Synthesis and antitumor characterization of pyrazolic analogues of the marine pyrroloquinoline alkaloids: Wakayin and tsitsikammamines

Article abstract of DOI:10.1021/jm051247f

A series of aza analogues of the marine alkaloids wakayin and tsitsikammamines A and B have been synthesized. The strategy used was based on [3 + 2] cycloaddition reactions involving 3-ethylamine-indole-4,7-dione and different diazo reagents. All the comp

Full text of DOI:10.1021/jm051247f

J. Med. Chem. 2006, 49, 2979-2988
2979
Synthesis and Antitumor Characterization of Pyrazolic Analogues of the Marine
Pyrroloquinoline Alkaloids: Wakayin and Tsitsikammamines
Laurent Legentil,† Laurent Benel,‡ Viviane Bertrand,‡ Brigitte Lesur,‡ and Evelyne Delfourne*,†
Laboratoire SPCMIB, UMR-CNRS 5068, UniVersite´ Paul Sabatier, 118 route de Narbonne, 31062, Toulouse Cedex 4, France, and
Laboratoire Cephalon France, Centre de Recherches et de De´Veloppement, 19 aVenue du Pr Cadiot, B.P. 22,
94701 Maisons-Alfort Cedex, France
ReceiVed December 15, 2005
A series of aza analogues of the marine alkaloids wakayin and tsitsikammamines A and B have been
synthesized. The strategy used was based on [3 + 2] cycloaddition reactions involving 3-ethylamine-indole-
4,7-dione and different diazo reagents. All the compounds were evaluated in vitro for antiproliferative activity
against five distinct cancer cell lines and for their inhibitory effect on topoisomerase isoenzymes I and II.
Some of the compounds inhibited the topoisomerase I and/or II catalyzed relaxation of supercoiled DNA at
a concentration comparable to the drugs camptothecin and etoposide. Only a few of them exhibited cytotoxic
activity with IC50 values in the micromolar range.
Introduction
Pyrrolo[4,3,2-de]quinolines, an important family of alkaloids
isolated from marine source, recently received considerable
attention due to their biological activities.1 Wakayin 1, isolated
in 1991 from the ascidian ClaVelina species, has been reported
to have a diverse array of bioactivities including murine cell
line cytotoxicity and topoisomerase I inhibition.2 The closely
structurally related tsitsikammamines A 2 and B 3, isolated from
a Latrunculid sponge, are cytotoxic but they inhibit neither
topoisomerases I nor II3 (Figure 1).
Synthesis of compounds structurally related to wakayin was
reported by Zhang et al.,4 Barret and Roue,5 and most recently
by Beneteau et al.6 who also reported on their antiproliferative
activity.
Figure 1. Natural compounds and analogues structures.
There are a wide range of topoisomerase I and II inhibitors
reported in the literature, but very few possess dual inhibitory
cleavage of the Boc-protective group of the amine of the side
chain (Scheme 1).
activities against I and II enzymes. Dual topoisomerase inhibitors
should have a broader cell cycle activity and antitumor
efficiency. They would prevent the frequently observed com-
pensation process: alterations in expression of one isoform when
the other is selectively inhibited; also they should display
broader spectrum of activity with regards to tumor type.
As part of our research program on new potential anticancer
drugs,7 we focused our efforts on the preparation of analogues
a and b of wakayin and tsitsikammamines in which one of the
two pyrrole rings has been replaced by a pyrazole ring. We
report herein the synthesis of these compounds and the results
of their topoisomerases I and II inhibition along with their in
vitro antitumor activity.
The synthesis of 4 (Scheme 2) was inspired by a recent work
of Beneteau and Besson who described the preparation of a
related compound 5 (Scheme 1).8 The tosyl group was preferred
to the Boc group as the protective group of the indolic nitrogen.
Indeed these authors demonstrated that the final cyclization of
similar N-protected indole only occurs if the N-indole is
substituted by a strong electron-withdrawing group. The nitro
derivative 14 precursor of compound 4 was prepared from 4,7-
dimethoxyindole 12 prepared in turn from 2,5-dimethoxyben-
zaldehyde according to a five-step procedure previously pub-
lished.9 In our hands, the last step of this procedure, the Henry
reaction on dimethoxyindole-3-carboxaldehyde 13, gives rise
to the dinitro derivative 15 as a side product. Compound 14
was obtained in 60% on a 1 g scale, but the yield drastically
decreased upon scale-up. The production of 15 could be limited
by using acetic acid as the solvent of the reaction. Unfortunately,
in this case any yield improvement was observed due to the
formation of several degradation products. The indolic nitrogen
of compound 14 was further tosylated in 78% yield by treatment
with potassium hydroxide and p-toluenesulfonyl chloride The
resulting compound 16 was reduced into the corresponding
amino derivative 17 by LAH in 95% yield.
Chemistry
The retrosynthetic analysis to prepare the desired analogues
was based on a [3 + 2] cycloaddition reaction between the fully
protected dioxotryptamine derivative 4 and adequately substi-
tuted diazo compounds 6 to give rise to the corresponding
tricyclic analogues 7-10. Final cyclization will take place after
* Corresponding author. Phone: 33 561 55 62 93. Fax: 33 561 55 60
11. E-mail: delfourn@chimie.ups-tlse.fr.
The side chain amino group of 17 was then protected as Boc
derivative 18 in 60% by treatment with sodium hydride and
† Universite´ Paul Sabatier.
‡ Centre de Recherches et de De´veloppement.
10.1021/jm051247f CCC: $33.50 © 2006 American Chemical Society
Published on Web 04/25/2006

2980 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 10
Legentil et al.
Scheme 1. Retrosynthetic Scheme for the Preparation of
Scheme 3a
Analogues
Scheme 2a
a (a) Me2NH, CH2O, AcOH, room temperature, 3 h. (b) 1: MeI, CH2Cl2,
toluene, room temperature, overnight. 2: TMSCN, 1 N TBAF in THF, THF,
room temperature, 1 h. (c) LAH, CH2Cl2, Et2O, room temperature, 1 h. (d)
Boc2O, CH2Cl2, room temperature, 1 h. (e) p-TsCl, NaH, room temperature,
30 min.
Scheme 4a
a (a) TrCl, Et3N, DMAP, THF, reflux overnight. (b) TsNHNH2, THF,
room temperature, 1 h. (c) KOH/MeOH, THF, 90 °C, 1 h. (d) TsNHNH2,
EtOH, room temperature, 1 h. (e) CH2N2, THF, room temperature, 30 min.
a (a) POCl3/DMF, DMF, 0 °C, 1 h 30. (b) CH3NO2, NH4OAc, AcOH,
reflux, 3 h. (c) p-TsCl, KOH, THF, room temperature, 1 h. (d) LAH, CH2Cl2,
Et2O, 0 °C, 15 min. (e) Boc2O, NaH, THF, room temperature, 1 h. (f) CAN,
CH3CN/H2O, room temperature, 15 min.
hydrazones of corresponding aryl aldehydes in alcohol solvents.
In this way, phenyldiazomethane 6b was obtained as previously
reported from the commercially available benzaldehyde tosyl-
hydrazone,13 whereas the diazoderivative 6c (Scheme 4) was
synthesized from 4-hydroxybenzaldehyde according to the
reaction sequence described in Scheme 4.
di-tert-butyl dicarbonate in THF along with the di-Boc com-
pound 19 in 5% yield. The overall yield of the synthesis of 18
from 4,7-dimethoxyindole 12 was 20%. The oxidation of 18
by CAN gave rise to quinone 4 in 83% yield.
