Synthesis of 5,6-Dimethyl-9-methoxy-1-phenyl-6H-pyrido[4,3-b]carbazole derivatives and their cytotoxic activity

Article abstract of DOI:10.1002/ardp.200500141

Starting from 2-(6-methoxy-1-methyl-9H-carbazol-2-yl)ethylamine 7 and mixed anhydrides of 4-nitrobenzoic acid or 4-methoxybenzoic acid, the corresponding 5,6-dimethyl-9-methoxy-1-(4-substituted phenyl)-6H-pyrido[4,3-b]carbazoles 11a-b, 5,6-dimethyl-9-hydr

Full text of DOI:10.1002/ardp.200500141

556
DOI 10.1002/ardp.200500141
Arch. Pharm. Chem. Life Sci. 2005, 338, 556−561
Synthesis of 5,6-Dimethyl-9-methoxy-1-phenyl-6H-
pyrido[4,3-b]carbazole Derivatives and their
Cytotoxic Activity
Ryszard Jasztold-Howorkoa, Marzena Pelczynskab, Anna Nasulewiczb,
Joanna Wietrzykb, Adam Opolskib,c
a Wroclaw University of Medicine, Faculty of Pharmacy, Department of Organic Chemistry, Wroclaw,
Poland
b Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland
c Jan Dlugosz Academy, Czestochowa, Poland
Starting from 2-(6-methoxy-1-methyl-9H-carbazol-2-yl)ethylamine 7 and mixed anhydrides of 4-nitro-
benzoic acid or 4-methoxybenzoic acid, the corresponding 5,6-dimethyl-9-methoxy-1-(4-substituted
phenyl)-6H-pyrido[4,3-b]carbazoles 11aϪb, 5,6-dimethyl-9-hydroxy-1-(4-substituted phenyl)-6H-
pyrido[4,3-b]carbazoles 12a, 12c, and their quaternary salts 13aϪd were obtained. The four new
pyridocarbazole derivatives 12aϪc and 13d satisfy the international activity criterion for synthetic
compounds, namely an ID50 value lower then 4 µg/mL in preliminary in vitro cytotoxic activity screen-
ing against the A549 cell line (non-small cell lung cancer).
Keywords: Olivacine; 1-Substituted 6H-pyrido[4,3-b]carbazole; Cytotoxicity
Received: May 25, 2005; Accepted: July 29, 2005
Introduction
It has been known for almost forty years that the natural
alkaloids Ellipticine 1 and Olivacine 2 (Figure 1) exert
anti-neoplastic activity. Structure modifications of the
above-mentioned alkaloids have since been conducted in the
search for new derivatives with advantageous antitumor ac-
tivity in the pyrido[4,3-b]carbazole ring system. Numerous
derivatives of pyrido[4,3-b] carbazole have shown strong
anticancer activity in in vitro and in vivo studies [1, 2]. So
far, only the acetate of 9-hydroxy-2-methylellipticinium 3
(Figure 2) has found application in clinical treatment [3].
Figure 2. Structure of acetate of 9-hydroxy-2-methylellipti-
cinium 3.
Much less attention has been paid to the synthesis of
analogues of the alkaloid Olivacine 2, which has also been
proved to possess potential anticancer properties. One of
Figure 3. Analogue of alkaloid Olivacine 2.
these (4, Figure 3) is in the course of clinical trials [4, 5].
1-Pyridylsubstituted pyrido[4,3-b]carbazole derivatives 5
(Figure 4) have shown strong cytostatic activity [6Ϫ8].
Figure 1. Structure of natural alkaloids Ellipticine 1 and
Olivacine 2.
