Cytotoxic activities of novel hexahydroindolizino[8,7-b]indole derivatives prepared by 1,3-dipolar cycloaddition reactions of 3,4-dihydro-β-carboline ylides

Article abstract of DOI:10.1016/S0968-0896(01)00119-5

A series of 1-cyano and 2-cyanohexahydroindolizino[8,7-b]indole derivatives was prepared by 1,3-dipolar cycloaddition of acrylonitrile with ylides derived from 3,4-dihydro-β-carboline and its 6-methoxy, 6-benzyloxy, 9-methyl and 9-benzyl analogues. The pr

Full text of DOI:10.1016/S0968-0896(01)00119-5

Bioorganic & Medicinal Chemistry 9 (2001) 2155–2164
Cytotoxic Activities of Novel Hexahydroindolizino[8,7-b]indole
Derivatives Prepared by 1,3-Dipolar Cycloaddition Reactions of
3,4-Dihydro-ꢀ-carboline Ylides
Martial Bertrand,a,y Guillaume Poissonnet,a,{ Marie-Helene Theret-Bettiol,a
Christiane Gaspard,a Georges H. Werner,a Bruno Pfeiffer,b Pierre Renard,b
Stephane Leoncec and Robert H. Dodda,*
aInstitut de Chimie des Substances Naturelles, Centre National de la Recherche Scientifique, 91198 Gif-sur-Yvette Cedex, France
b
´
A.D.I.R., 1, rue Carle Hebert, 92415 Courbevoie cedex, France
c
´
Institut de Recherches Servier, Division de Cancerologie Experimentale, 11, rue des Moulineaux, 92150 Suresnes, France
´
Received 19 February 2001; accepted 11 April 2001
Abstract—A series of 1-cyano and 2-cyanohexahydroindolizino[8,7-b]indole derivatives was prepared by 1,3-dipolar cycloaddition
of acrylonitrile with ylides derived from 3,4-dihydro-b-carboline and its 6-methoxy, 6-benzyloxy, 9-methyl and 9-benzyl analogues.
The products, together with their reduced 1- or 2-aminomethyl derivatives, were evaluated for cytotoxic activity in L1210 cancer
cells. Compounds derived from 6-benzyloxy or 9-benzyl-3,4-dihydro-b-carboline were found to be the most active, with IC50’s in
the 2–50 mM range. Of these, two compounds, the 1- and 2-cyano 8-benzyloxyindolizino[8,7-b]indole derivatives 20a and 20c,
respectively, were found by cytometric flux analysis to stop cancer cell growth at the G2M and 8N (>G2M) stage of the cell cycle.
These two compounds also showed no loss of cytotoxic activity in K562Rcancer cells resistant to doxorubicin. # 2001 Elsevier
Science Ltd. All rights reserved.
The b-carboline nucleus is a recurring motif in a wide
variety of cytotoxic compounds, both natural and syn-
thetic. In addition to simple, substituted harman and
norharman derivatives,1 more complex structures such
as the manzamines (e.g., 1),2 eudistomine K (2),3 aza-
toxin (3),4 fascaplysine (4),5 picrasidine L (5)6 and java-
carboline (6)7 display cytotoxic activities in various
cancer cell lines. Structural analogues of javacarboline
in the form of 1- and/or 2-methyl (or phenyl)indoli-
zino[8,7-b]indoles (e.g., 7) were recently synthesized by
Nikaido and coworkers and shown to possess activity
against P-388 and PC-6 cell lines.8 The compounds of
type 7 were prepared by base-promoted cyclizations of
1-alkyl-2-acyl-b-carbolinium bromides.
lylmethyl-b-carbolinium salts, with electron-deficient
dipolarophiles.9ꢀ11 In this communication, we wish to
report the synthesis and activity against L1210 murine
leukemia cells of several indolizino[8,7-b]indoles pre-
pared by our cycloaddition methodology. Some of these
compounds were shown to induce significant accumula-
tion of the L1210 cells in the G2M and 8N (>G2M)
phases of the cell cycle and, moreover, to be active
against resistant strains of K562 cancer cells.
The starting materials for this study were 3,4-dihydro-b-
carboline (8) and its 6-methoxy and 6-benzyloxy analo-
gues (9 and 10, respectively) prepared from the corre-
sponding Nb-formyltryptamine derivatives by
a
Bischler–Napieralski reaction.12 The N-9 position of
compound 8 was further methylated (11)13 or benzy-
lated (12)14 by the action of sodium hydride in DMF
followed by addition of the appropriate alkylating agent
(methyl tosylate or benzyl bromide). The dihydro-b-
carbolines 8–12 were then efficiently transformed into
their corresponding 2-N-trimethylsilylmethyl-b-carboli-
nium triflate salts 13–17,15 respectively, by treatment
with 1.1 equiv of trimethylsilylmethyl triflate in di-
chloromethane.11 In the presence of cesium fluoride, the
We ourselves have reported the synthesis of substituted
indolizino[8,7-b]indoles by 1,3-dipolar cycloadditions of
b-carboline ylides, generated from N-2-trimethylsi-
*Corresponding author. Tel.: +33-1-69-82-45-94; fax: +33-1-69-07-
72-47; e-mail: robert.dodd@icsn.cnrs.gif.fr
yPresent address: Eli Lilly, Dunderrow, Co. Cork, Ireland.