To improve the yield of the preparation of intermediate 18,
an alternative route derived from the method reported by Iwao
et al. was examined (Scheme 3).10
It also started with compound 12, which was further converted
quantitatively into the gramine derivative 20 using a Mannich
type reaction. This last compound was reacted with methyl
iodide to give the quaternary salt, which was subsequently
transformed into the cyano derivative 21 by action of cyanot-
rimethylsilane in the presence of tetrabutylammonium fluoride
(90% yield). Despite what was reported by the authors, a
silylated activating group on the nitrogen of the indole was not
necessary to do this reaction.11 It should be noted that
compounds 20 and 21 have been previously described.12
Compound 21 was then reduced by LAH in 86% yield to
provide indole derivative 22; the amino group of the side chain
was further protected as a Boc derivative (compound 23). The
overall yield is 55% from 12 to 18 with no scale-up limitation.
Aryldiazomethane derivatives 6b (R ) Ph) and 6c were
readily prepared by Banford-Stevens pyrolysis of the tosyl-
The hydroxyl group was protected as a triphenylmethyl group
(trityl or Tr) to give compound 24, which was reacted with
hydrazine to form the corresponding hydrazone 25. In alkaline
conditions, this last compound yielded the diazo derivative 6c.
Similarly, indole carboxaldehyde was easily transformed into
the corresponding tosylhydrazone derivative 26, but attempts
to convert it into the diazo compound 6d failed. An alternative
way to introduce an indole moiety via a cycloaddition reaction
was to generate the diazo species 6e by action of diazomethane
on isatine as previously reported by Eistert and Ganster.14
The [3 + 2] cycloaddition reactions of quinone 4 with the
diazo derivatives diazomethane or aryldiazomethanes led as
expected to a mixture of the two regioisomers (Scheme 5).
Reaction of diazomethane 6a with quinone 4 gave in 88%
global yield the mixture of regioisomers 7c and 7d in a ratio of
60/40, the methylated analogues 8c/8d being side products of
the reaction. 8c/8d were obtained in 97% yield when compounds
7c/7d were reacted with excess diazomethane in methanol. The
other diazo reagents including 6b, 6c, and 6e led to the mixtures
9c/9d, 10c/10d, and 11c/11d, respectively. It should be noted
that the reaction leading to compounds 10c/10d also gave a

Wakayin and Tsitsikammamines Pyrazolic Analogues
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 10 2981
Scheme 5a
a (a) THF, 30 min. (b) 1: TFA, CH2Cl2, room temperature, 30 min. 2: EtOH, 4Α molecular sieve, reflux. (c) 1 N TBAF in THF, THF, reflux or 1 N
NaOH, dioxane, room temperature, 24 h.
Table 1. Intramolecular Cyclization Reaction, Yields, and Ratios of
respectively. For compounds 10e and 10f this reaction was
Isomers (See Scheme 5)
performed in the presence of tetrabutylammonium fluoride in
THF in order to recover the phenolic form of the final product.
In the case of compounds 11e and 11f, attempts to further
convert the oxo-dihydroindolyl group into an indolic group16
were unsuccessful. Further cleavage of the tosyl group under
standard conditions as reported above give rise to corresponding
11a and 11b derivatives.
Structural Assignment. The structure assignment of each
tetracyclic isomer 7e/7f was first studied by 2D-NMR. Unfor-
tunately, we were unable using HMBC experiments to show a
J3 long-distance coupling between the hydrogen on the pyrazole
ring and CdN in one isomer or CdO in the other isomer.
Finally, the structure of each isomer was assigned using
comparison with related compounds. As we have previously
reported in the case of quinolinic17 or isoquinolinic7c derivatives,
a The ratio is indicated in the order of elution in HPLC.
the proton close to the iminoquinone function (isomer 7e) is
deshielded compared to the same proton in the other isomer
(7f). On that basis, the major isomer (which is also the more
polar) in 7e/7f mixture, with the proton of the pyrrazole being
downfield shifted, would correspond to structure 7e. The same
analysis can be applied to 8e/8f, compound 8f being the major
isomer of the two products. In the case of compounds 9e/9f,
10e/10f, and 11e/11f, which do not have any proton on the
pyrrazole, the structural assignment of each isomer has not been
possible so far. Experiments are currently in progress to obtain
a monocrystal of at least one component of each couple in order
to perform X-ray analysis.
small amount (10%) of the corresponding deprotected analogues
10′c/10′d. Regioisomeric mixtures were not separated at this
stage of our synthetic pathway.
Cycloadducts were further converted to the final cyclized
derivatives as previously described for products of the pyrrolo-
quinoline family.15 The cleavage of the Boc protection of the
side chain amino group with trifluoroacetic acid in diclo-
romethane gave the corresponding trifluoroacetate in good yields
(above 80%). For compounds 10c/10d, the trityl-protective
group was cleaved upon cleavage of the Boc group (10e/10f).
The next cyclization step was achieved by reflux in ethanol in
the presence of both 4 Α molecular sieve and sodium bicarbon-
ate. The yields of these reactions as well as the ratio of the
different regioisomers which were separated are reported in
Table 1. Finally, cleavage of the tosyl-protective group of
compounds 7e, 7f, 8e, 8f, 9e, and 9f was performed with sodium
hydroxide in dioxane to give 7a, 7b, 8a, 8b, 9a, and 9b,
Pharmacology
Inhibition of Topoisomerases. Final cyclized derivatives 7a
and 7b, 8a and 8b, 9a and 9b, and 10a and 10b and their
tosylated precursors 7e and 7f, 8e and 8f, 9e and 9f, and 10e
and 10f, respectively, and compound 11f were evaluated in vitro
for their ability to inhibit enzymatic cleavage of DNA by

2982 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 10
Table 2. Effect on Topoisomerases I and II Enzymatic Activitya
Legentil et al.
a Values are expressed as the micromolar concentration of compound which induced complete, partial (∼), or no observable (>) inhibition of DNA
cleavage activity.
topoisomerase I and II isoenzymes. The results reported in Table
2 are the lowest concentration to achieve complete inhibition
of topoisomerase I or II activity compared to those of etoposide
and camptothecin (specific topoisomerase II and I inhibitor,
respectively).
Activities for the natural compound wakayin reported in the
literature are, respectively, 10 and 250 µM on topoisomerase I
and II; unfortunately, this compound was not available for us
to include in our test.
Compounds 10e and 10f are the most active compounds,
demonstrating a 3-10-fold better activity, respectively, 30 and
10 µM on topoisomerase I compared to camptothecin and a
topoisomerase II activity similar to the one of etoposide for 10f
(100 µM), while 10e is less active (>100 µM).
Compounds 7a, 10a, 7b, and 8b have topoisomerase II
activity (around 100 µM) equivalent to the one observed with
etoposide but are less active (>100 mM) than camptothecin on
topoisomerase I activity.
and 100 µM, respectively, being more or equally potent than
the selective topoisomerase I inhibitor camptothecin. Com-
pounds 9e, 8e, and 7f have shown, but to a lesser extent,
topoisomerase I inhibitory effect, with an active concentration
of 300 µM. Among all the compounds cited above, 10f is the
sole one to also inhibit human topoisomerase II activity at a
concentration comparable to that of the control drug etoposide
(100 µM). For all the other tested compounds no relevant
inhibition of topoisomerase I or II activity was observed at the
maximum tested concentrations.
In Vitro Determination of the Drug-Induced Inhibition
of Cancer Cell Line Growth. Compounds were further
evaluated in vitro for their antiproliferative activity on three
human cell lines (HCT15 and HT29, colon; PC3, prostate) and
two murine cell lines (CT-26, colon; Renca, kidney). Camp-
tothecin and etoposide selective topoisomerase I and II inhibi-
tors, respectively, were included in the same experiment as
comparators.