According to existing results, the sole attempt to synthesize
1-(4-chlorophenyl)-substituted derivative of the above men-
tioned ring system, resulted in 1-phenyl-5-methyl-6H-
pyrido[4,3-b] carbazole, which was inactive against the
Correspondence: Ryszard Jasztold-Howorko, Wroclaw University of
Medicine, Faculty of Pharmacy, Department of Organic Chemistry,
ul. Grodzka 9, 50-137 Wroclaw, Poland. Phone: ϩ48 71 784-0347,
Fax: ϩ48 71 784-0341, e-mail: howorko@bf.uni.wroc.pl
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Arch. Pharm. Chem. Life Sci. 2005, 338, 556−561
1-Phenyl-6H-pyrido[4,3-b]carbazole derivatives
557
Figure 5. Structure of 1-phenyl-substituted derivative of
type 6.
Figure 4. Structure of 1-pyridylsubstituted pyrido[4,3-b]carb-
azole derivative 5.
L1210 mouse leukemia cell line [9]. Because the above-
mentioned 1-pyridylsubstituted pyridocarbazoles, having
substituents in the aromatic pyridyl group, exerted signifi-
cant activity in identical research, it was decided to synthe-
size new 1-phenyl-substituted derivatives of type 6 (Figure
5) and verify their cytotoxic activity. These pyrido[4,3-b]-
carbazole derivatives
Scheme 1.
6
were obtained according to
Scheme 1. Synthesis route for pyrido[4,3-b]carbazole derivative 6.
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Arch. Pharm. Chem. Life Sci. 2005, 338, 556−561
Table 1. Cell growth inhibition of A549 cells (non-small cell
lung cancer), ID50 values (µg/mL) of 1-substituted-6H-
pyrido[4,3-b]carbazole 11aϪ13d compared to 5c.
Results and discussion
Synthetic chemistry
The starting compound, 2-(6-methoxy-1-methyl-9H-carb-
azol-2-yl)ethylamine 7, has already been described [4]. It
was allowed to react with mixed anhydride of 4-nitrobenzoic
acid. Cyclization of the resulting amide 8a with phosphor-
ous oxychloride in boiling toluene gave 9-methoxy-5-
methyl-1-(4-nitrophenyl)-3,4-dihydro-6H-pyrido[4,3-b]carb-
azole 9a, which was aromatized to derivative 10a by de-
hydrogenation over 10% palladium on charcoal in boiling
diphenyl ether. N-6-Methylation of 10a to 11a was per-
formed using an excess of dimethyl carbonate in dimethyl-
formamide, in the presence of potassium carbonate and
18-Crown-6. The compound 11a was 9-O-demethylated to
5,6-dimethyl-9-hydroxy-1-(4-nitrophenyl)-6H-pyrido[4,3-b]-
carbazole 12a by heating with an 48% aqueous solution of
hydrobromic acid.
Compound No
ID50 (µg/mL)
IC50 (µM)
5c
11a
11b
12a
12b
12c
13a
13b
13c
13d
3.38 0.54
3.78 2.06
4.925 2.22
0.84 1.21
2.305 0.43
0.625 0.02
10.25 1.04
3.05 1.15
inactive
9.56 1.52
9.51 5.18
12.87 5.80
2.37 3.41
6.25 1.16
1.76 0.056
20.69 2.099
5.97 2.25
2.51 0.93
4.78 1.77
sulted. It was possible to see a tripling in growth activity
only in the case of compound 13d compared with the start-
ing derivative 11b. Compound 13c was inactive in this con-
centration range.
An analogous sequence of reactions was carried out using
mixed anhydride of 4-methoxybenzoic acid as the acylating
agent for amine 7 and adequate amide 8b was obtained.
Compound 8b was boiled in toluene with POCl3 and the
cyclization product a 3,4-dihydroderivative of the pyr-
ido[4,3-b]carbazole ring system 9b was obtained, which was
aromatized to derivative 10b and then N-6 methylated to 9-
methoxy-1-(4-methoxyphenyl)-5,6-dimethyl-6H-pyrido[4,3-
b]carbazole 11b. The same protocol for the demethylation
of 11a was used and led to a mixture of the 1-(4-hydroxy-
phenyl)-9-methoxy derivative 12b and the 1-(4-hydroxy-
phenyl)-9-hydroxy derivative 12c. Quaternization of the
pyridinic nitrogens of compounds 11b and 12aϪc into the
corresponding salts 13aϪd was achieved with methyl iodide
in DMF [10].
