Synthesis and antiproliferative activity of clausine E, mukonine, and koenoline bioisosteres

Article abstract of DOI:10.1016/j.bmc.2007.05.033

Aza-analogues of clausine E, mukonine and koenoline were prepared from 1-(benzenesulfonyl)-1H-pyrrolo[2,3-b]pyridine-3-carboxaldehyde and their antiproliferative activity was evaluated against miscellaneous cancer cell lines and compared to those obtained

Full text of DOI:10.1016/j.bmc.2007.05.033

Bioorganic & Medicinal Chemistry 15 (2007) 5615–5619
Synthesis and antiproliferative activity of clausine E,
mukonine, and koenoline bioisosteres
Franc¸ois Liger,a Florence Popowycz,a Thierry Besson,b Laurent Picot,b
a,
Carlos M. Galmarinic, and Benoıt Joseph
*
*
ˆ
a
ˆ
Laboratoire de Chimie Organique 1, UMR – CNRS 5181, Universite´ Claude Bernard – Lyon 1, Batiment 308 – CPE,
43 Boulevard du 11 Novembre 1918, 69622 Villeurbanne Cedex, France
bLaboratoire de Biotechnologies et Chimie Bio-organique, FRE-CNRS 2766, UFR Sciences Fondamentales et Sciences
ˆ
´
pour l’Inge´nieur, Batiment Marie Curie, Universite de La Rochelle, F-17042 La Rochelle Cedex 1, France
cENS-CNRS UMR 5239, U.F.R. de Me´decine Lyon-Sud, 165 chemin du Grand Revoyet, BP 12,69921 Oullins, France
Received 10 January 2007; accepted 4 May 2007
Available online 18 May 2007
Abstract—Aza-analogues of clausine E, mukonine and koenoline were prepared from 1-(benzenesulfonyl)-1H-pyrrolo[2,3-b]pyri-
dine-3-carboxaldehyde and their antiproliferative activity was evaluated against miscellaneous cancer cell lines and compared to
those obtained with clausine E and mukonine.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
biological activities, physicochemical and pharmacoki-
netic properties.7
The 1-oxygenated carbazole alkaloids like clausine E,
mukonine, and koenoline (Fig. 1) have been isolated
from higher plants of the Rutaceae family.1 The cyto-
toxic activity of this alkaloid class has been roughly
reported in the literature.2,3 Nevertheless, koenoline was
screened in the NCI in vitro anticancer drug discovery
(NCS-654286) on sixty cancer cell lines.4 The compound
showed low antiproliferative activity (mean log GI50 va-
lue over all cell lines = À4.63). The broad range of useful
biological activities exhibited by many carbazole alka-
loids prompted several research groups to develop
chemical strategies (Fischer indolization, oxidative cycli-
zation of diarylamine, transition metal-mediated and
-catalyzed processes) for their total synthesis.1 Amongst
them, two close and practical syntheses were reported
by Bringmann and Brenna to prepare 1-oxygenated
carbazoles.5,6
With regard to the pharmacological potential of these
1-oxygenated carbazole alkaloids, we have designed
aza-analogues 1–3 (Fig. 1) in order to evaluate their
antiproliferative activities against miscellaneous tumor
cell lines and compare it with the natural products clau-
sine E and mukonine prepared in our Laboratory (in
CO2Me
CH2OH
N
H
N
H
OR
OMe
clausine E R = H
mukonine R = Me
koenoline
CO2Me
Pyrrolo[2,3-b]pyridine is an indole surrogate of interest
in medicinal chemistry. It has been used as a new
scaffold to prepare new drug candidates with improved
CH2OH
N
N
N
H
N
H
OR
OMe
1 R = H, 2 R = Me
3
Keywords: Aza-carbazole; Natural product; Bioisostere; Cancer.
*
Figure 1. Clausine E, mukonine, koenoline, and bioisosteres 1–3.