Compounds 10f, 10e, and 9f exhibited complete inhibition
of DNA cleavage activity of human topoisomerase I at 10, 30,
Compounds were tested for 72 h at nine concentrations
ranging from 1 nM to 10 µM with a 0.1% DMSO final

Wakayin and Tsitsikammamines Pyrazolic Analogues
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 10 2983
Table 3. Characterization of the in Vitro Cytotoxic-Related Antitumor Effects
concentration in the growth medium. The results, expressed as
IC50 (concentration reducing the cell proliferation rate by 50%),
are reported in Table 3.
pyrazole ring are the most active. This structural change seems
to have increased the inhibitory effect on topoisomerase I, as
the natural product tsitsikammamine is not an inhibitor of the
enzyme. If compared to wakayin, the new analogues trend to
show lower potency with regard to the topoisomerase inhibitory
activities.
Cell growth inhibitory activities were reported in the literature
for wakayin and tsitsikammamines in the HCT-116 cell line;
IC50s of 1.53 µM for wakayin and of 1.4 and 2.38 µM for
tsitsikammamines A and B were reported, respectively.
As presented in Table 3, compounds 8e, 8f, and 11f inhibited
the growth of the five cell lines with IC50 values in the
micromolar range like what is observed for etoposide in the
same conditions. Camptothecin inhibited the growth of these
human and mouse tumoral cell lines at lower nanomolar range
concentrations. Compound 8b showed activity on three out the
five tested cell lines. Quite unexpectedly, the most potent
topoisomerase I inhibitors 10e and 10f are not inhibiting cell
proliferation in any of the cell lines tested.
For the Cancer Cell Proliferation Inhibition (Table 3).
On the basis of data from Table 3, camptothecin is 4- to 100-
fold more efficient than etoposide in inhibiting, after a 72 h
incubation period, the growth of the five tested tumoral cell
lines. This may be due to a predominant topoisomerase I pattern
of expression in those cell lines.
It has been reported that wakayin differs from camptothecin
in that its cleavage complexes were much less stable than those
of camptothecin and that wakayin poorly stabilizes cleavage
complexes.2 This could be a reason compounds such as 10e
and 10f despite their interesting topoisomerases inhibitory
activity profile are not inhibiting cell growth of the tested
tumoral cell lines.
Discussion
For the Topoisomerase Inhibition (Table 2). We have
described a new series of aza analogues of the marine alkaloids
wakayin and tsitsikammamines A and B and evaluated their
inhibitory activities with regard to topoisomerases I and II.
Compounds 10e and 10f structurally corresponding to tsitsika-
mmamine A in which one pyrrole ring has been replaced by a
The noncorrelation of enzyme versus cell line inhibitory
activities for 10e and 10f could also be due to a poor cell
penetration of those two compounds. To investigate that
hypothesis, in silico physicochemical properties calculations
were performed for all tested compounds, etoposide and

2984 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 10
Table 4. Calculated Values for log P and PSA
Legentil et al.
solvents serving as internal standards. IR spectra were obtained on
a Perkin-Elmer (1600 series FTIR) spectrometer. Mass spectra (MS)
were recorded on an automass Unicam spectrometer. Reagents were
purchased from commercial sources and used as received. Chro-
matography was performed on silica gel (15-40 µm) by means of
the solvent systems indicated below.
A.2. 4,7-Dimethoxy-3-(2-nitro-1-nitromethyl-ethyl)-1H-indole
(15). To a suspension of compound 138 (4 g, 19.5 mmol) in
nitromethane (100 mL) was added ammonium acetate (900 mg,
11.6 mmol). The mixture was refluxed for 1 h. After concentration
on vacuum, CH2Cl2 (50 mL) was added to the residue. The mixture
was separated, washed with H2O, and dried over MgSO4. After
concentration the crude product was purified by flash chromatog-
raphy (CH2Cl2) to give the expected product as an orange solid
(3.62 g, 60%), mp 145 °C.
A.3. 4,7-Dimethoxy-3-(2-nitro-vinyl)-1-(toluene-4-sulfonyl)-
1H-indole (16). To a solution of compound 14 (1.12 g, 20 mmol)
in anhydrous THF (40 mL) was added at 0 °C, under nitrogen
atmosphere, powder KOH (967 mg, 24.2 mmol). After 5 min of
stirring, p-toluenesulfonyl chloride (850 mg, 4.45 mmol) was added,
and the mixture was stirred for additional 1 h. AcOEt (80 mL)
was added, and the organic layer was washed with H2O (3 × 50
mL). After drying over MgSO4 and concentration, the crude product
was purified by flash chromatography (CH2Cl2) to give the product
as an orange solid (880 mg, 54%), mp 195 °C.
camptothecin being included in the data set. Calculated values
for log P and PSA (polar surface area) are presented in Table
4. PSA values for compounds 10e and 10f are above the
acceptable threshold value of 160. Camptothecin (nanomolar
inhibition activity on cell proliferation) is in the acceptable range
for the PSA, while etoposide (micromolar inhibition activity
on cell proliferation) also has a very high PSA value. Also,
10e and 10f with calculated log P values of 3.6 are more
lipophilic which may correlate with a lower cell penetration
than for camptothecin.
It is also interesting to mention that new compounds designed
to target topoisomerase I were reported in the literature to be a
substrate for P-glycoprotein (P-gp), while camptothecin and its
neutral derivatives are not.18 However, we have not tested our
newly synthesized compounds with regard to P-gp.
Finally, compounds 8e, 8f, 11f showed antiproliferative effect,
blocking cell proliferation in the micromolar range, while no
relevant inhibition of topoisomerase was detectable suggesting
another mechanism for their cytotoxic activity against cancer
cells. Further studies, based on the above findings, are in
progress in our laboratory and will be reported in due course.
Experimental Section
A. Chemistry. A.1. Chemical Synthesis. 1H and 13C NMR
spectra were performed on a JEOL 400 MHz spectrometer with
the chemical shifts of the remaining protons of the deuterated
A.4. 2-[4,7-Dimethoxy-1-(toluene-4-sulfonyl)-1H-indol-3-yl]-
ethylamine (17). To a solution of LiAlH4 (283 mg, 7.46 mmol) in
diethyl ether (10 mL) was added dropwise, at 0 °C, a solution of

Wakayin and Tsitsikammamines Pyrazolic Analogues
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 10 2985
compound 16 (500 mg, 1.24 mmol) in CH2Cl2 (30 mL). The mixture
was stirred for 15 min, and H2O (0.5 mL) was added. After filtration
over Celite, the filtrate was dried over MgSO4 and concentrated to
give the product as an unstable brown solid (402 mg, 86%).
concentrated in vacuum to give the expected compound as an orange
oil (3.60 g, 95%).
A.11. 4-Trityloxy-benzaldehyde (24). A solution of hydroxy-
benzaldehyde (500 mg, 4.09 mmol), trytil chloride (1.37 g, 4.91
mmol), triethylamine (1.71 mL, 12.28 mmol), and DMAP (50 mg,
0.41 mmol) in THF (30 mL) was refluxed overnight. CH2Cl2 (50
mL) was added, and this organic layer was washed with water (3
× 50 mL). The organic layer was dried over MgSO4 and
concentrated. The crude product was purified by flash chromatog-
raphy (pentane/AcOEt 80:20) to give the expected product as a
white solid (1.57 mg, 92%), mp 119 °C.