{Present address: Institut de Recherches Servier, 11, rue des Mouli-
neaux, 92150 Suresnes, France.
0968-0896/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00119-5

2156
M. Bertrand et al. / Bioorg. Med. Chem. 9 (2001) 2155–2164
Scheme 1.
triflate salts were converted into the ylides which reacted
in situ with acrylonitrile to afford the products of
1,3-dipolar cycloaddition 18–22 (Scheme 1).15,16
corresponding 8-hydroxy compounds 27a–d in 72–83%
yield.
The hexahydroindolizino[8,7-b]indole derivatives syn-
thesized were then tested for cytotoxic activity in vitro
in L1210 cancer cells.17,18 As seen from Table 1, none of
the four cyano derivatives having an unsubstituted
indole nucleus (18a–d) or the two cyano derivatives
having an N-methyl substituent (21a,b) inhibited the
growth of L1210 cells at 100 mM concentrations. How-
ever, replacement of the N-methyl group by an N-benzyl
group (compounds 22a–d) produced cytotoxic com-
pounds having IC50’s in the 45–56 mM range. Interest-
ingly, all four isomers showed approximately equivalent
cytotoxicities. While introduction of an 8-methoxy
group (compounds 19a–d) also led to inactive com-
pounds, the 8-benzyloxy group had a favorable effect on
cytotoxicities with compounds 20a and 20c displaying
IC50’s of 15.6 and 16.9 mM, respectively. Both these cis
regioisomeric cyano derivatives were 2–3 times more
active than their trans isomers (20b and 20d). Removal
of the 8-benzyl group to give, by analogy with the ellip-
ticine family of cytotoxic compounds, the 8-hydroxy
derivatives 23a–d led to complete loss of activity for all
four isomers.
In practically all cases (except for 16) both possible C-1
and C-2 regioisomers were obtained. Each regioisomer
was, in turn accompanied by both diastereomeric forms
(a, b and c, d). The four isomers produced in each reac-
tion could be easily separated either by fractional crys-
tallization or by chromatography on silica gel. Regio-
and relative stereochemistry were determined by 1H-
and 13C NMRspectroscopy correlated with the struc-
ture of compound 18c determined by X-ray crystal-
lography. Overall yields were in the 70–85% range for
each cycloaddition reaction. All compounds shown are
racemic mixtures.
Some of the products of cycloaddition were subjected to
further chemical transformations. Thus, treatment of
the 8-benzyloxy derivatives 20a–d with triflic acid in the
presence of thioanisole provided approximately 60% of
the corresponding analogues (23a–d) (Scheme 2). The
nitrile functions of compounds 18a–d, 20a–d and 22a–d
could be selectively reduced to the primary amines (24a–
d, 25a–d and 26a–d, respectively) in 65–76% yields by
the action of aluminum hydride (prepared in situ from
lithium aluminum hydride and aluminum chloride) in
THF at 0 ꢁC (Scheme 3). Use of lithium aluminum
hydride alone led to epimerization of the substrates.
Finally, catalytic hydrogenolysis of the 8-benzyloxy-1-
(and 2-) aminomethyl derivatives 25a–d afforded the
Scheme 2.
Scheme 3.

M. Bertrand et al. / Bioorg. Med. Chem. 9 (2001) 2155–2164
2157
Attention was then turned to the primary amine deri-
vatives corresponding to reduction of the nitrile func-
tions. In contrast to the unsubstituted 1- and 2-cyano-
indolizino[8,7-b]indoles 18a–d, which were completely
inactive, the amino counterparts 24a–d showed sig-
nificant cytotoxic activities, with the most potent isomer
being the cis-1-aminomethyl derivative 24a which dis-
played an IC50 of 17.5 mM. Even more active were the
C-8 benzyloxy 1- and 2-aminomethyl derivatives 25a–d
as well as the N-11 benzyl analogues 26a–d which all
displayed IC50 values in the 2–5 mM range. Again, in
both series of molecules, activity was little affected by
the regio- or stereochemistry of the aminomethyl group.
In contrast, in the 8-hydroxy series 27a–d, only the cis-
1-aminomethyl derivative 27a showed cytotoxic activity
(IC50=7.6 mM), the other three isomers being
completely inactive.
The effects of the most active compounds on the differ-
ent phases of the cell cycle were then determined using
cytometric flux. L1210 cells, in contact with the test
substances, could thus be sorted according to their
levels of nuclear DNA, thereby indicating at which
stage of the cell cycle growth was inhibited.19 All the
aminomethyl derivatives tested (the unsubstituted deri-
vative 24a, the C-8 benzyloxy derivatives 25a–d, the N-
11 benzyl derivatives 26a–d and the C-8 hydroxy deri-
vative 27a) showed non-selective action against L1210
cells (data not shown). In the case of the two most
active members of the 1- and 2-cyano derivatives (20a
and 20c), these were found to induce significant accu-
mulation of the cells in the G2M and 8N (>G2M) pha-
ses of the cell cycle at a concentration of 25 mM.