Corresponding authors. E-mail: benoit.joseph@univ-lyonl.fr
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.05.033

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F. Liger et al. / Bioorg. Med. Chem. 15 (2007) 5615–5619
our hands, koenoline was found particularly labile as
already reported in the literature8 and was not tested).
116, SW-48, and SW-480). IC50 values for 1–3 and
5-FU (for comparison) are reported in Table 1. Clausine
E inhibited the proliferation of MCF-7 breast cancer
line at high nanomolar concentrations, and of Mes-sa
sarcoma and colon cancer cell lines at low micromolar
concentrations. Mukonine was much less potent than
clausine E in all the cancer cell lines with antiprolifera-
tive activity obtained at high micromolar concentra-
2. Results and discussion
2.1. Chemistry
The preparation of derivatives 1–3 is summarized in
Scheme 1. Following the synthetic pathway of clausine
E disclosed by Bringmann,5 1-(benzenesulfonyl)-1H-
pyrrolo[2,3-b]pyridine-3-carboxaldehyde 49 was engaged
in a Horner–Wadsworth–Emmons reaction in the pres-
ence of diester phosphonate 510 to afford alkene 6.
Tert-butyl ester hydrolysis of 6 in acidic medium fol-
lowed by intramolecular cyclization of monoester acid
led to the desired aza-carbazole 7 in 80% yield (two
steps). Deacetylation of 7 occurred in the presence of
EtONa/EtOH at 0 ꢁC to give alcohol 8 in 89% yield.
Benzenesulfonyl group of 8 was removed in MeONa/
MeOH to provide aza-clausine E 1 in 80% yield.
O-Methylation (K2CO3, Me2SO4, acetone) of 7 led to
the intermediate 9 which was then treated by MeONa/
MeOH to afford aza-mukonine 2. Finally, the reduction
of the ester group was performed on compound 9 to give
aza-koenoline 3 in 87% yield.
tions. Compound 1 displayed almost the same
antiproliferative activity in all the cancer cell lines used
here. Derivative 2 showed a preferential growth inhibi-
tory activity in the breast and colorectal cancer cell lines
(concentration range 1–10 lM). The pattern of activities
of compounds 1 and 2 looked similar to that of clausine
E. However, compound 1 was more active than clausine
E in the Mes-sa sarcoma line, while both compounds
showed higher antiproliferative activity in the SW-48
colorectal cancer line. More importantly, both com-
pounds showed similar activity profiles to that of the
clinically used 5-FU in colorectal cancer models.
Finally, compound 3 showed a weak antiproliferative
activity in all cellular models compared to clausine E
and compounds 1 and 2. This weak antiproliferative
activity was similar to that observed for mukonine.
The cell cycle effects of clausine E, mukonine, 1, 2, and 3
are shown in Table 2. Clausine E induced a moderate
G2/M arrest in all colorectal cell lines. Differently, treat-
ment of HCT-116 and SW-48 with mukonine induced a
slight increase of cells in the G1 phase; however, this
drug showed no major perturbations of the cell cycle
in the SW-480 cells. Incubation of HCT-116, SW-48,
2.2. Antiproliferative activity
Clausine E, mukonine, and compounds 1–3 were tested
for their antiproliferative activities against MCF-7
breast adenocarcinoma cell line, Mes-sa sarcoma cell
line, and three colorectal carcinoma cell lines (HCT-
(EtO)2P(O)
5
CO2Et
CO2Et
CO2Et
CO2t-Bu
CHO
1) TFA, H2O, CH2Cl2, rt
2) Ac2O, AcONa, rflx
CO2t-Bu
LDA, THF, 0 °C
N
OAc
SO2Ph
N
77%
80%
N
N
N
N
SO2Ph
SO2Ph
4
7
6
89% Na, EtOH, 0 °C
CO2Et
CO2Me
OMe
CO2Et
K2CO3, Me2SO4
acetone, rflx
Na, MeOH, rflx
OMe
SO2Ph
N
OH
N
N
H
75%
N
80%
N
N
SO2Ph
8
2
9
1) LiAlH4, THF, rflx
2) NH4Cl, H2O
80% Na, MeOH, rflx
87%
CO2Me
5
CH2OH
4
7
3
2
OH
N
H
N
OMe
N
N
H
1
3
Scheme 1. Synthetic pathway to bioisosteres 1, 2, and 3.