A.12. 4-Trityloxy-benzaldehyde-(4-toluenesulfonyl)-hydra-
zone (25). To a solution of compound 24 (13.7 g, 41.4 mmol) in
THF (500 mL) was added tosylhydrazine (7.7 g, 41.4 mmol). The
mixture was stirred at room temperature for 30 min. After
concentration, the crude product was purified by flash chromatog-
raphy (petroleum ether/AcOEt 70:30) to give quantitatively the
expected product as a white solid, mp 76 °C.
A.13. 4-Methyl-N′-[(1E)-(1-methyl-1H-indol-3-yl)methylene]-
benzenesulfonohydrazide (26). A solution of 1-methylindole-3-
carboxaldehyde (500 mg, 3.24 mmol) and tosylhydrazine (585 mg,
3.24 mmol) in EtOH (50 mL) was stirred at room temperature for
30 min. Filtration of the mixture gave the expected product as a
pale-yellow solid (850 mg, 83%), mp 143 °C.
A.14. tert-Butyl(2-{7-[(4-methylphenyl)sulfonyl]-4,8-dioxo-
1,4,7,8-tetrahydro-pyrrolo[3,2-f]indazol-5-yl}ethyl)carbamate (7c)
and tert-Butyl(2-{5-[(4-methylphenyl)sulfonyl]-4,8-dioxo-1,4,5,8-
tetrahydro-pyrrolo[2,3-f]indazol-7-yl}ethyl)carbamate (7d). To
a solution of compound 4 (500 mg, 1.13 mmol) in THF (50 mL)
was added, at 0 °C in the dark, the solution of diazomethane (1
equiv), and the mixture was stirred for 30 min. The solvent was
evaporated over vacuum, and the residue was purified by flash
chromatography (CH2Cl2/MeOH 98:2) to give the mixture of the
two isomers as an orange solid (480 mg, 88%): isomer 1, 60%;
isomer 2, 40%.
A.5. tert-Butyl(2-{4,7-dimethoxy-1-[(4-methylphenyl)sulfonyl]-
1H-indol-3-yl}ethyl)carbamate (18) and Di-tert-butyl(2-{4,7-
dimethoxy-1-[(4-methylphenyl)sulfonyl]-1H-indol-3-yl}ethyl)-
imidodicarbonate (19). To a solution of compound 17 (260 mg,
0.69 mmol) in anhydrous THF (10 mL) was added sodium hydride
(60% dispersion in oil, 55 mg, 1.39 mmol) under nitrogen
atmosphere. After 5 min of stirring, di-tert-butyl-dicarbonate (227
mg, 1.04 mmol) was added, and the stirring was maintained for 1
h. Water (1 mL) and CH2Cl2 (10 mL) were added, and the organic
layer was separated. It was then washed with water (3 × 10 mL),
dried over MgSO4, and concentrated on vacuum. The crude product
was purified by flash chromatography (CH2Cl2/MeOH 99:1) to give
18 as an oil (200 mg, 60%) and 19 as an oil (20 mg, 5%).
A.5.1. Compound 18 Was Also Obtained from Compound
23. To a solution of compound 21 (1.15 g, 3.59 mmol) in anhydrous
THF (30 mL) was added under nitrogen atmosphere NaH (60%
dispersion in oil, 720 mg, 17.97 mmol). The mixture was stirred
for 1 h. p-TsCl (753 mg, 3.95 mmol) was added, and stirring was
maintained for 30 min. H2O (2 mL) and CH2Cl2 (50 mL) were
added. After drying over MgSO4 and concentrating, the crude
product was purified by flash chromatography (CH2Cl2/MeOH 99:
1) to give the product (1.53 g, 90%).
A.6. 1-(4,7-Dimethoxy-1H-indol-3-yl)-N,N-dimethylmeth-
anamine (20). A 50% aqueous solution of dimethylamine (3.81
mL, 29.94 mmol), acetic acid (4 mL), and formaldehyde (37% in
H2O, 2.19 mL, 28.81 mmol) was added to compound 12 (5 g, 28.25
mmol). The reaction media was stirred at room temperature for 3
h. The solution was poured into a mixture of 10% NaOH (50 mL)/
ice (50 g), and it was extracted with CH2Cl2 (3 × 50 mL). The
organic layers were dried over MgSO4 and concentrated to give
the expected product as an oil (6.61 g, 100%).
A.15. tert-Butyl(2-{1-methyl-7-[(4-methylphenyl)sulfonyl]-4,8-
dioxo-1,4,7,8-tetrahydro-pyrrolo[3,2-f]indazol-5-yl}ethyl)-
carbamate (8c) and tert-Butyl(2-{1-methyl-5-[(4-methylphenyl)-
sulfonyl]-4,8-dioxo-1,4,5,8-tetrahydro-pyrrolo[2,3-f]indazol-7-
yl}ethyl)carbamate (8d). To a solution of compounds 7c/7d (1 g,
2.07 mmol) in MeOH (20 mL) was added diazomethane (2 equiv).
The mixture was stirred for 2 h and then was concentrated on
vacuum to give the expected products as a yellow solid (998 mg,
97%): isomer 1, 60%; isomer 2, 40%.
A.16. tert-Butyl(2-{7-[(4-methylphenyl)sulfonyl]-4,8-dioxo-3-
phenyl-1,4,7,8-tetrahydro-pyrrolo[3,2-f]indazol-5-yl}ethyl)-
carbamate (9c) and tert-Butyl(2-{1-methyl-5-[(4-methylphenyl)-
sulfonyl]-4,8-dioxo-3-phenyl-1,4,5,8-tetrahydro-pyrrolo[2,3-
f]indazol-7-yl}ethyl)carbamate (9d). To a solution of benzaldehyde
tosylhydrazone (670 mg, 2.43 mmol) in THF (40 mL) was added
a solution of KOH (340 mg, 6.08 mmol) in MeOH (10 mL). The
mixture was warmed at 90 °C for 1 h. Et2O (60 mL) was added,
and the solution was washed with brine (3 × 50 mL). This
phenyldiazomethane 6b solution was added to a solution of quinone
4 (900 mg, 203 mmol) in THF (100 mL). After 30 min of stirring,
the solution was concentrated in vacuum. The crude product was
purified by flash chromatography (CH2Cl2/MeOH 99:1) to give the
two expected isomers as a yellow solid (555 mg, 49%): isomer 1,
90%; isomer 2, 10%.
A.7. 4,7-Dimethoxy-1H-indol-3-yl)acetonitrile (21). To a sus-
pension of compound 20 (6.61 g, 28.25 mmol) in a mixture CH2-
Cl2/toluene (80/150 mL) was added methyl iodide (7.02 mL, 56.49
mmol). The mixture was stirred at room temperature overnight.
The solution was then concentrated, and THF (130 mL) was added
to the residue. TMSCN (4.91 mL, 42.37 mmol) was added to the
resulted suspension, and TBAF (1 N in THF, 85 mL, 85 mmol)
was also added dropwise. After 1 h of additional stirring, the
solution was concentrated in vacuum. CH2Cl2 (200 mL) was added
to the residue. The mixture was washed with H2O (3 × 100 mL),
dried over MgSO4, and evaporated. The residue was purified by
flash chromatography (CH2Cl2) to give the expected product as a
white solid (5.67 g, 94%), mp 140 °C.