Approximately 45% of the L1210 cells were in the G2M
phase and between 17 and 33% in the 8N phase (tetra-
ploid cells) compared to 28 and 1%, respectively, for
untreated controls (Table 2). The cytotoxic activity of
these two nitrile compounds thus appears to be due to
an interaction with specific cellular components, unlike
the aminomethyl derivatives which are non-specifically
toxic.
Table 1. Antiproliferative activity of the indolizino[8,7-b]indoles with
respect to L1210 tumor cells
In view of this apparently selective action observed with
nitriles 20a and 20c, these compounds were further
investigated for cytotoxic activity in a multidrug resis-
tant cancer cell line. Thus, in K562Rhuman ery-
throleukemia cells resistant to doxorubicin, both
compounds 20a and 20b displayed cytotoxic activities
(IC50 9.0 and 8.2 mM, respectively) similar or identical
to activities in the doxorubicin-sensitive K562S strain
(IC50 8.0 and 8.2 mM, respectively; Table 3).20,21 By
comparison, doxorubicin undergoes a 2000-fold loss of
potency in the K562Rcells with respect to the K562S
cells (IC50 20 and 0.01 mM, respectively).
Compound
R1
R2
R3
R4
IC50 (mM)
Adriamycin
18a
18b
18c
18d
19a
19b
19c
19d
20a
20b
20c
20d
21a
21b
22a
22b
22c
22d
23a
23b
23c
23d
24a
24b
24c
24d
25a
25b
25c
25d
26a
26b
26c
26d
27a
27b
27c
27d
—
H
H
H
H
OMe
OMe
OMe
OMe
OBn
OBn
OBn
OBn
H
—
H
H
H
H
H
H
H
H
H
H
H
H
Me
Me
Bn
Bn
Bn
Bn
H
H
H
H
H
H
H
H
H
H
H
H
Bn
Bn
Bn
Bn
H
—
c-CNa
t-CN
H
H
c-CN
t-CN
H
H
c-CN
t-CN
H
H
c-CN
t-CN
c-CN
t-CN
H
H
c-CN
t-CN
H
H
c-CN2NH2
t-CN2NH2
H
H
c-CN2NH2
t-CN2NH2
H
—
H
H
c-CN
t-CN
H
H
c-CN
t-CN
H
H
c-CN
t-CN
H
H
H
H
c-CN
t-CN
H
H
c-CN
t-CN
0.021
>100
>100
>100
>100
>100
>100
>100
>100
15.6
49.5
16.9
36.6
In summary, the present results show that substituted
hexahydroindolizino[8,7-b]indoles should be a profitable
>100
>100
55.8
H
H
H
H
Table 2. Effect of compounds 20a and 20c on the L1210 cell cycle
50.0
45.1
47.6
Compound
Effect on cell cyclea,b
H
G2M
8N (>G2M)
c (mM)
OH
OH
OH
OH
H
H
H
H
OBn
OBn
OBn
OBn
H
H
H
H
>100
>100
>100
>100
17.5
Adriamycin
20a
20c
83
47
46
6
17
33
0.1
25
25
H
H
37.3
42.3
32.1
2.5
2.5
2.1
2.5
4.4
4.85
1.8
2.4
7.6
aResults expressed as percentage of the cells found in the G2M and 8N
(>G2M) phases of the cell cycle.
bUntreated control L1210 cells: 40% G1; 31% S; 28% G2M; 1% 8N
(>G2M).
c-CN2NH2
t-CN2NH2
H
H
c-CN2NH2
t-CN2NH2
H
H
c-CN2NH2
t-CN2NH2
H
H
c-CN2NH2
t-CN2NH2
H
Table 3. Comparison of the growth inhibitory potency of com-
pounds 20a and 20c in K562 tumor cells sensitive (S) and resistant (R)
to doxorubicin
H
c-CN2NH2
t-CN2NH2
H
Compound
IC50 (mM)
OH
OH
OH
OH
H
H
H
H
>100
>100
>100
K562S
K562R
c-CN2NH2
t-CN2NH2
H
Doxorubicin
20a
20c
0.01
8.0
8.2
20
9.0
8.2
aa-CN and c-CH2NH2 refer to these groups being cis to H-11b while t-
CN and t-CH2NH2 refer to these groups being trans to H-11b.

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M. Bertrand et al. / Bioorg. Med. Chem. 9 (2001) 2155–2164
Chart 1.
class of compounds to exploit in the search for new
antitumor agents. While cytotoxic activities of the com-
pounds presented here remain relatively modest (i.e.,
with IC50’s in the low mM range) they are comparable to
those of the compounds shown in Chart 1, many of
which have served as starting points for the develop-
ment of more active compounds. More significant,
however, is the observation that some of the compounds
prepared in this study (e.g., 20a, 20c) appear to act on
specific (though as yet unknown) cell targets rather than
via generalized non-specific toxic mechanisms. Finally
the fact that both these compounds are also active in a
multidrug resistant cancer cell line lends strong impetus
to further structure–activity relationship studies in this
class of compounds, using the dipolar cycloaddition
methodology which we have developed. The results of
these studies will be reported in due course.
or with a 3.5% solution of phosphomolybdic acid in
ethanol. Column chromatography was conducted on
Merck 60 silica gel (230–400 mesh) at medium pressure
(200 mbar). Reagents were purchased from the Aldrich
Chemical Co. and were used without further purifica-
tion. All solvents were distilled and stored over 4 A
molecular sieves before use. Elemental analyses were
performed at the ICNS, CNRS, Gif-sur-Yvette.