F. Liger et al. / Bioorg. Med. Chem. 15 (2007) 5615–5619
5617
Table 1. In vitro cytotoxicity of clausine E, mukonine, 1, 2, 3, and 5-FU against human cancer cell lines
a
Compound
IC50 (lM)
HCT-116
1.9 0.2
MCF-7
Mes-sa
SW-48
21
SW-480
4.8 3
Clausine E
Mukonine
0.6 0.3
35
80 38
1
7
30
11
9
4
43
5
62 21
9.0 1.3
5.6 2.6
21.1 2.2
5.3 2.8
72 26
8.8 1.8
4.7 2.4
63.1 9.5
3.5 3.6
1
16
8
7.1 1.4
8.6 1.8
2
4.4 3.4
58 14
0.3 0.1
144 32
65 26
ND
3
5-FU
36.6 11.6
2.8 1.8
a Values are expressed as means standard deviation (SD) of five different experiments (ND, not determined).
Table 2. Percentage of cells (%) in the different phases of the cell cycle
Compound
HCT-116
S
SW-48
SW-480
a
G1
G2/M
11 1.9
G1
S
G2/M
G1
S
G2/M
Control
Clausine E
Mukonine
61.8 8.7
60 10
27.2 6.9
12.1 0.1
17.4 6.8
29.1 5.4
31 7.2
66.1 2.9
65.6 7.9
21 1.3
8
12.9 1.9
21.9 1.6
61.8 2.8
59.9 14.1
55.8 1.5
58.7 2.9
60.7 5.6
52.3 0.5
27.5 2.3
17.5 10.5
33.8 4.8
26.5 2.1
10.7 2.8
25.7 3.6
10.3 3.3
14.7 1.3
11.9 1.2
14.7 2.5
28 6.2
11.7 0.4
14.3 4.2
16.4 2.6
29.3 1.5
12.5
70.8 7.1
56.6 5.5
52.6 5.5
53.3 1.4
76.7
62.4 3.1
59
57.4 0.9
2
14.1 1.4
18.9 1.4
23.0 1.4
9.7 2.6
9
18.7
3.4
2
1
2
3
2
18.1 2.5
32.4
27.3
33
6
2
17.4 1.1
3
a Values are expressed as means SD of three different experiments.
and SW-480 colorectal cancer cells with 1 and 2 induced
a slight increase in G2/M phases suggesting induction of
a modest mitotic arrest. For compound 3, a higher G2/
M arrest pattern was observed in HCT-116 and SW-48
cells that may be ascribed to a moderate mitotic arrest.
This mitotic arrest was not so evident in the SW-480
cells.
flame-dried apparatus. Clausine E (mp 207–209 ꢁC, lit.
mp 203ꢁC), mukonine (mp 195–197 ꢁC, lit. mp 201 ꢁC)
were synthesized according to the procedure described
by Bringmann et al.5 IR, NMR, and MS data were iden-
tical with those reported in the literature.5
4.1. Tert-butyl 4-(1-benzenesulfonyl-1H-pyrrolo[2,3-b]-
pyridin-3-yl)-3-(ethoxycarbonyl)but-3-enoate (6)
3. Conclusion
At 0 ꢁC and under argon atmosphere, a solution of 2 M
lithium diisopropylamide (3.49 mL, 6.98 mmol) in THF
was added dropwise to a solution of diester phospho-
nate 5 (2.36 g, 6.98 mmol) in THF (30 mL). The solution
was stirred at 0 ꢁC for 20 min. A solution of 1-(benze-
nesulfonyl)-1H-pyrrolo[2,3-b]pyridine-3-carboxaldehyde
4 (1.33 g, 4.65 mmol) was added dropwise at 0 ꢁC. The
final mixture was stirred at room temperature for 4 h.