A.8. 2-(4,7-Dimethoxy-1H-indol-3-yl)ethanamine (22). To a
suspension of LiAlH4 (2.25 g, 59.2 mmol) in Et2O (90 mL) was
added dropwise at 0 °C a solution of compound 21 (1.83 g, 8.47
mmol) in CH2Cl2 (30 mL). The mixture was stirred at room
temperature for 30 min. After this time, H2O (2 mL), 10% NaOH
(2 mL), and again H2O (5 mL) were added, and stirring was
maintained for an additional 10 min. The mixture was filtered over
Celite, and the filtrate was concentrated on vacuum to give the
expected product as an oil (1.6 g, 86%). This product, which is
unstable, was engaged in the next step without further purification.
A.9. tert-Butyl(2-{4,7-dimethoxy-1H-indol-3-yl}ethyl)carbamate
(23). To a solution of compound 22 (1.76 g, 8.02 mmol) in CH2-
Cl2 (45 mL) was added di-tert-butyl dicarbonate (1.75 g, 8.02
mmol). The mixture was stirred at room temperature for 1 h, and
it was left in the refrigerator overnight. After filtration, the expected
product was obtained as a white solid (2.30 g, 90%), mp 180 °C.
A.10. tert-Butyl(2-{1-[(4-methylphenyl)sulfonyl]-4,7-dioxo-4,7-
dihydro-1H-indol-3-yl}ethyl)carbamate (4). A mixture of com-
pound 18 (4 g, 8.4 mmol) and CAN (13.88 g, 25.3 mmol) in
CH3CN/H2O (280 mL/140 mL) was stirred at room temperature
for 15 min. The solution was then extracted with CH2Cl2 (3 × 200
mL). The combined organic layers were dried over MgSO4 and
A.17. tert-Butyl(2-{7-[(4-methylphenyl)sulfonyl]-4,8-dioxo-3-
(4-hydroxyphenyl)-1,4,7,8-tetrahydro-pyrrolo[3,2-f]indazol-5-
yl}ethyl)carbamate (10c), tert-Butyl(2-{1-methyl-5-[(4-meth-
ylphenyl)sulfonyl]-4,8-dioxo-3-(4-hydroxyphenyl)-1,4,5,8-
tetrahydro-pyrrolo[2,3-f]indazol-7-yl}ethyl)carbamate (10d), tert-
Butyl(2-{7-[(4-methylphenyl)sulfonyl]-4,8-dioxo-3-(4-
trityloxyphenyl)-1,4,7,8-tetrahydro-pyrrolo[3,2-f]indazol-5-
yl}ethyl)carbamate (10′c), and tert-Butyl(2-{1-methyl-5-[(4-
methylphenyl)sulfonyl]-4,8-dioxo-3-(4-trityloxyphenyl)-1,4,5,8-
tetrahydro-pyrrolo[2,3-f]indazol-7-yl}ethyl)carbamate (10′d). To
a solution of compound 25 (1.80 g, 3.38 mmol) in THF (60 mL)
was added a solution of KOH (380 mg, 6.78 mmol) in MeOH (40

2986 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 10
Legentil et al.
mL). The mixture was warmed at 90 °C for 1 h. Et2O (100 mL)
was added, and the solution was washed with brine (3 × 100 mL).
This solution of trityloxyphenyldiazomethane 6c was added to a
solution of compound 4 (1 g, 2.25 mmol) in THF (100 mL). After
30 min of stirring, the solution was concentrated on vacuum. The
crude product was purified by flash chromatography (petroleum
ether/AcOEt 70:30 and 50:50) to give 10c and 10d as an orange
solid (920 mg, 50%).
7f involving compounds 10c/10d (580 mg, 0.71 mmol), CH2Cl2
(30 mL), TFA (3 mL), 1 h of stirring; 4 A molecular sieve (600
mg), NaHCO3 (100 mg), and EtOH (50 mL), 12 h of reflux.
Purification by flash chromatography (CH2Cl2/MeOH 98:2) gave
the two separated isomers: isomer 1, pale-yellow solid (130 mg,
40%), mp 186 °C, Anal. (C24H18N4O4S) C, H, N; isomer 2, pale-
yellow solid (40 mg, 12%), mp 192 °C, Anal. (C24H18N4O4S) C,
H, N.
Isomer 1 (60%), Isomer 2 (40%). And (10′c and 10′d): separated
Isomer 1 (30 mg, 0.6%), mp 187 °C. Isomer 2: (20 mg, 0.4%),
mp 198 °C.
A.23. 9-(3-Hydroxy-1-methyl-2-oxo-2,3-dihydro-1H-indol-3-
yl)-5-[(4-methylphenyl)sulfonyl]2,3,5,7-tetrahydro-6H-pyrazolo-
[3,4-h]pyrrolo[4,3,2-de]quinolin-6-one (11e) and 9-(3-Hydroxy-
1-methyl-2-oxo-2,3-dihydro-1H-indol-3-yl)-5-[(4-methylphenyl)-
sulfonyl]2,3,5,9-tetrahydro-6H-pyrazolo[4,3-h]pyrrolo[4,3,2-de]-
quinolin-6-one (11f). From isomer 1: the same procedure was used
as for compounds 7e/7f involving the mixture comprising isomer
1 of compound 11 (700 mg which corresponded to 1.08 mmol of
isomer 1), CH2Cl2 (20 mL), TFA (2 mL), 30 min of stirring; 4 A
molecular sieve (100 mg), NaHCO3 (100 mg), and EtOH (25 mL),
1 h 30 min of reflux. Purification by flash chromatography (CH2-
Cl2/MeOH 97:3) gave isomer 1 of compound 11 as a yellow solid
(400 mg, 70%), mp 172 °C, Anal. (C27H21N5O5S) C, H, N. From
isomer 2: the same procedure was used as for compounds 7e/7f
involving isomer 2 of compound 11 (250 mg, 0.39 mmol), CH2Cl2
(10 mL), TFA (1 mL), 30 min of stirring; 4 A molecular sieve
(100 mg), NaHCO3 (50 mg), and EtOH (10 mL), 1 h 30 min of
reflux. Purification by flash chromatography (CH2Cl2/MeOH 97:
3) gave isomer 2 of compound 11 as a yellow solid (100 mg, 49%),
mp 182 °C, Anal. (C27H21N5O5S) C, H, N.
A.24. 2,3,5,7-Tetrahydro-6H-pyrazolo[3,4-h]pyrrolo[4,3,2-de]-
quinolin-6-one (7a). A solution of compound 7e (80 mg, 0.22
mmol) and tetrabutylammonium fluoride (200 µL of a 1 N solution
in THF) in THF (10 mL) was refluxed for 2 h. After concentration
to dryness, H2O (20 mL) was added to the residue. The precipitate
was filtered and washed with H2O and finally with Et2O to give
the expected compound as a dark yellow solid (25 mg, 55%), mp
182 °C, Anal. (C11H9N4O) C, H, N.
A.25. 2,3,5,9-Tetrahydro-6H-pyrazolo[4,3-h]pyrrolo[4,3,2-de]-
quinolin-6-one (7b). The same procedure was used as for com-
pound 7a involving compound 7f (60 mg, 0.16 mmol) and
tetrabutylammonium fluoride (150 µL of a solution 1 N in THF)
in THF (10 mL), reflux time of 2 h. After concentration to dryness,
H2O (20 mL) was added to the residue. After workup, the expected
product was obtained (20 mg, 60%), mp 198 °C, Anal. (C11H9N4O)
C, H, N.
A.26. 7-Methyl-2,3,5,7-tetrahydro-6H-pyrazolo[3,4-h]pyrrolo-
[4,3,2-de]quinolin-6-one (8a). A solution of compound 8e (500
mg, 1.31 mmol) and 1 N NaOH (10 mL) in dioxane (10 mL) was
stirred at room temperature for 24 h. After concentration to dryness,
the residue was purified by flash chromatography on C8 silica gel
(H2O/MeOH 80:20, 0.1% TFA) to give the expected product as a
red solid (220 mg, 74%), mp 186 °C, Anal. (C12H11N4O) C, H, N.