Conclusion
The results of the in vitro study may be summarized as
follows:
The compounds 11aϪb and 12b, containing a methoxy
group at position 9 in the pyrido[4,3-b] carbazole ring sys-
tem, exhibit lower cytotoxic activity than their 9-hydroxy
equivalents 12a and 12c.
The compounds 12aϪc, 13b, and 13d were more active than
the reference derivative 5c.
The four new pyridocarbazole derivatives 12aϪc and 13d
satisfy the international activity criterion for synthetic com-
pounds, namely an ID50 value lower then 4 µg/mL [11].
Biology
The nine new 1-substituted-6H-pyrido[4,3-b]carbazole de-
rivatives 11aϪ13d were subjected to preliminary in vitro
cytotoxic activity screening against the A549 cell line (non-
small cell lung cancer). All the new tested compounds exhi-
bited different biological properties, depending on the kind
of substituent at positions 1 and 9 in the main heterocyclic
system.
Acknowledgments
´
The technical assistance of B. Biadun is acknowledged. The
presented study was supported by the Wrocław University
of Medicine (internal project number 434).
Based on the results assembled in Table 1, it can be seen
that replacement of the studied ring system of the phenyl
group at the 1-position by p-hydroxy, p-methoxy, or p-nitro-
phenyl groups has caused reactivation of the stripped ac-
tivity of 1-phenylsubstituted pyrido[4,3-b] carbazole. The
boost in the hydrophilic properties of the studied com-
pounds (11b, 12aϪc) by their transformation on quaternary
salts (13aϪd) did not cause the expected increase in activity.
A decline in the cytotoxic activity of the obtained com-
pounds in the studied range of concentrations generally re-
Experimental
Chemistry
Melting points were determined on a Köfler apparatus (C. Reichert,
Vienna, Austria) and are uncorrected; 1H NMR spectra were
recorded on a Tesla BS 587 A at 80 MHz or on a Bruker 300 at
300 MHz (Bruker, Rheinstetten, Germany). 14 MHz, using TMS
as the internal standard. Column chromatography was carried out
on silica gel (Merck Kieselgel 100; Merck, Darmstadt, Germany).
All of the newly obtained compounds were analyzed for C, H, and
N, and the analytical results were within 0.4% of the theoretical
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Arch. Pharm. Chem. Life Sci. 2005, 338, 556−561
1-Phenyl-6H-pyrido[4,3-b]carbazole derivatives
559
values. The starting 2-(6-methoxy-1-methylcarbazol-2-yl)ethylamine
General procedure for the synthesis of 9-methoxy-5-methyl-1-(4-sub-
7 (Scheme 1) was prepared according to a described procedure [4].
stituted phenyl)-6H-pyrido[4,3-b] carbazoles 10
The required compound 9a-b (3 mmol) was refluxed in diphenyl
ether (50 mL) in the presence of 10% palladized charcoal (0.1 g)
for 45 min. The catalyst was filtered off and the filtrate was cooled
and diluted with hexane. The resulting precipitate was collected and
washed with hexane and recrystallized in the given solvent.
General procedure for the synthesis of N-[2-(6-methoxy-1-methyl-
9H-carbazol-2-yl)ethyl]-4-substituted benzamides 8
Triethylamine (0.506 g, 5 mmol) was added to the appropriate 4-
nitro-or 4-methoxybenzoic acid (4.4 mmol) in dry THF (100 mL).
After cooling to Ϫ10°C, a solution of ethyl chloroformiate (0.543
g, 5 mmol) in dry THF (10 mL) was added to the resulting mixture
under stirring. The mixture was stirred for a further 30 min and
then a solution of 2-(6-methoxy-1-methylcarbazol-2-yl)ethylamine 7
(1.016 g, 4 mmol) in THF (100 mL) was added drop-wise at Ϫ10°C.