After evaporation of solvent and addition of water,
the aqueous phase was extracted with EtOAc
(2· 10 mL). The organic phases were combined, dried
over MgSO4, and evaporated in vacuo. The residue
was purified by flash chromatography (PE/EtOAc
85:15) to give 6 (1.68 g, 77%) as a solid. Mp 102–
104 ꢁC (EtOH); IR (KBr) m 3063, 2980, 1712, 1640,
In summary, we have developed an efficient and
straightforward route to 1-oxygenated aza-carbazoles.
Aza-clausine E 1 and aza-mukonine 2 exhibited signifi-
cant human colorectal cancer cell growth inhibitory
activity in the micromolar range. These compounds rep-
resent promising new anticancer agents and structure–
activity relationship studies are in progress to reach
more potent derivatives.
4. Experimental
Melting points were measured with a Buchi Tottoli
¨
1
SMP-20 heating unit and are uncorrected. IR spectra
were recorded with a Perkin-Elmer spectrum one spec-
trophotometer. NMR spectra were recorded at 300 K
with an AVANCE 300 Bruker spectrometer at
1537, 1450, 1252, 1179 cmÀ1; H NMR (CDCl3) d 1.39
(t, 3H, J = 7.2 Hz, CH3), 1.56 (s, 9H, CH3), 3.61 (s,
2H, CH2), 4.34 (q, 2H, J = 7.2 Hz, CH2), 7.29 (dd, 1H,
J = 4.6, 7.9 Hz, HAr), 7.53 (t, 2H, J = 7.5 Hz, HAr),
7.63 (t, 1H, J = 7.5 Hz, HAr), 7.88 (s, 1H, HAr), 8.00
(dd, 1H, J = 1.3, 7.9 Hz, HAr), 8.02 (s, 1H, @CH), 8.25
(d, 2H, J = 7.5 Hz, HAr), 8.51 (dd, 1H, J = 1.3, 4.6 Hz,
HAr); 13C NMR (CDCl3) d 14.4 (CH3), 28.1 (3 CH3),
35.9 (CH2), 61.4 (C), 81.8 (CH2), 114.1 (C), 119.4
(CH), 122.6 (C), 125.7 (CH), 127.4 (C), 128.2 (2 CH),
128.3 (CH), 129.2 (2 CH), 129.9 (CH), 134.4 (CH),
138.0 (C), 145.9 (CH), 147.0 (C), 167.0 (CO), 169.8
(CO); MS (ESI) m/z 471 (M+H)+; Anal. Calcd for
C24H26N2O6S: C, 61.26; H, 5.57; N, 5.95. Found: C,
60.88; H, 5.33; N, 6.05.
300 MHz for H and 75 MHz for 13C. Chemical shifts
1
are expressed in parts per million (ppm) relative to
TMS. Mass spectra were recorded with a Thermo Finn-
igan Mat 95 XL. Elemental analyses were performed on
a Thermoquest Flash 1112 series EA analyser. TLC was
conducted on precoated silica gel plates (Merck 60F254
)
and the spots were visualized under UV light. Flash
chromatography was carried out on column using flash
silica gel 60 Merck (40–63 mm) using the indicated sol-
vents (petroleum ether (PE): bp 40–60 ꢁC). All reactions
requiring anhydrous conditions were conducted in

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F. Liger et al. / Bioorg. Med. Chem. 15 (2007) 5615–5619
4.2. Ethyl 8-acetoxy-9-benzenesulfonyl-8-hydroxy-9H-
pyrido[2,3-b]indole-6-carboxylate (7)
for 12 h. After addition of H2O and evaporation of sol-
vent, the residue was taken up in H2O and extracted
with EtOAc (2· 5 mL). The organic phases were com-
bined, dried over MgSO4, and evaporated in vacuo.