A.27. 9-Methyl-2,3,5,9-tetrahydro-6H-pyrazolo[4,3-h]pyrrolo-
[4,3,2-de]quinolin-6-one (8b). The same procedure was used as
for compound 8a involving compound 8f (100 mg, 0.26 mmol), 1
N NaOH (5 mL), and dioxane (5 mL). Flash chromatography on
C8 silica gel (H2O/MeOH 80:20, 0.1% TFA) gave the expected
compound as a red solid (30 mg, 50%), mp 196 °C, Anal.
(C12H11N4O) C, H, N.
A.28. 9-Phenyl-2,3,5,7-tetrahydro-6H-pyrazolo[3,4-h]pyrrolo-
[4,3,2-de]quinolin-6-one (9a) and 7-Phenyl-2,3,5,9-tetrahydro-
6H-pyrazolo[4,3-h]pyrrolo[4,3,2-de]quinolin-6-one (9b). From
isomer 1: the same procedure was used as for compound 8a
involving compound 9e (80 mg, 0.18 mmol), 1 N NaOH (5 mL),
and dioxane (5 mL). Flash chromatography on C8 silica gel (H2O/
MeOH 80:20, 0.1% TFA) gave the expected compound as yellow
solid (39 mg, 75%), mp 171 °C, Anal. (C17H13N4O) C, H, N. From
isomer 2: the same procedure was used as for compound 8a
involving compound 9f (50 mg, 0.11 mmol), 1 N NaOH (5 mL),
and dioxane (5 mL). Flash chromatography on C8 silica gel (H2O/
MeOH 80:20, 0.1% TFA) gave the expected compound as a yellow
solid (27 mg, 83%), mp 165 °C, Anal. (C17H13N4O) C, H, N.
A.18. tert-Butyl(2-{3-(3-hydroxy-1-methyl-2-oxo-2,3-dihydro-
1H-indol-3-yl)-7-[(4-methylphenyl)sulfonyl]-4,8-dioxo-1,4,7,8-tet-
rahydro-pyrrolo[3,2-f]indazol-5-yl}ethyl)carbamate (11c) and
tert-Butyl(2-{3-(3-hydroxy-1-methyl-2-oxo-2,3-dihydro-1H-in-
dol-3-yl)-5-[(4-methylphenyl)sulfonyl]-4,8-dioxo-3-(4-hydroxy-
phenyl)-1,4,5,8-tetrahydropyrrolo[2,3-f]indazol-7-yl}ethyl)carba-
mate (11d). To a solution of N-methylisatine (599 mg, 3.72 mmol)
in THF (30 mL) was added, at 0 °C in the dark, diazomethane (1.5
equiv) in Et2O (60 mL). The mixture was stirred for 1 h. After
concentration up to 1/4, the solution was added to a solution of
quinone 4 (1.5 g, 3.38 mmol) in THF (30 mL). The mixture was
stirred for an additional 1 h. After concentration in vacuum, the
crude product was purified by flash chromatography (CH2Cl2/
MeOH 98:2) to give a mixture of the first isomer with an impurity
as an orange solid (730 mg) comprising 70% of isomer 1 (23%
yield) and isomer 2 as an orange solid (360 mg, 17%), mp 185 °C.
A.19. 5-[(4-Methylphenyl)sulfonyl]-2,3,5,7-tetrahydro-6H-
pyrazolo[3,4-h]pyrrolo[4,3,2-de]quinolin-6-one (7e) and 5-[(4-
Methylphenyl)sulfonyl]-2,3,5,9-tetrahydro-6H-pyrazolo[4,3-h]-
pyrrolo[4,3,2-de]quinolin-6-one (7f). To a solution of compounds
7c/7d (700 mg, 1.40 mmol) in CH2Cl2 (30 mL) was added dropwise
TFA (3 mL). After 30 min of stirring the mixture was concentrated
over vacuum to yield the corresponding trifluoroacetate salt. A
suspension of this compound and NaHCO3 (300 mg) and 4 A
molecular sieve (600 mg) in EtOH (50 mL) was refluxed in the
dark for 3 h. The molecular sieve was filtered off, and the filtrate
was concentrated over vacuum. The crude product was purified by
flash chromatography (CH2Cl2/MeOH 97:3) to give the two
separated isomers: 7f (80 mg, 12%), mp 264 °C, Anal. (C18H14-
N4O3S) C, H, N; 7e, white solid (180 mg, 28%), mp 248 °C, Anal.
(C18H14N4O3S) C, H, N.
A.20. 7-Methyl-5-[(4-methylphenyl)sulfonyl]-2,3,5,7-tetrahy-
dro-6H-pyrazolo[3,4-h]pyrrolo[4,3,2-de]quinolin-6-one (8e) and
9-Methyl-5-[(4-methylphenyl)sulfonyl]-2,3,5,9-tetrahydro-6H-
pyrazolo[4,3-h]pyrrolo[4,3,2-de]quinolin-6-one (8f). The same
procedure was used as for compounds 7e/7f involving compounds
8c/8d (800 mg, 1.61 mmol), CH2Cl2 (50 mL), TFA (5 mL), 30
min of stirring; NaHCO3 (300 mg), 4 A molecular sieve (600 mg),
and EtOH (100 mL), 3 h of reflux. Purification by flash chroma-
tography (CH2Cl2/MeOH 99:1) gave the two separated isomers:
8f, yellow solid (130 mg, 21%), mp 117 °C, Anal. (C19H16N4O3S)
C, H, N; 8e, yellow solid (170 mg, 28%), mp 120 °C, Anal.
(C19H16N4O3S) C, H, N.
A.21. 5-[(4-Methylphenyl)sulfonyl]-9-phenyl-2,3,5,7-tetrahy-
dro-6H-pyrazolo[3,4-h]pyrrolo[4,3,2-de]quinolin-6-one (9e) and
5-[(4-Methylphenyl)sulfonyl]-7-phenyl-2,3,5,9-tetrahydro-6H-
pyrazolo[4,3-h]pyrrolo[4,3,2-de]quinolin-6-one (9f). The same
procedure was used as for compounds 7e/7f involving compounds
9c/9d (630 mg, 1.13 mmol), CH2Cl2 (40 mL), TFA (4 mL), 1 h of
stirring; NaHCO3 (200 mg), 4 A molecular sieve (400 mg), and
EtOH (50 mL), 4 h of reflux. Purification by flash chromatography
(CH2Cl2/MeOH 98:2) gave the two separated isomers: isomer 1,
pale-yellow solid (50 mg, 10%), mp 236 °C, Anal. (C24H18N4O3S)
C, H, N; isomer 2, pale-yellow solid (200 mg, 40%), mp 254 °C,
Anal. (C24H18N4O3S) C, H, N.