The resulting mixture was left to reach room temperature (20 h)
under stirring and was then evaporated to dryness. The residue was
taken up in water (50 mL), basified with concentrated aqueous
ammonia, extracted with methylene dichloride, and then dried over
magnesium sulfate. Evaporation of the solvent provided a solid resi-
due, which was recrystallized in the given solvent.
10a: Yield 51.6%, mp 298°C. Anal. calcd. for C23H17N3O3: C,
72.05; H, 4.47; N, 10.96. Found: C, 71.87; H, 4.66; N, 10.73. 1H
NMR (DMSO-d6) δ: 2.87 (s, 3H, 5-CH3), 3.82 (s, 3H, 9-OCH3),
7.14 (dd, J8-7 ϭ 8.8 Hz, J8-10 ϭ 2.4 Hz, 1H, 8-H), 7.45 (d, J7-8 ϭ 8.8
Hz, 1H, 7-H), 7.84 (d, J10-8 ϭ 2.4 Hz, 1H, 10-H), 8.00Ϫ8.05 (m,
3H, 4-H ϩ Phenyl-H), 8,46 (d, J ϭ 8.8 Hz, 2H, Phenyl-H), 8.51 (d,
J3-4 ϭ 6.3 Hz, 1H, 3-H), 8.61 (s, 1H, 11-H), 11.30 (s, 1H, 6-H).
10b: Yield 80.4%, mp 250-251°C (ethyl acetate). Anal. calcd. for
C24H20N2O2: C, 78.24; H, 5.47; N, 7.60. Found: C, 78.03; H, 5.66;
N, 7.51. 1H NMR (DMSO-d6) δ: 2.85 (s, 3H, 5-CH3), 3.84 (s, 3H,
4Ј-OCH3), 3.90 (s, 3H, 9-OCH3), 7.13 (dd, J8-7 ϭ 8.8 Hz, J8-10
ϭ
2.4 Hz, 1H, 8-H), 7.17 (d, J ϭ 8.8 Hz, 2H, Phenyl-H), 7.44 (d,
J7-8 ϭ8.6 Hz, 1H, 7-H), 7.69 (d, J ϭ 8.6 Hz, 2H, Phenyl-H), 7.74
(d, J10-8 ϭ 2.4 Hz, 1H, 10-H), 7.90 (d, J4-3 ϭ 6.1 Hz, 1H, 4-H), 8.43
(d, 1H, 3-H), 8.68 (s, 1H, 11-H), 11.20 (s, 1H, 6-H).
8a: Yield 52.9%, mp 223Ϫ224°C (from ethanol). Anal. calcd. for
C24H24N3O4: C, 68.47; H, 5.25; N, 10.42. Found: C, 68.60; H, 5.40;
N, 10.13. 1H NMR (DMSO-d6) δ: 2.53 (s, 3H, 1-CH3), 3.03 (m,
2H, α-CH2), 3.50 (m, 2H, β-CH2), 3.83 (s, 3H, 6-OCH3), 6.98 (m,
2H, 3-H ϩ 7-H), 7.37 (d, J8-7 ϭ 8.8 Hz, 1H, 8-H), 7.58 (d, J5-7
ϭ
General procedure for the synthesis of 5,6-dimethyl-9-methoxy-1-(4-
substituted phenyl)-6H-pyrido[4,3-b]carbazoles 11
2.4 Hz, 1H, 5-H), 7.83 (d, J4-3 ϭ 7.8 Hz, 1H, 4-H), 8.07 (d, J ϭ 8.8
Hz, 2H, Phenyl-H), 8.32 (d, Jϭ 8.8 Hz, 2H, Phenyl-H), 8.97 (t, 1H,
β- NH), 10.82 (s, 1H, 9-H).
A mixture of the required compound 10aϪb (2 mmol), finely
powdered dry potassium carbonate (500 mg), dimethyl carbonate
(15 mL), dimethylformamide (2 mL), and 18-crown-6-ether
(3 drops) was heated at reflux under stirring for a 12-h period. After
evaporation to dryness, the residue was taken up in water. The solid
was collected, air-dried, and recrystallized in the given solvent.