The crude solid was washed with MeOH to afford 1
(49 mg, 80%) as a solid. Mp > 210 ꢁC (washing MeOH);
IR (KBr) m 3300–3100, 2851, 1677, 1638, 1586, 1353,
A
solution of 6 (570 mg, 1.21 mmol), TFA/H2O
(8.2 mL, 8/0.2) in CH2Cl2 (8 mL) was stirred at room
temperature for 2 h. The solvents were evaporated and
the residue was dissolved in acetic anhydride (10 mL)
and sodium acetate (220 mg, 2.68 mmol) was added.
The mixture was heated at reflux for 3 h. After cooling,
the solvent was evaporated. The crude solid was purified
by flash chromatography (PE/EtOAc 8:2) to afford 7
(340 mg, 80%) as a solid. Mp = 178–180 ꢁC (EtOH);
IR (KBr) m 3070, 2987, 1776, 1728, 1590, 1370, 1260,
1
1255, 1089 cmÀ1; H NMR (DMSO-d6) d 3.87 (s, 3H,
CH3), 7.23 (dd, 1H, J = 4.9, 7.7 Hz, H3), 7.51 (d, 1H,
J = 1.3 Hz, H5), 8.35 (d, 1H, J = 1.3 Hz, H7), 8.45 (dd,
1H, J = 1.5, 4.9 Hz, H2), 8.60 (dd, 1H, J = 1.5, 7.7 Hz,
H4), 10.27 (s, 1H, OH), 12.08 (s, 1H, NH); 13C NMR
(DMSO-d6) d 51.8 (CH3), 111.2 (CH), 114.7 (CH),
115.6 (CH), 115.8 (C), 121.4 (C), 121.5 (C), 129.1
(CH), 131.8 (C), 143.1 (C), 146.7 (CH), 152.2 (C),
166.9 (CO); MS (ESI) m/z 243 (M+H)+; Anal. Calcd
for C13H10N2O3: C, 64.46; H, 4.16; N, 11.56. Found:
C, 64.76; H, 3.99; N, 11.40.
1202 cmÀ1
;
1H NMR (CDCl3)
d
1.43 (t, 3H,
J = 7.2 Hz, CH3), 2.47 (s, 3H, CH3), 4.43 (q, 2H,
J = 7.2 Hz, CH2), 7.32 (dd, 1H, J = 4.9, 7.7 Hz, H3),
7.39 (t, 2H, J = 7.4 Hz, HAr), 7.51 (t, 1H, J = 7.4 Hz,
HAr), 7.94 (d, 1H, J = 1.5 Hz, H7), 7.99 (br d, 2H,
J = 7.4 Hz, HAr), 8.22 (dd, 1H, J = 1.5, 7.7 Hz, H4),
8.49 (d, 1H, J = 1.5 Hz, H5), 8.56 (dd, 1H, J = 1.5,
4.9 Hz, H2); 13C NMR (CDCl3) d 14.4 (CH3), 21.4
(CH3), 61.6 (CH2), 118.9 (C), 119.8 (CH), 120.2 (CH),
125.6 (CH), 127.3 (C), 127.7 (C), 127.7 (2 CH), 128.8
(CH), 128.9 (2 CH), 132.9 (C), 134.0 (CH), 138.6 (C),
138.7 (C), 148.1 (CH), 152.9 (C), 165.3 (CO), 169.7
(CO); MS (ESI) m/z 439 (M+H)+; Anal. Calcd for
C22H18N2O6S: C, 60.27; H, 4.14; N, 6.39. Found: C,
60.01; H, 4.05; N, 6.27.