A.22. 5-[(4-Methylphenyl)sulfonyl]-9-(4-hydroxyphenyl)-2,3,5,7-
tetrahydro-6H-pyrazolo[3,4-h]pyrrolo[4,3,2-de]quinolin-6-one
(10e) and 5-[(4-Methylphenyl)sulfonyl]-7-(4-hydroxyphenyl)-
2,3,5,9-tetrahydro-6H-pyrazolo[4,3-h]pyrrolo[4,3,2-de]quinolin-
6-one (10f). The same procedure was used as for compounds 7e/

Wakayin and Tsitsikammamines Pyrazolic Analogues
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 10 2987
A.29. 9-(4-Hydroxyphenyl)-2,3,5,7-tetrahydro-6H-pyrazolo-
[3,4-h]pyrrolo[4,3,2-de]quinolin-6-one (10a) and 7-(4-Hydroxy-
phenyl)-2,3,5,9-tetrahydro-6H-pyrazolo[4,3-h]pyrrolo[4,3,2-de]-
quinolin-6-one (10b). From isomer 1: the same procedure was used
as for compound 7a involving isomer 1 of compound 10e/10f (90
mg, 0.2 mmol) and tetrabutylammonium fluoride (420 µL of a
solution 1 N in THF) in THF (5 mL), reflux time of 4 h. Purification
by flash chromatography on C8 silica gel (H2O/MeOH 80:20, 0.1%
TFA) gave the product as a brown solid (30 mg, 50%), mp > 260
°C, Anal. (C17H13N4O2) C, H, N. From isomer 2: the same procedure
was used as for compound 7a involving isomer 2 of compound
10e/10f (50 mg, 0.2 mmol) and tetrabutylammonium fluoride (5
mL of a solution 1 N in THF) in THF (5 mL), reflux time of 4 h.
Purification by flash chromatography on C8 silica gel (H2O/MeOH
80:20, 0.1% TFA) gave the product as a yellow solid (15 mg, 45%),
mp > 260 °C, Anal. (C17H13N4O2) C, H, N.
A.30. 9-(3-Hydroxy-1-methyl-2-oxo-2,3-dihydro-1H-indol-3-
yl)-2,3,5,7-tetrahydro-6H-pyrazolo[3,4-h]pyrrolo[4,3,2-de]quino-
lin-6-one (11a) and 9-(3-Hydroxy-1-methyl-2-oxo-2,3-dihydro-
1H-indol-3-yl)-2,3,5,9-tetrahydro-6H-pyrazolo[4,3-h]pyrrolo[4,3,2-
de]quinolin-6-one (11b). From isomer 1: the same procedure was
used as for compound 8a involving compound 11e (100 mg, 0.19
mmol), 1 N NaOH (5 mL), and dioxane (5 mL). Flash chroma-
tography on C8 silica gel (H2O/MeOH 70:30, 0.1% TFA) gave the
expected compound as a red solid (51 mg, 72%), mp 193 °C, Anal.
(C20H16N5O3) C, H, N. From isomer 2: the same procedure was
used as for compound 8a involving compound 11f (90 mg, 0.17
mmol), 1 N NaOH (5 mL), and dioxane (5 mL). Flash chroma-
tography on C8 silica gel (H2O/MeOH 70:30, 0.1% TFA) gave the
expected compound as a red solid (51 mg, 72%), mp 199 °C, Anal.
(C20H16N5O3) C, H, N.
B.2.1. Topoisomerase II. An amount of 4 units of human
topoisomerase II was incubated with 0.25 µg of pRYG DNA
supplemented with ATP (0.5 mM final), at 37 °C, for 30 min in
the absence or presence of the tested compound. The reactions were
stopped by addition of SDS (1% final). After digestion with
proteinase K (50 µg/mL final) at 37 °C for 15 min and addition of
loading buffer, samples were extracted twice with CIA and loaded
onto a 1% agarose gel. The gel was run for 3.5 h at 75 V; gels
were run in the absence of ethidium bromide but stained for 45
min in a solution of ethidium bromide (0.5 µg/mL) and, finally,
photographed and digitalized in a jpg image format.
B.2.2. Topoisomerase I. An amount of 2 units of topoisomerase
I was incubated with 0.25 µg of DNA I at 37 °C for 30 min in the
absence or presence of the compound. The reactions were stopped
with SDS (1% final). After digestion with proteinase K (50 µg/mL
final) at 37 °C for 15 min and addition of loading buffer, samples
were extracted twice with CIA and loaded onto a 1% agarose gel.
The gel was run for 3.5 h at 75 V like for topoisomerase II, in the
absence of ethidium bromide, and stained, photographed, and
digitalized in a jpg image format.
Addition of DMSO from 0.1% to 3% had no incidence on the
activities of both enzymes (data not shown).
B.3. Active concentration was considered as the lowest
concentration of test or reference compound for which complete
inhibition of the enzymatic activity was observed, i.e., the absence
of relaxed or linear DNA form on the gel.
Supporting Information Available: MS, NMR, IR, and
elemental analysis data. This material is available free of charge
via the Internet at http://pubs.acs.org.
B. Biological Studies. All compounds were weighed and
prepared as a 10 mM stock solution in pure, analytical grade,
DMSO. Small aliquots were stored at -80 °C and used on demand.
B.1. Evaluation of the Effects on Cell Proliferation. The cell
lines were obtained from the American Type Culture Collection
(ATCC, Manassas, VA); HCT15 and HT29 colon carcinoma, PC3
prostate cancer, or mouse CT26 colon carcinoma or Renca renal
cancer. All cell lines were checked for the absence of mycoplasma
by an immunological method at the time of the experiments
(mycoplasma detection kit, Boehringer Roche, Germany).
The cells were grown in RPMI (Invitrogen) medium supple-
mented with 10% heat-inactivated fetal calf serum (FCS), in absence
of antibiotics to avoid any interference on growth, at 37 °C in a
95% air/5% CO2 humidified atmosphere.
References
(1) (a) Ding, Q.; Chichak, K.; Lown, J. W. Pyrroloquinoline and
pyridoacridine alkaloids from marine source. Curr. Med. Chem. 1999,
6, 1-27. (b) Urban, S.; Hickford, S. J. H.; Blunt, J. W.; Munro, M.
H. G. Bioactive marine alkaloids. Curr. Org. Chem. 2000, 4, 765-
807. (c) Antunes, E. M.; Copp, B. R.; Davies-Coleman, M. T.;
Samaai, T. Pyrroloiminoquinone and related metabolites from marine
sponges. Nat. Prod. Rep. 2005, 22, 62-72.
(2) Kokoshka, J. M.; Capson, T. L.; Holden, J. A.; Ireland, C. M.;
Barrows, L. R. Differences in the topoisomerase I cleavage complexes
formed by camptothecin and wakayin, a DNA-intercalating marine
natural product. Anti-Cancer Drugs 1996, 7, 758-765.
(3) Hooper, G. J.; Davies-Coleman, M. T.; Kelly-Borges, M.; Coetzee,
P. S. New alkaloids from South African Latrunculid sponge.
Tetrahedron Lett. 1996, 37, 7135-7138.
Exponentially growing cells (seeding density ) 2000 cells/well
for HCT15, PC3, CT26, RENCA; 1000 cells/well for HT29) were
incubated, 24 h after seeding, for 72 h in the presence of the growth
medium without phenol red containing 0.1% DMSO (control
condition) or with nine concentrations of test compounds ranging
from 1 nM to 10 µM with a fixed 0.1% final DMSO concentration.
Cell proliferation was then assayed by the XTT reduction assay.
Briefly, the cells were stained for 4 h with an XTT/PMS solution
(XTT Sigma ×4626; PMS Sigma P9625). The absorbance was read
using a microtiter plate reader Labsystem Multiskan (Ascent version
2.4.2) at 492 nm for four wells per concentration. The cell growth
was expressed as percent (%) of the control (0.1% DMSO in the
growth medium) and plotted as a function of compound concentra-
tion. Whenever possible, IC50 values were determined using a
logistic four-parameter regression model (Sigmaplot, Jandel).