General procedure for the preparation of 3,4-dihydro-
ꢀ-carbolines 8, 9 and 10. To phosphorus oxychloride (5
mL) was added, in small portions with rapid stirring
under argon, the appropriate solid Nb-formyltryptamine
(10 mmol), the reaction mixture being kept at room
temperature by means of an ice-water bath. Stirring was
continued until complete disappearance of the starting
material (20–60 min). The bright yellow suspension was
slowly poured into anhydrous diethyl ether (70 mL)
with rapid stirring. The hydrochloride salt of the dihy-
dro-b-carboline which precipitated was then collected
by filtration and washed several times with ether.
Recrystallization of the solid in a mixture of ethyl ace-
tate and 95% ethanol afforded the pure 3,4-dihydro-b-
carboline hydrochloride (which, for 10, was used for
elemental analysis). The latter was dissolved in water
(45 mL) and the solution was made basic by slow addi-
tion of aqueous sodium hydroxide (1 M), leading to
precipitation of the b-carboline. The mixture was
extracted with ether (3ꢂ20 mL), the organic extracts
were combined, washed with saturated aqueous NaCl
(30 mL), dried over sodium sulfate and the solvent
removed under reduced pressure, leaving a pale yellow
amorphous solid which was crystallized in the solvent
specified below. The following 3,4-dihydro-b-carbolines
were prepared in this manner.
Experimental
Chemistry
General. Melting points were determined on a Buchi
apparatus and are uncorrected. IRspectra of samples
were obtained either as KBr pellets or as films with a
Nicolet 205 FT-IRspectrometer. 1H NMRand 13C
NMRwere determined on a Bruker 200, 250 or
300 MHz instrument. Chemical shifts are given as d
values with reference to Me4Si as internal standard.
Electron impact (EI) and chemical ionization (CI) mass
spectra were recorded on an AEI MS-50 nd AEI MS-9
spectrometer, respectively. High-resolution (HR), fast
atom bombardment (FAB) and liquid secondary ion (L-
SI) mass spectra were obtained using a Kratos MS-80
spectrometer. Thin-layer chromatography was per-
formed on Merck silica gel plates with fluorescent indi-
cator. The plates were visualized with UV light (254 nm)
3,4-Dihydro-ꢀ-carboline (8). Obtained in 82% yield
from Nb-formyltryptamine22 after crystallization in

M. Bertrand et al. / Bioorg. Med. Chem. 9 (2001) 2155–2164
2159
ethyl acetate : mp 87–90 ꢁC (lit. mp 93–97 ꢁC)21; 1H
NMR(200 MHz, CDCl 3+10% CD3OD) d 2.89 (t,
J=8.6 Hz, 2H), 3.87 (t, J=8.6 Hz, 2H), 7.12 (t, J=7.4
Hz, 1H), 7.26 (t, J=7.4 Hz, 1H), 7.38 (d, J=8.1 Hz,
1H), 7.57 (d, J=8.1 Hz, 1H), 8.29 (s, 1H); IR(KBr)
3185, 1634 cmꢀ1; L-SIMS m/z 171 (MH)+.
12.52; Cl, 16.00. Found: C, 64.66; H, 6.29; N, 12.27; Cl,
16.15.
9-N-Benzyl-3,4-dihydro-ꢀ-carboline (12). Following the
same procedure as for the preparation of compound 11,
treatment of compound 8 (3.0 g, 17.6 mmol) with
sodium hydride (1.2 g of a 60% dispersion in oil; 29.9
mmols) and then with benzyl bromide (3.32 g, 19.4
mmol) afforded, after workup, compound 12 as a col-
orless amorphous solid (3.7 g, 81%): mp 132–133 ꢁC (lit.
3,4-Dihydro-6-methoxy-ꢀ-carboline (9). Obtained in
91% yield from 5-methoxy-Nb-formyltryptamine23 after
crystallization in ethyl acetate: mp 204 ꢁC; 1H NMR
(200 MHz, CDCl3) d 2.81 (t, J=6.0 Hz, 2H), 3.83 (br s,
5H), 6.91 (d, J=8.4 Hz, 1H), 6.93 (s, 1H), 7.28 (d, J=8.4
Hz, 1H), 8.35 (s, 1H), 8.56 (br s, 1H, exchangeable with
D2O); IR(KBr): 3300, 1625 cm ꢀ1; HREIMS calcd for
C12H12N2O m/z 200.0947, found 200.0959.
mp 138–139 ꢁC);14 1H NMR(200 MHz, CD OD) d 2.92
3
(t, J=8.0 Hz, 2H), 3.75 (t, J=8.0 Hz, 2H), 5.42 (s, 2H),
7.03–7.66 (m, 9H), 8.34 (br s, 1H). Anal. calcd for
C18H16N2:C, 81.91; H, 6.26; N, 10.26. Found: C, 82.01;
H, 6.53; N, 10.35.