4.5. Ethyl 9-benzenesulfonyl-8-methoxy-9H-pyrido[2,3-
b]indole-6-carboxylate (9)
A solution of 8 (160 mg, 0.40 mmol), potassium carbon-
ate (56 mg, 0.41 mmol), and dimethylsulfate (80 mL,
0.82 mmol) in acetone (10 mL) was stirred at reflux for
7 h. After cooling, the solvent was evaporated. The res-
idue was taken up in H2O and extracted with EtOAc
(2· 10 mL). The organic phases were combined, dried
over MgSO4, and evaporated in vacuo. The crude solid
was recrystallized from EtOH to give 9 (132 mg, 80%) as
a solid. Mp = 161–162 ꢁC (EtOH); IR (KBr) m 2979,
4.3. Ethyl 9-benzenesulfonyl-8-hydroxy-9H-pyrido[2,3-
b]indole-6-carboxylate (8)
1
1721, 1590, 1499, 1345, 1260 cmÀ1; H NMR (CDCl3)
At 0 ꢁC and under argon atmosphere, sodium (18 mg,
0.78 mmol) was added portionwise to a solution of 7
(340 mg, 0.77 mmol) in EtOH/THF (15 mL, 1/2). The
solution was stirred at 0 ꢁC for 2 h. At 0 ꢁC, the reaction
was hydrolyzed by addition of H2O and the solvents
were evaporated in vacuo. The residue was taken up in
H2O and extracted with EtOAc (2· 5 mL). The organic
phases were combined, dried over MgSO4, and evapo-
rated in vacuo. The crude solid was recrystallized from
EtOH to give 8 (275 mg, 89%) as a white solid.
Mp = 198–200 ꢁC (EtOH); IR (KBr) m 3301, 2963,
d 1.44 (t, 3H, J = 7.2 Hz, CH3), 3.92 (s, 3H, CH3),
4.43 (q, 2H, J = 7.2 Hz, CH2), 7.38 (dd, 1H, J = 4.9,
7.7 Hz, H3), 7.55–7.65 (m, 3H, HAr), 7.68 (d, 1H,
J = 1.5 Hz, H7), 8.26 (br s, 1H, H5), 8.28-8.33 (m, 3H,
HAr + H4), 8.68 (dd, 1H, J = 1.5, 4.9 Hz, H2); 13C
NMR (CDCl3) d 14.5 (CH3), 56.1 (CH3), 62.5 (CH2),
112.4 (CH), 115.0 (CH), 119.7 (C), 119.9 (CH), 126.0
(C), 127.6 (2 CH), 127.8 (C), 128.8 (2 CH), 128.9
(CH), 131.0 (C), 133.5 (CH), 140.9 (C), 147.7 (CH),
148.7 (C), 153.7 (C), 166.2 (CO); MS (ESI) m/z 411
(M+H)+; Anal. Calcd for C21H18N2O5S: C, 61.45; H,
4.42; N, 6.83. Found: C, 61.66; H, 4.52; N, 6.97.
1
1716, 1585, 1377, 1261 cmÀ1; H NMR (CDCl3) d 1.44
(t, 3H, J = 7.2 Hz, CH3), 4.43 (q, 2H, J = 7.2 Hz,
CH2), 7.32 (dd, 1H, J = 4.9, 7.7 Hz, H3), 7.41 (t, 2H,
J = 7.4 Hz, HAr), 7.54 (t, 1H, J = 7.4 Hz, HAr), 7.87 (d,
1H, J = 1.5 Hz, H7), 7.98 (br d, 2H, J = 7.4 Hz, HAr),
8.15 (d, 1H, J = 1.5 Hz, H5), 8.19 (dd, 1H, J = 1.5,
7.7 Hz, H4), 8.55 (dd, 1H, J = 1.5, 4.9 Hz, H2), 9.51 (s,
1H, OH); 13C NMR (CDCl3) d 14.4 (CH3), 61.4
(CH2), 113.9 (CH), 119.4 (CH), 119.6 (C), 120.5 (CH),
126.7 (C), 127.9 (2 CH), 128.0 (C), 128.7 (C), 129.0
(CH), 129.2 (2 CH), 134.6 (CH), 136.9 (C), 145.2 (C),
147.8 (CH), 152.0 (C), 165.9 (CO); MS (ESI) m/z 397
(M+H)+; Anal. Calcd for C20H16N2O5S: C, 60.60; H,
4.07; N, 7.07. Found: C, 60.55; H, 3.98; N, 6.99.