8b: Yield 86.4%, mp 243Ϫ244°C (from ethanol). Anal. calcd. for
C24H24N2O3: C, 74.21; H, 6.23; N, 7.21. Found: C, 73.96; H, 6.38;
1
N, 6.97. H NMR (DMSO-d6) δ: 2.52 (s, 3H, 1-CH3), 2.99 (m, 2H,
α-CH2), 3.45 (m, 2H, β-CH2), 3.80 (s, 3H, 4Ј-OCH3), 3.83 (s, 1H,
6-OCH3), 6.98 (m, 4H, 3H ϩ 7-H ϩ Phenyl-H), 7.37 (d, J8-7 ϭ 8.8
Hz, 1H, 8-H), 7.58 (d, J5-7 ϭ 2.2 Hz, 1H, 5-H), 7.83 (m, 3H, 4-H
ϩ Phenyl-H), 8.47 (t, 1H, β-NH), 10.80 (s, 1H, 9-H).
11a: Yield 91.1%, mp 304°C (ethyl acetate). Anal. calcd. for
C24H19N3O3: C, 72.53; H, 4.82; N, 10.57. Found: C, 78.19; H, 5.69;
1
N, 11.57. H NMR (DMSO-d6) δ: 3.15 (s, 3H, 5-CH3), 3.83 (s, 3H,
9-OCH3), 4.19 (s, 3H, 6-CH3), 7.21 (dd, J8-7 ϭ 8.8 Hz, J8-10 ϭ 2.4
Hz, 1H, 8-H), 7.57 (d, 1H, 7-H), 7.85 (d, J10-8 ϭ 2.4 Hz, 1H, 10-
H), 8.02 (d, J ϭ 8.8 Hz, 2H, Phenyl-H), 8.14 (d, J4-3 ϭ 5.8 Hz, 1H,
4-H), 8.47 (d, J ϭ 8.8 Hz, 2H, Phenyl-H), 8.54 (d, 1H, 3-H), 8.64
(s, 1H, 11-H).
General procedure for the synthesis of 9-methoxy-5-methyl-1-(4-sub-
stituted phenyl)-3,4-dihydro-6H-pyrido[4,3-b]carbazoles 9
Preceding amide 8aϪb (3 mmol) was dissolved in boiling toluene
(150 mL) and treated drop-wise with phosphorous oxychloride
(12 mL). Reflux was continued for a 12-h period, and evaporation
under reduced pressure afforded a residue, which was taken up in
water (100 mL), basified to pH 9-10 with concentrated aqueous
ammonia, and extracted with methylene dichloride. Evaporation of
the solvent provided a solid residue, which was recrystallized in the
given solvent.
11b: Yield 90.3%, mp 210Ϫ211°C (from cyclohexane). Anal. calcd.
for C25H22N2O2: C, 78.51; H, 5.80; N, 7.32. Found: C, 78.19; H,
1
5.95; N, 7.13. H NMR (DMSO-d6) δ: 3,10 (s, 3H, 5-CH3), 3.84 (s,
3H, 4Ј-OCH3), 3.89 (s, 3H, 9-OCH3), 4.15 (s, 3H, 6-CH3), 7.17 (m,
3H, 8-H ϩ Phenyl-H), 7.54 (d, J7-8 ϭ 8.8 Hz, 1H, 7-H), 7.70 (d,
J ϭ 8.6 Hz, 2H, Phenyl-H), 7.73 (d, J10-8 ϭ 2.4 Hz, 1H, 10-H), 7.98
(d, J4-3 ϭ 6.4 Hz, 1H, 4-H), 8.45 (d, 1H, 3-H), 8.68 (s, 1H, 11-H).