6-Benzyloxy-3,4-dihydro-ꢀ-carboline (10). Obtained in
72% yield from 5-benzyloxy-Nb-formyltryptamine24
after crystallization in ethyl acetate–95% ethanol : mp
6-Benzyloxy-3,4-dihydro-2-N-[(trimethylsilyl)methyl]-ꢀ-
carbolinium triflate (15). To a solution of compound 10
(2.76 g, 10 mmol) in anhydrous dichloromethane (30
mL) was added dropwise at 0 ꢁC under argon a solution
of (trimethylsilyl)methyl triflate (2.2 mL, 11 mmol) in
dichloromethane (10 mL). The reaction mixture was
stirred for 20 min at 0 ꢁC and then for 1 h at rt. The
solvent was removed under reduced pressure and the
residue was crystallized in ethanol, affording compound
15 as bright yellow needles (3.78 g, 95%) : mp 168–
82–83 ꢁC (hygroscopic); H NMR(250 MHz, CD OD)
1
3
d 2.75 (t, J=8.5 Hz, 2H), 3.74 (t, J=8.5 Hz, 2H), 4.96
(s, 2H), 6.93–7.00 (m, 2H), 7.25–7.40 (m, 6H), 8.22 (s,
1H); 13C NMR(62.5 MHz, CDCl ) d 19.2, 48.3, 70.8,
102.3, 112.2, 113.1, 115.5, 116.3, 125.5, 127.6, 127.9,
3
128.5, 132.7, 137.4, 152.2, 153.6; IR(KBr) 3398, 1602
cmꢀ1
;
CIMS m/z 277 (MH)+. Anal. calcd for
170 ꢁC; H NMR(250 MHz, acetone- d6) d 0.30 (s, 9H),
1
C18H16N2O.HCl.H2O: C, 65.35; H, 5.79; N, 8.47; Cl,
10.72. Found: C, 65.08; H, 5.99; N, 8.31; Cl, 10.68.
3.42 (m, 2H), 3.56 (s, 2H), 4.25 (m, 2H), 5.15 (s; 2H),
7.20–7.57 (m, 8H), 8.94 (s, 1H), 11.29 (br s, 1H); 13C
NMR(62.5 MHz, acetone- d6) d-2.6, 20.5, 53.2, 53.6,
70.9, 102.5, 115.6, 122.8, 127.2, 128.5, 128.7, 129.4,
130.7, 132.4, 143.5, 144.1, 158.1, 160.5; IR(KBr) 3206,
1616, 1289 cmꢀ1; L-SIMS m/z 363 (MH)+. Anal. calcd
for C23H27F3N2O4SSi.0.25H2O:C, 53.42; H, 5.36; N,
5.42; S, 6.20. Found: C, 53.23; H, 5.22; N, 5.19; S, 5.93.
3,4-Dihydro-9-N-methyl-ꢀ-carboline (11). To a suspen-
sion of sodium hydride (1.13 g of a 60% dispersion in
oil; 28.2 mmol) in anhydrous DMF (20 mL) was added
dropwise with stirring at ꢀ10 ꢁC under argon a solution
of compound 8 (2.84 g, 16.7 mmol) in DMF (10 mL).
The mixture was stirred for 1 h at ꢀ10 ꢁC and then
cooled to ꢀ60 ꢁC before dropwise addition of a solution
of methyl tosylate (3.41 g, 18.4 mmol) in DMF (10 mL).