4.6. Methyl 8-methoxy-9H-pyrido[2,3-b]indole-6-carbox-
ylate (2)
A solution of 9 (120 mg, 0.29 mmol) and catalytic
amount of sodium in MeOH (6 mL) was heated over-
night at reflux. After addition of H2O and evaporation
of solvent, the residue was taken up in H2O and
extracted with EtOAc (2· 5 mL). The organic phases
were combined, dried over MgSO4, and evaporated in
vacuo. The crude solid was washed with MeOH to
afford 2 (56 mg, 75%) as a white solid. Mp > 210 ꢁC
(washing MeOH); IR (KBr) m 3122, 2990, 1698, 1584,
1
1406, 1227 cmÀ1; H NMR (DMSO-d6) d 3.90 (s, 3H,
4.4. Methyl 8-hydroxy-9H-pyrido[2,3-b]indole-6-carbox-
ylate (1)
CH3), 4.04 (s, 3H, CH3), 7.26 (dd, 1H, J = 4.7, 7.7 Hz,
H3), 7.56 (s, 1H, H5), 8.47 (d, 1H, J = 4.7 Hz, H2),
8.48 (s, 1H, H7), 8.65 (d, 1H, J = 7.7 Hz, H4), 12.38 (s,
1H, NH); 13C NMR (DMSO-d6) d 51.9 (CH3), 55.7
(CH3), 107.1 (CH), 115.5 (C), 115.9 (CH), 116.4 (CH),
A solution of 8 (100 mg, 0.25 mmol) and catalytic
amount of sodium in MeOH (6 mL) was heated at reflux

F. Liger et al. / Bioorg. Med. Chem. 15 (2007) 5615–5619
5619
121.0 (C), 121.5 (C), 129.3 (CH), 132.0 (C), 145.3 (C),
146.9 (CH), 152.2 (C), 166.8 (CO); MS (ESI) m/z 257
(M+H)+; Anal. Calcd for C14H12N2O3: C, 65.62; H,
4.72; N, 10.93. Found: C, 65.44; H, 4.66; N, 11.05.
IC50 values were defined as drug doses resulting in
50% cell growth inhibition relative to untreated cells.
4.9. Cell cycle assay
4.7. (8-Methoxy-9H-pyrido[2,3-b]indol-6-yl)methanol (3)
For analysis of DNA content and cell cycle distribution,
colorectal cancer cell lines were treated with compounds
1, 2, and 3 for 72 h. Based on cytotoxicity assay, a con-
centration of 100 lM (aprox IC80 values for these cell
lines) was chosen for drug exposure experiments. After
drug exposure, 106 cells/mL were resuspended in 2 mL
of propidium iodide solution (50 lL/mL), incubated at
4 ꢁC overnight, and then analyzed by flow cytometry.
Flow cytometry was performed on a FACScalibur (Bec-
ton–Dickinson, San Jose, California). Cell cycle distri-
bution and DNA ploidy status were calculated after
exclusion of cell doublets and aggregates on a FL2-
area/FL2-width dot plot using Modfit LT 2.0TM software
(Verity Software Inc. Topsham. ME).