9a: Yield 94.2%, mp 279°C (from ethanol). Anal. calcd. for
C23H19N3O3: C, 71.68; H, 4.97; N, 10.90. Found: C, 71.53; H, 5.12;
N, 10.57. 1H NMR (DMSO-d6) δ: 2.53 (s, 3H, 5-CH3), 2.87 (t,
J4-3 ϭ 6.4Hz, 2H, 4-H), 3.76 Ϫ 3.79 (m, 5H, 9-OCH3 ϩ 3-H), 6.99
(dd, J8-7 ϭ 8.6 Hz, J8-10 ϭ 1.9 Hz, 1H, 8-H), 7.41 (d, J7-8 ϭ 8.5 Hz,
1H, 7-H), 7.57 (d, J10-8 ϭ1.8 Hz, 1H, 10-H), 7.76 (s, 1H, 11-H),
7.85 (d, J ϭ 8.8 Hz, 2H, Phenyl-H), 8.34 (d, J ϭ 8.8 Hz, 2H, Phenyl-
H) 11.27 (s, 1H, 6-H).
5,6-Dimethyl-9-hydroxy-1-(4-nitrophenyl)-6H-pyrido[4,3-b]carb-
azole 12a
A mixture of the 9-methoxy derivative 11a (1 mmol) and hydro-
bromic acid 48% (15 mL) was heated at reflux under stirring for
2 h. After evaporation to dryness, the residue was taken up in water.
The resulting mixture was basified with concentrated ammonia, ex-
tracted with methylene dichloride, and then dried over magnesium
sulfate. After evaporation of the solvent, the solid was recrystallized
in the given solvent.
9b: Yield 89.8%, mp 261-262°C (from ethanol). Anal. calcd. for
C24H22N2O2: C, 77.81; H, 5.99; N, 7.56. Found: C, 77.53; H, 6.07;
1
N, 7.35. H NMR (DMSO-d6) δ: 2.52 (s, 3H, 5-CH3), 2.88 (m, 2H,
12a: Yield 71.2%; mp >300°C (from methylene chloride). Anal.
calcd. for C23H17N3O3: C, 72.05; H, 4.47; N, 10.96. Found: C, 71.93;
H, 4.67; N, 10.75. 1H NMR (DMSO-d6) δ: 3.11 (s, 3H, 5-CH3),
4.13 (s, 3H, 6-CH3), 7.05 (dd, J8-7 ϭ 8.5 Hz, J8-10 ϭ 2.2 Hz, 1H, 8-
H), 7.45 (d, 1H, 7-H), 7.53 (d, J10-8 ϭ 2.2 Hz, 1H, 10-H), 8.02 (d,
4-H), 3.69 (m, 2H, 3-H), 3.78 (s, 3H, 4Ј-OCH3), 3.85 (s, 1H, 9-
OCH3), 7.00 (dd, J8-7 ϭ 8.6 Hz, J8-10 ϭ 2.4 Hz, 1H, 8-H), 7.07 (d,
J ϭ 8.6 Hz, 2H, Phenyl-H), 7.42 (d, 1H, 7-H), 7.58 (m, 3H, 10-H ,
Phenyl-H), 7.89 (s, 1H, 11-H), 11,35 (s, 1H, 6-H).
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Arch. Pharm. Chem. Life Sci. 2005, 338, 556−561
J ϭ 8.8 Hz, 2H, Phenyl-H), 8.12 (d, J4-3 ϭ 6.0 Hz, 1H, 4-H), 8.46
(m, 3H, Phenyl-H ϩ 11-H), 8.53 (d, 1H, 3-H), 9.13 (s, 1H, 9-OH).
(d, J7-8 ϭ 8.8 Hz, 1H, 7-H), 8.17 (s, 1H, 11-H), 8.60 (m, 2H, 3-Hϩ
4-H), 9.33 (s, 1H, 4’-OH), 10.34 (s, 1H, 9-OH).