Stirring was maintained for 2 h at ꢀ60 ꢁC and methanol
(5 mL), water (5 mL) and saturated aqueous sodium
chloride (250 mL) were added successively. The solution
was extracted with dichloromethane (3ꢂ75 mL), the
organic extracts were combined, washed with water (30
mL) and dried over sodium sulfate. The solvents were
removed under reduced pressure, the residue was taken
up in dichloromethane and the solution was filtered
through a pad of silica gel. Evaporation of the filtrate
afforded compound 11 which was crystallized in ethyl
acetate–95% ethanol (1.87 g, 61%). For analysis, the
hydrochloride salt was prepared by dissolving 11 in
ethyl acetate and adding ethanolic HCl until the solu-
tion turned acid. Addition of diethyl ether to the solu-
tion then led to precipitation of the hydrochloride salt
which was collected by filtration and washed with ether:
mp 148–150 ꢁC (lit. mp 153–154 ꢁC),13 hydrochloride,
220–222 ꢁC; 1H NMR(hydrochloride, 250 MHz,
DMSO-d6) d 3.37 (t, J=9.0 Hz, 2H), 4.05 (s, 3H), 4.06
(t, J=9.0 Hz, 2H), 7.33 (t, J=7.5 Hz, 1H), 7.67 (t,
J=7.5 Hz, 1H), 7.75 (d, J=8.2 Hz, 1H), 7.89 (d, J=8.2
Hz, 1H), 9.46 (br s, 1H), 13.67 (br s, 1H); 13C NMR
(hydrochloride, 62.5 MHz, DMSO-d6) d 18.5, 30.1, 41.2,
111.6, 121.4, 121.9, 123.3, 126.9, 128.5, 141.5, 154.1; IR
(KBr) 1639 cmꢀ1; CIMS m/z 185 (MH)+. Anal. calcd
for C12H12N2.HCl.0.15 H2O: C, 64.41; H, 5.99; N,
3,4-Dihydro-9-N-methyl-2-N-[(trimethylsilyl)methyl]-ꢀ-
carbolinium triflate (16). Following the same procedure
as for the preparation of compound 15, reaction of
compound 11 (1.84 g, 10 mmol) with (trimethylsi-
lyl)methyl triflate (2.2 mL, 11 mmol) afforded, after
crystallization in ethanol, compound 16 as bright yellow
needles (2.54 g, 94%) : mp 174–175 ꢁC; 1H NMR
(250 MHz, CD3OD) d 0.28 (s, 9H), 3.37 (t, J=8.8 Hz,
2H), 3.80 (s, 2H), 3.92 (s, 3H), 4.03 (t, J=8.8 Hz, 2H),
7.23 (t, J=7.5 Hz, 1H), 7.40 (d, J=8.6 Hz, 1H), 7.50 (t,
J=7.5 Hz, 1H), 7.65 (d, J=8.6 Hz, 1H), 9.07 (s, 1H);
13C NMR(200 MHz, DMSO- d6) d-2.7, 19.1, 23.0, 51.4,
52.4, 111.4, 120.9, 121.4, 121.7, 123.3, 127.5, 128.2,
141.4, 151.3; IR(KBr) 1630, 1264 cm ꢀ1; L-SIMS m/z
271 (MH)+. Anal. calcd for C17H23F3N2O3SSi:C, 48.55;
H, 5.51; N, 6.66. Found: C, 48.67; H, 5.38; N, 6.61.
(1R,11bR)- and (1S, 1 1Sb)-1,2,3,5,6,11b-Hexahydro-8-
methoxyindolizino[8,7-b]indole-1-carbonitrile [(ꢃ)-19a],
(1S,11bR)- and (1R,11bS)-1,2,3,5,6,11b-hexahydro-8-
methoxyindolizino[8,7-b]indole-1-carbonitrile [(ꢃ)-19b],
(2S,11bR)- and (2R,11bS)-1,2,3,5,6,11b-hexahydro-8-
methoxyindolizino[8,7-b]indole-2-carbonitrile [(ꢃ)-19c]
and (2R,11bR)- and (2S,11bS)-1,2,3,5,6,11b-hexahydro-
8-methoxyindolizino[8,7-b]indole-2-carbonitrile [(ꢃ)-19d].
Cesium fluoride (0.61 g, 4 mmol) was placed in a round
bottom flask and heated under vacuum at 300–400 ꢁC

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M. Bertrand et al. / Bioorg. Med. Chem. 9 (2001) 2155–2164
for 1–2 min. The flask was cooled and, under a stream
of dry argon, anhydrous acetonitrile (3 mL) was intro-
duced through a septum followed by a solution of
acrylonitrile (58 mg, 1.1 mmol) in acetonitrile (2 mL).
The mixture was cooled to 0 ꢁC and a solution of com-
pound 1411 (316 mg, 1.1 mmol) in acetonitrile (2 mL)
was added dropwise. The reaction mixture was stirred
for 30 min at 0 ꢁC and then for 1 h at rt. The solvent was
removed under reduced pressure, the residue was taken
up in dichloromethane (50 mL) and the mixture was
washed with water (50 mL). The organic phase was then
extracted with aqueous hydrochloric acid (2ꢂ50 mL of
a 1 M solution), the aqueous extracts were combined,
washed with dichloromethane (50 mL) and neutralized
by addition of aqueous sodium hydroxide (10 M). The
white precipitate which formed was extracted with di-
chloromethane (2ꢂ50 mL), the organic extracts were
combined, washed with water (50 mL), dried over
sodium sulfate and evaporated under reduced pressure.
The residue was first purified by filtration through a pad
of silica gel (dichloromethane containing 5% methanol),
affording, after evaporation of the filtrate, a mixture of
19a–d (250 mg, 85% overall yield), the Rf’s of each
compound on silica gel being: 0.61 (19a), 0.51 (19c),
0.36 (19b) and 0.30 (19d) (CH2Cl2/MeOH 95:5). Crys-
tallization of the mixture in ethyl acetate provided 19b
(59 mg, 20%): mp 144–145 ꢁC; 1H NMR(250 MHz,
CDCl3) d 2.09–2.18 (m, 1H), 2.21–2.39 (m, 1H), 2.59–
2.72 (m, 1H), 2.88–3.39 (m, 6H), 3.86 (s, 3H), 4.35 (d,
J=6.5 Hz, 1H), 6.85 (d, J=8.7 Hz, 1H), 6.95 (s, 1H),
7.24 (d, J=8.7 Hz, 1H), 8.23 (br s, 1H); 13C NMR
(62.5 MHz, CDCl3) d 18.2, 28.4, 31.2, 46.1, 48.5, 56.1,
60.0, 100.6, 110.8, 112.2, 112.4, 121.1, 127.3, 130.5,
131.6, 154.3; IR(KBr) 3547, 2238 cm ꢀ1; CIMS m/z 268
(MH)+. Anal. calcd for C16H17N3O. 0.8 H2O: C,
68.23; H, 6.61; N, 14.92. Found: C, 68.53; H, 6.82; N,
14.63.