A solution of 9 (60 mg, 1.46 mmol), LiAlH4 (14 mg,
0.29 mmol) in THF (3 mL) was heated at reflux for
2 h. After addition of NH4Cl, the aqueous phase was ex-
tracted with EtOAc (2· 10 mL). The organic phases
were combined, dried over MgSO4, and evaporated in
vacuo. The crude solid was recrystallized from EtOAc
to afford 3 (29 mg, 87%) as a white solid. Mp > 210 ꢁC
(EtOAc); IR (KBr) m 3400–3100, 2994, 1607, 1586,
1407, 1279 cmÀ1 1H NMR (CD3OD + D2O) d 4.05
;
(s, 3 H, CH3), 4.76 (s, 2H, CH2), 7.08 (s, 1H, H5),
7.20 (dd, 1H, J = 4.7, 7.7 Hz, H3), 7.68 (s, 1H, H7),
8.35 (dd, 1H, J = 1.5, 5.1 Hz, H2), 8.43 (dd, 1H,
J = 1.5, 7.7 Hz, H4); 13C NMR (CD3OD) d 56.1
(CH3), 66.0 (CH2), 108.1 (CH), 112.8 (CH), 116.1
(CH), 118.2 (C), 122.7 (C), 129.9 (C), 130.1 (CH),
135.4 (C), 146.2 (CH), 147.4 (C), 152.8 (C); MS (ESI)
m/z 229 (M+H)+; Anal. Calcd for C13H12N2O2:
C, 68.41; H, 5.30; N, 12.27. Found: C, 68.66; H, 5.15;
N, 12.13.
References and notes
1. (a) Kno¨lker, H.-J.; Reddy, K. R. Chem. Rev. 2002, 102,
4303; (b) Kno¨lker, H.-J. Top. Curr. Chem 2005, 244, 115.
2. Fiebig, M.; Pezzuto, J. M.; Soejarto, D. D.; Kinghorn, A.
D. Phytochemistry 1985, 24, 3041.
3. Ito, C.; Katsuno, S.; Itoigawa, M.; Ruangrungsi, N.;
Mukainaka, T.; Okuda, M.; Kitagawa, Y.; Tokuda, H.;
Nishino, H.; Furukawa, H. J. Nat. Prod. 2000, 63, 125.
4. http://dtp.nci.nih.gov.
5. Bringmann, G.; Tasler, S.; Endress, H.; Peters, K.; Peters,
E.-M. Synthesis 1998, 1501.
6. Brenna, E.; Fuganti, C.; Serra, S. Tetrahedron1998, 54, 1585.
4.8. Cell proliferation assay
In vitro antiproliferative activity was determined in
five separate experiments, each of which was performed
in triplicate as previously described.11 Briefly, asynchro-
nously growing cells were transferred into 96-well
culture plates (Costarꢂ, Corning Inc., New York) in
100 lL of medium at a final cell concentration of
5 · 103 cells/well and incubated in media for 24 h.
Corresponding drug concentrations were then added
to each plate. After 72 h of drug exposure, 20 lL of 3-
[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bro-
mide (MTT) reagent (5 mg/mL) was added to each well.
Cell growth was expressed as the percent of absorbance
of treated wells relative to the untreated control wells.
´
7. (a) Merour, J.-Y.; Joseph, B. Curr. Org. Chem. 2001, 5, 471;
(b) Popowycz, F.; Routier, S.; Joseph, B.; Merour, J.-Y.
´
Tetrahedron 2007, 63, 1031.
8. Bringmann, G.; Tasler, S. Tetrahedron 2001, 57, 2337.
´
9. Mouaddib, A.; Joseph, B.; Hasnaoui, A.; Merour, J.-Y.
Tetrahedron Lett. 1999, 40, 5853.
10. Owton, W. M.; Gallagher, P. T.; Juan-Montesinos, A.
Synth. Commun. 1993, 23, 2119.
11. Galmarini, C. M.; Clarke, M. L.; Falette, N.; Puisieux, A.;
Mackey, J. R.; Dumontet, C. Int. J. Cancer 2002, 97, 439.