13d: Yield 95%; mp 297-298°C (from ethanol). Anal. calcd. for
C26H25N2O2I: C, 59.55; H, 4.81; N, 5.34. Found: C, 59.23; H, 4.97;
N, 5.17. 1H NMR (DMSO-d6) δ: 3.18 (s, 3H, 5-CH3), 3.81 (s, 3H,
4’-OCH3), 3.94 (s, 3H, 9-OCH3), 4.05 (s, 3H, 6-CH3), 4.20 (s, 3H,
2-CH3), 7.28 (dd, J8-7ϭ 8.9 Hz, J8-10 ϭ 2.5 Hz, 1H, 8-H), 7.33 (d,
J ϭ 8.7 Hz, 2H, Phenyl-H), 7.65 (m, 3H, 7-Hϩ Phenyl-H), 7.87 (d,
J10-8 ϭ 2.4 Hz, 1H, 10-H), 8.38 (s, 1H, 11-H), 8.55 (d, J4-3 ϭ 7.4
Hz, 1H, 4-H), 8.62 (d, 1H, 3-H).
9-Methoxy-1-(4-hydroxyphenyl)-5,6-dimethyl-6H-pyrido[4,3-b]-
carbazole (12b) and 9-methoxy-1-(4-hydroxyphenyl)-5,6-dimethyl-
6H-pyrido[4,3-b]carbazole 12c
A mixture of the 9-methoxy derivative 11b (1 mmol) and hydro-
bromic acid 48% (15 mL) was heated at reflux under stirring for
2 h. After evaporation to dryness, the residue was taken up in water.
The resulting mixture was basified with concentrated ammonia, ex-
tracted with methylene dichloride, and then dried over magnesium
sulfate. After evaporation of the solvent, the residue was purified
by column chromatography over a silica gel column. Elution with a
methylene chloride/methanol 98:2 v/v mixture gave two products: a
more mobile one, which corresponding to the monohydroxy deriva-
tive 12b and a less mobile corresponding to the dihydroxy deriva-
tive 12c.
Biological test procedures
Human tumor cell lines
Test solutions of all the tested compounds 5c and 11aϪ13d
(1 mg/mL) were prepared ex tempore for each test by dissolving
them in 100 µL of DMSO ϩ 900 µL of culture medium. After that,
the compounds were diluted in culture medium (described below)
to reach the final concentrations of 100, 10, 1, and 0.1 µg/mL.
12b: Yield 9.2%, mp 183-185°C (from isopropyl acetate). Anal.
calcd. for C24H20N2O2: C, 78.24; H, 5.47; N, 7.60. Found: C, 78.03;
H, 5.63; N, 7.47.1H NMR (DMSO-d6) δ: 3.09 (s, 3H, 5-CH3), 3.89
(s, 3H, 9-OCH3), 4.12 (s, 3H, 6-CH3), 7.03 (dd, J8-7 ϭ 8.8 Hz,
J8-10 ϭ 2.2 Hz, 1H, 8-H), 7.17 (d, J ϭ 8.2 Hz, 2H, Phenyl-H), 7.43
(m, 2H, 7H ϩ 10H), 7.69 (d, J ϭ 7.9 Hz, 2H, Phenyl-H), 7.98 (d,
J4-3 ϭ 6.3 Hz, 1H, 4-H), 8.43 (d, 1H, 3-H), 8.52 (s, 1H, 11-H), 9.14
(s, 1H, 4Ј-OH).
Cell lines
Cells of the human A549 cell line (non-small cell lung cancer) were
used. This line was obtained from American Type Culture Collec-
tion (Rockville, MD, USA) and cultured at the Cell Culture Collec-
tion of the Department of Tumor Immunology, Institute of
Immunology and Experimental Therapy, Wrocław, Poland. 24 h
before adding the tested agents, the cells were plated in 96-well
plates (Sarstedt, Newton, NC, USA) at a density of 104 cells per
well. The cells were cultivated in opti-MEM medium supplemented
with 2 mM glutamine (Gibco, Warsaw, Poland), streptomycin
(50 µg/mL), penicillin (50 U/mL) (both antibiotics from Polfa, Tar-
chomin, Poland), and 5% fetal calf serum (Gibco, Grand Island,
USA). The cell cultures were maintained at 37°C in a humid atmos-
phere with 5% CO2.