19d. 68 mg, 23%); 1H NMR(250 MHz, CDCl ) d 2.05–
3
2.16 (m, 1H), 2.60–3.31 (m, 7H), 3.85 (s, 3H), 4.22 (t,
J=6.8 Hz, 1H), 6.80 (d, J=8.7 Hz, 1H), 6.93 (s, 1H),
7.17 (d, J=8.7 Hz, 1H), 7.97 (br s, 1H); 13C NMR
(62.5 MHz, CDCl3) d 18.0, 26.5, 34.6, 45.7, 53.3, 56.1, 56.4,
100.7, 107.6, 111.8, 112.9, 121.4, 127.4, 131.4, 134.9, 154.2;
IR(KBr) 3386, 2239 cm ꢀ1; CIMS m/z 268 (MH)+. Anal.
calcd for C16H17N3O. 0.6 CH3CO2C2H5: C, 69.04; H, 6.82;
N, 13.13. Found: C, 68.96; H, 6.72; N, 12.95.
(1R,11bR)- and (1S, 1 1S)b-8-Benzyloxy-1,2,3,5,6,11b-
hexahydroindolizino[8,7-b]indole-1-carbonitrile [(ꢃ)-20a],
(1S,11bR)- and (1R,11bS)-8-benzyloxy-1,2,3,5,6,11b-hex-
ahydroindolizino[8,7-b]indole-1-carbonitrile
[(ꢃ)-20b],
(2S,11bR)- and (2R,11bS)-8-benzyloxy-1,2,3,5,6,11b-hexa-
hydroindolizino[8,7-b]indole-2-carbonitrile [(ꢃ)-20c] and
(2R,11bR)- and (2S,11bS)-8-benzyloxy-1,2,3,5,6,11b-hexa-
hydroindolizino[8,7-b]indole-2-carbonitrile [(ꢃ)-20d]. Fol-
lowing the same procedure as for the preparation of
compounds 19a–d, compound 15 (398 mg, 1.1 mmol)
was reacted with acrylonitrile (58 mg, 1.1 mmol) in the
presence of cesium fluoride (0.61 g, 4 mmol) in acetoni-
trile to afford compounds 20a–d (309 mg, 82%) whose
Rf’s on silica gel (CH2Cl2/MeOH 98:2) were 0.45 (20a),
0.33 (20c), 0.24 (20b) and 0.15 (20d). Compound 20b
was isolated by crystallization of the crude product in
ethyl acetate (75 mg, 20%) : mp 198–199 ꢁC; H NMR
1
(300 MHz, acetone-d6) d 2.37–2.56 (m, 2H), 2.72–2.81
(m, 3H), 2.89–2.98 (m, 1H), 3.22–3.29 (m, 2H), 3.49–
3.52 (m, 1H), 3.89 (d, J=6.3 Hz, 1H), 5.09 (s, 2H), 6.84
(d, J=8.7 Hz, 1H), 7.04 (s, 1H), 7.21 (d, J=8.7 Hz,
1H), 7.28–7.38 (m, 4H), 7.47 (d, J=7.6 Hz, 1H), 9.64
(br s, 1H); 13C NMR(75 MHz, acetone- d6) d 18.1, 26.3,
34.5, 45.7, 53.6, 56.5, 70.9, 102.2, 108.0, 111.5, 112.3,
122.1, 127.3, 127.4, 127.7, 128.4, 132.7, 133.8, 153.2; IR
(KBr) 3363, 2237 cmꢀ1; CIMS m/z 344 (MH)+. Anal.
calcd for C22H21N3O: C, 76.94; H, 6.16; N, 12.24.
Found: C, 76.93; H, 6.25; N, 12.16.
The mother liquor was concentrated and the residue
was purified by preparative TLC on silica gel (CH2Cl2/
MeOH 98:2) affording, in order of elution.