B.2. Inhibition of Human Topoisomerase Isoenzymes. This
assay (adapted from refs 19 and 20) determines the ability of the
test compounds to interfere with the cleavage catalyzed on specific
sequences by the human topoisomerase isoenzymes I or II. The
experiments were performed using the topoisomerase I kit (Topo-
gen, 1018-1) + the human topoisomerase I enzyme (Topogen,
20005H-1) and the topoisomerase II kit (Topogen, 1009-1) + the
human topoisomerase II enzyme (Topogen, 2000H-1), obtained
from Topogen U.S.A., through Biovalley in France.
(4) Zhang, L.; Cava, M. P.; Rogers, R. D.; Rogers, L. M. Synthesis of
a wakayin model compound: Oxidative formation of a new pyrrole
ring in the indol-3-yl-indoloquinone system. Tetrahedron Lett. 1998,
39, 7677-7678.
(5) Barret, R.; Roue, N. Synthesis of a bis-pyrrolo-quinone structure
analogue to wakayin. Tetrahedron Lett. 1999, 40, 3889-3890.
(6) Be´ne´teau, V.; Pierre´, A.; Pfeiffer, B.; Renard, P.; Besson, T. Synthesis
and antiproliferative evaluation of 7-aminosubstituted pyrroloimino-
quinone derivatives. Bioorg. Med. Chem. Lett. 2000, 10, 2231-2234.
(7) (a) Delfourne, E.; Darro, F.; Bontemps-Subielos, N.; Decaestecker,
C.; Bastide, J.; Frydman, A.; Kiss, R. Synthesis and characterization
of the antitumor activities of analogues of meridine, a marine
pyridoacridine alkaloid. J. Med. Chem. 2001, 44, 3275. (b) Delfourne,
E.; Darro, F.; Portefaix, P.; Galaup, C.; Bayssade, S.; Bouteille, A.;
Le Corre, L.; Bastide, J.; Collignon, F.; Lesur, F.; Frydman, A.; Kiss,
R. Synthesis and in vitro antitumor activity of novel ring D analogues
of the marine pyridoacridine ascididemin: structure activity relation-
ship. J. Med. Chem. 2002, 45, 3765. (c) Brahic, C.; Darro, F.; Belloir,
M.; Bastide, J.; Kiss, R.; Delfourne, E. Synthesis and cytotoxic
evaluation of analogues of the marine pyridoacridine amphimedine.
Bioorg. Med. Chem. 2002, 10, 2845. (d) Delfourne, E.; Kiss, R.; Le
Corre, L.; Dujols, F.; Bastide, J.; Collignon, F.; Lesur, B.; Frydman,
A.; Darro, F. Synthesis and in vitro antitumor activity of phenan-
throlin-7-one derivatives, analogues of the marine pyridoacridine
alkaloids ascididemin and meridine: structure-activity relationship.
J. Med. Chem. 2003, 46, 3536. (e) Delfourne, E.; Kiss, R.; Le Corre,
L.; Dujols, F.; Bastide, J.; Collignon, F.; Lesur, B.; Frydman, A.;
Darro, F. Synthesis and in vitro antitumor activity of ring C and
D-substituted phenanthrolin-7-one derivatives, analogues of the
marine pyridoacridine alkaloids ascididemin and meridine. Bioorg.
After preliminary experiments, procedures recommended by the
manufacturer were modified to be as follows:

2988 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 10
Legentil et al.
Med. Chem. 2004, 12, 3987. (f) Legentil, L.; Lesur, B.; Delfourne,
E. Aza-analogues of the marine pyrroloquinoline alkaloids wakayin
and tsitsikammamines: synthesis and topoisomerase inhibition.
Bioorg Med. Chem. Lett. 2006, 16, 427-429.
dins C and E and dethiadiscorhabdin D. J. Org. Chem. 1999, 64,
16-22.
(16) (a) Katz, A. H.; Demerson, C. A.; Shaw, C.-C.; Asselin, A. A.;
Humber, L. G.; Conway, K. M.; Gavin, G.; Guinosso, C.; Jensen,
N. P.; Mobilio, D.; Noureldin, R.; Schmid, J.; Shah, U.; Van Engen,
D.; Chau, T. T.; Weichman, B. M. Synthesis and analgesic activity
of pemedolac [cis-1-ethyl-1,3,4,9-tetrahydro-4-(phenylmethyl)pyrano-
[3,4-b]indole-1-acetic acid]. J. Med. Chem. 1988, 31, 1244-1250.
(b) Berens, U.; Brown, J. M.; Long, J.; Selke, R. Synthesis and
resolution of 2,2′-bis-diphenylphosphino[3,3′]indolyl, a new atropi-
someric ligand for transition metal catalysis. Tetrahedron: Asymmetry
1995, 7, 285-292.
(8) Be´ne´teau, V.; Besson, T. Synthesis of novel pentacyclic pyrrolothia-
zolobenzoquinolones, analogues of natural marine alkaloids. Tetra-
hedron Lett. 2001, 42, 2673-2676.
(9) Showalter, H. D. H.; Pohlmann, G. An improved synthesis of 4,7-
dime´thoxy-1H-idole. Org. Prep. Proced. Int. 1992, 24, 484-488.
(10) Iwao, M.; Motoi, O.; Fukuda, T.; Ishibashi, M. New synthetic
approach to pyrroloiminoquinone marine alkaloids. Total synthesis
of makaluvamines A, D, I, and K. Tetrahedron 1998, 54, 8999-
9010.
(11) Iwao, M.; Motoi, O. Methodology for the efficient synthesis of 3,4-
differentially substituted indoles. Fluoride ion-induced elimination-
addition reaction of 1-triisopropylsilylgramine methiodides. Tetra-
hedron Lett. 1995, 36, 5929-5932.
(12) (a) Rodighiero, G.; Malesani, G.; Fornasier, U. Synthesis of 4,7-
dimethoxyindole derivatives. Gazz. Chim. Ital. 1961, 91, 214-221.
(b) Malesani, G.; Rodighiero, G. 4,7-Dimethoxyindole-3-acetic acid
and DL-4,7-dimethoxytryptophan. Gazz. Chim. Ital. 1967, 97, 214-
221.
(17) Legentil, L.; Bastide, J.; Delfourne, E. Total synthesis of the marine
pyridoacridine alkaloid sebastianine A. Tetrahedron Lett. 2003, 44,
2473-2475.
(18) Pommier, Y.; Leteurtre, F.; Fesen, M.; Fujimori, A.; Bertrand, R.;
Solary, E.; Kohlagen, G.; Kohn, K. W. Cellular determinants of
sensitivity and resistance to DNA topoisomerase inhibitors. Cancer
InVest. 1994, 12, 530-542.
(19) Champoux, J. J.; Aronoff, R. The effect of camptothecin on the
reaction and specificity of the wheat germ type I topoisomerase. J.
Biol. Chem. 1989, 264, 1010-1015.
(20) Dassonneville, L.; Wattez, N.; Baldeyrou, B.; Mahieu, C.; Lansiaux,
A.; Banaigs, B.; Bonnard, I.; Bailly, C. Inhibition of topoisomerase
II by the marine alkaloid ascididemin and induction of apoptosis in
leukemia cells. Biochem. Pharmacol. 2000, 60, 527-537.
(13) Bhalay, G.; Dunstan, A. R. Facile solid-phase synthesis of an activated
diazo linker. Tetrahedron Lett. 1998, 39, 7803-7806.
(14) Eistert, B.; Ganster, O. Abfangen intermedia¨rer 1:1-additiosproduckt
aus diazomethane und isatinen bzw. indandionen-(1.2). Chem. Ber.
1971, 104, 78-86.
(15) See, for example: Aubart, K. M.; Heathcock, C. H. A biomimetique
approach to the discorhabdin alkaloids: total syntheses of discorhab-
JM051247F