12c: Yield 26.4%; mp >300°C (from isopropyl acetate). Anal. calcd.
for C23H18N2O2: C, 77.95; H, 5.12; N, 7.90. Found: C, 77.73; H,
1
5.39; N, 7.73. H NMR (DMSO-d6) δ: 3.08 (s, 3H, 5-CH3), 4.12 (s,
3H, 6-CH3), 6.98 (d, J ϭ 8.3 Hz, 2H, Phenyl-H), 7.05 (dd,
J8-7 ϭ 8.6 Hz, J8-10 ϭ 2.2 Hz, 1H, 8-H), 7.44 (m, 1H, 7-H ϩ 10H),
7.57 (d, J ϭ 8.2 Hz, 2H, Phenyl-H), 7.96 (d, J4-3 ϭ 6.1 Hz, 1H, 4-
H), 8.42 (d, J3-4ϭ 6.1 Hz, 1H, 3-H), 8.54 (s, 1H, 11-H), 9.10 (s, 1H,
4Ј-OH), 9.77 (s, 1H, 9-OH).
General procedure for quaternization
The corresponding 1-(4-substituted phenyl)-6H-pyrido[4,3-b]carb-
azole (11b, 12aϪ12c) (0.1 mmol) was dissolved in 3 mL of DMF
(anhydrous) at room temperature and 0.06 mL (1 mmol) of iodome-
thane was added. The mixture was stirred for 2 h at room tempera-
ture, usually with the formation of an orange precipitate, and con-
centrated in vacuo. The precipitate was recrystallized from ethanol.
SRB assay
The cytotoxicity assays were performed after 72-h exposure of the
cultured cells to varying concentrations (from 0.1 to 100 µg/mL) of
the tested agents. The SRB method was used as described by Skehan
et al. [12]. The optical densities of the samples were measured on a
Multiskan RC photometer (Labsystems, Helsinki, Finland) at
570 nm.
13a: Yield 96%; mp >300°C (from ethanol). Anal. calcd. for
C
24H20N3O3I: C, 54.87; H, 3.84; N, 8.00. Found: C, 54.63; H, 3.97;
N, 7.73. 1H NMR (DMSO-d6) δ: 3.22 (s, 3H, 5-CH3), 4.02 (s, 3H,
6-CH3), 4.25 (s, 3H, 2-CH3), 7.15 (dd, J8-7 ϭ 8.9 Hz, J8-10 ϭ 2.5
Hz, 1H, 8-H), 7.59 (d, 1H, 7-H), 7.66 (d, J10-8 ϭ 2.5 Hz, 1H, 10-H),
8.05 (d, J ϭ 8.5 Hz, 2H, Phenyl-H), 8.19 (s, 1H, 11-H), 8.61Ϫ8.66
(m, 3H, Phenyl-H ϩ 4-H), 8.73 (d, J3-4 ϭ 7.4 Hz, 1H, 3-H), 9.33
(s, 1H, 9-OH).
The results were expressed as IC50 values (inhibitory concentration
50%), the dose of the compound which inhibits the proliferation
rate of the tumor cells by 50% compared with untreated control
cells, and these are summarized in Table 1 in comparison to
compound 5c. Each compound was tested in triplicate at every
concentration. Each experiment was repeated 3 times.
13b: Yield 49%; mp >300°C (from ethanol). Anal. calcd. for
C25H23N2O2I: C, 58.83; H, 4.54; N, 5.49. Found: C, 58.68; H, 4.73;
N, 5.27. 1H NMR (DMSO-d6) δ: 3.19 (s, 3H, 5-CH3), 3.95 (s, 3H,
References
9-OCH3), 4.03 (s, 3H, 6-CH3), 4.24 (s, 3H, 2-CH3), 7.13 (dd, J8-7
8.7 Hz, 1H, 8-H), 7.33 (d, J ϭ 8.7 Hz, 2H, Phenyl-H), 7.49 (d,
10-8 ϭ 2.3 Hz, 1H, 10-H), 7.59 (d, J7-8 ϭ 8.9 Hz, 1H, 7-H), 7.66 (d,
ϭ
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´
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10-8 ϭ 2.3 Hz, 1H, 10-H), 7.51 (d, J ϭ 8.5 Hz, 2H, Phenyl-H), 7.57
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