–5146
5145
325 °C; 1H NMR (DMSO-d6, 300 MHz) d 2.81 (s, 3H, CH3–Ar), 3.23
(s, 3H, CH3–Ar), 4.47 (s, 3H, CH3–N), 7.20 (td, J = 9.42, 9.40,
2.38 Hz, 1H, Ar–H), 7.36 (dd, J = 9.42, 2.33 Hz, 1H, Ar–H), 8.38–
8.44 (m, 3H, Ar–H), 10.01 (s, 1H, Ar–H), 12.19 (s, 1H, NH); 13C
115.92, 119.56, 121.21, 121.94, 123.02, 123.84, 126.05, 126.36,
127.70, 132.44, 140.10, 140.60, 140.86, 149.44; IR (KBr) mmax
3448, 3255, 3197, 3107, 3048, 2963, 2928, 2869, 2346, 1618,
1599, 1592, 1560, 1534, 1522, 1499, 1491, 1458, 1443, 1407,
1383, 1350, 1322, 1312, 1303, 1264, 1235, 1178, 1130, 1080,
1058, 1027, 1000, 916 cmÀ1; ESI-HRMS m/z calcd. for [M+(H)]+
275.1548, found 275.1543. Anal. Calcd. for C19H18N2 Â 0.3 H2O:
C, 81.57; H, 6.70; N, 10.01. Found C, 81.49; H, 6.46; N, 9.76.
NMR spectra for compounds that were not previously charac-
terized in literature are available in Supplementary data.
NMR (DMSO-d6, 300 Hz)
d 11.94, 14.81, 46.69, 98.18 (d,
2
2JF,C = 26.22 Hz), 108.38 (d, JF,C = 23.90 Hz), 109.63, 110.52,
3
118.77, 120.25, 125.23, 126.01 (d, JF,C = 10.53 Hz), 131.93,
132.23,
143.65,
143.82,
144.60,
147.19,
162.38
(d,
1JF,C = 244.28 Hz); IR (KBr) mmax 3436, 3169, 2346, 1637, 1603,
1508, 1492, 1458, 1451, 1420, 1399, 1364, 1322, 1293, 1242,
1185, 1144, 1109, 1070, 1032, 963, 922 cmÀ1; ESI-HRMS m/z calcd.
for [M+(H)]+ 279.1298, found 279.1287.
Acknowledgments
4.4.1.25. 8-Ethyl-1,4-dimethyl-9H-carbazole (18e). White crys-
tals. Yield = 76%, mp 114–116 °C; 1H NMR (CDCl3, 300 MHz) d 1.49
(t, J = 7.6 Hz, 3H, CH3–CH2), 2.61 (s, 3H, CH3–Ar), 2.89 (s, 3H, CH3–
Ar), 3.02 (q, J = 7.6 Hz, 2H, CH2), 6.98 (d, J = 7.3 Hz, 1H, Ar–H), 7.17
(d, J = 7.3 Hz, 1H, Ar–H), 7.24–7.33 (m, 2H, Ar–H), 7.88 (br s, 1H,
NH), 8.09 (d, J = 7.6 Hz, 1H, Ar–H); 13C NMR (DMSO-d6, 300 Hz) d
13.74, 16.68, 20.48, 24.18, 116.96, 119.63, 120.23, 120.86, 121.85,
123.58, 124.15, 125.62, 125.97, 130.82, 137.92, 138.56; IR (KBr)
mmax 3469, 3078, 3052, 3019, 2972, 2934, 2890, 2862, 2735,
1889, 1846, 1780, 1706, 1605, 1584, 1534, 1520, 1496, 1456,
1438, 1423, 1388, 1376, 1349, 1333, 1308, 1294, 1258, 1232,
1162, 1119, 1076, 1038, 1022, 988, 959, 948 cmÀ1; ESI-HRMS m/z
calcd. for [M+(H)]+ 224.1439, found 224.1430. Anal. Calcd. for
This work was supported by the European Union FP6 Integrated
Project EUR-INTAFAR (project no. LSHM-CT-2004-512138) under
the thematic priority of Life Sciences, Genomics, and Biotechnology
for Health. The support from the Ministry of Higher Education,
Science, and Technology of the Republic of Slovenia and the
Slovenian Research Agency is also acknowledged. We would like
to thank the Drug Synthesis and Chemistry Branch, Developmental
Therapeutics Program, Division of Cancer Treatment and Diagnosis,
National Cancer Institute for supplying compounds for testing. We
thank Noah Affan for technical assistance.
Supplementary data
C16H17N: C, 86.05; H, 7.67; N, 6.27. Found C, 85.85; H, 7.70; N, 6.20.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.07.020.
4.4.1.26. 8-Ethyl-1,4-dimethyl-9H-carbazole-3-carbaldehyde
(19e). White solid. Yield = 35%, mp 191–194 °C; 1H NMR (CDCl3,
300 MHz) d 1.47 (t, J = 7.6 Hz, 3H, CH3–CH2), 2.62 (s, 3H, CH3–Ar),
3.02 (q, J = 7.7 Hz, 2H, CH2), 3.21 (s, 3H, CH3–Ar), 7.31–7.37 (m,
2H, Ar–H), 7.78 (s, 1H, NH), 8.16 (d, J = 7.4 Hz, 2H, Ar–H), 10.48
(s, 1H, CHO); IR (KBr) mmax 3752, 3274, 2962, 1654, 1648, 1608,
1578, 1339, 1266, 1231, 1206, 1179, 1114 cmÀ1; ESI-HRMS m/z
calcd. for [M+(H)]+ 252.1388, found 252.1380. Anal. Calcd. for
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