The mother liquor was then purified by preparative
TLC on silica gel (CH2Cl2/MeOH 98:2) to provide, in
order of elution:
19a. (70 mg, 24%): mp 136–138 ꢁC; 1H NMR
(250 MHz, CDCl3) d 2.18–2.23 (m, 1H), 2.35–2.41 (m,
1H), 2.75–3.09 (m, 7H), 3.85 (s, 3H), 4.23 (d, J=7.1 Hz,
1H), 6.83 (d, J=8.8 Hz, 1H), 6.94 (s, 1H), 7.23 (d,
J=8.8 Hz, 1H), 8.20 (br s, 1H); 13C NMR(62.5 MHz,
CDCl3) d 20.1, 29.1, 32.2, 47.1, 51.2, 56.0, 62.4, 100.7,
109.1, 112.0, 112.2, 121.9, 127.1, 131.5, 132.3, 154.3; IR
(KBr) 3372, 2241 cmꢀ1; CIMS m/z 268 (MH)+. Anal.
calcd for C16H17N3O: C, 71.89; H, 6.41; N, 15.72.
Found: C, 71.81; H, 6.41; N, 15.75.
Compound 20a (98 mg, 26%): mp 172–173 ꢁC; 1H
NMR(300 MHz, acetone- d6) d 2.15–2.22 (m, 1H), 2.24–
2.34 (m, 1H), 2.67–3.17 (m, 7H), 4.34 (d, J=7.64 Hz,
1H), 5.10 (s, 2H), 6.88–7.47 (m, 8H), 8.97 (br s, 1H); 13C
NMR(75 MHz, acetone- d6) d 19.1, 29.1, 32.0, 46.5,
50.2, 62.1, 70.8, 102.1, 109.6, 111.8, 112.5, 126.9, 127.4,
127.6, 128.3, 131.7, 132.4, 137.6, 153.4; IR(KBr) 3352,
2246 cmꢀ1; CIMS m/z 344 (MH)+. Anal. calcd for
C22H21N3O: C, 76.94; H, 6.16; N, 12.24. Found: C,
76.69; H, 6.11; N, 12.04.
19c. (53 mg, 18%): mp 170–172 ꢁC; 1H NMR
(250 MHz, CDCl3) d 2.25–2.29 (m, 1H), 2.52–3.27 (m,
8H), 3.85 (s, 3H), 4.33 (t, J=6.8 Hz, 1H), 6.81 (d,
J=8.7 Hz, 1H), 6.93 (s, 1H), 7.20 (d, J=8.7 Hz, 1H),
Compound 20c (79 mg, 21%): mp 171–174 ꢁC; 1H
NMR(300 MHz, CDCl 3) d 2.18–2.24 (m, 1H), 2.43–
2.51 (m, 1H), 2.52–3.25 (m, 7H), 4.29 (t, J=6.4 Hz, 1H),
5.09 (s, 2H), 6.88 (dd, J=8.7 Hz and 2.3 Hz, 1H), 6.90
(s, 1H), 7.16 (d, J=8.7 Hz, 1H), 7.30–7.47 (m, 5H), 7.80
7.69 (br s, 1H); 13C NMR(62.5 MHz, CDCl ) d 18.4,
3
26.6, 34.8, 45.9, 53.9, 56.1, 56.7, 100.7, 108.4, 111.7,
111.9, 122.3, 127.1, 131.3, 134.0, 154.4; IR(KBr) 3150,
2233 cmꢀ1; CIMS m/z 268 (MH)+. Anal. calcd for
C16H17N3O. 0.3 CH3CO2C2H5: C, 70.35; H, 6.61; N,
14.31. Found: C, 70.12; H, 6.51; N, 14.67.
(br s, 1H); 13C NMR(75 MHz, CDCl ) d 18.4, 26.5,
3
34.7, 45.9, 53.8, 56.7, 71.1, 102.4, 108.2, 111.7, 112.5,
122.3, 127.6, 127.9, 128.6, 131.5, 134.1, 137.7, 153.4; IR
(KBr) 3360, 2239 cmꢀ1; CIMS m/z 344 (MH)+. Anal.

M. Bertrand et al. / Bioorg. Med. Chem. 9 (2001) 2155–2164
2161
calcd for C22H21N3O: C, 76.94; H, 6.16; N, 12.24.
Found: C, 77.10; H, 6.18; N, 11.96.
min. The reaction mixture was then slowly neutralized
by addition of saturated aqueous sodium hydrogen car-
bonate, the resulting emulsion was taken up in saturated
aqueous sodium chloride (15 mL) and the mixture was
extracted with diethyl ether (3ꢂ10 mL). The organic
extracts were combined, washed with saturated aqueous
sodium chloride (15 mL), dried over sodium sulfate and
evaporated under reduced pressure. The residue was
taken up in dichloromethane and filtered through a pad
of silica gel. After evaporation of the filtrate under
reduced pressure, the crude product was crystallized in
ethyl acetate/95% ethanol. The following compounds
were prepared in this manner:
Compound 20d (57 mg, 15%): mp 168–170 ꢁC; 1H
NMR(250 MHz, CDCl 3) d 2.04–2.19 (m, 1H), 2.60–
3.30 (m, 8H), 4.30 (t, J=6.5 Hz, 1H), 5.11 (s, 2H), 6.90
(dd, J=8.7 Hz and 2.4 Hz, 1H), 7.04 (d, J=2.4 Hz,
1H), 7.22–7.49 (m, 6H), 7.68 (br s, 1H); 13C NMR
(62.5 MHz, CDCl3) d 17.9, 26.6, 34.7, 45.7, 53.2, 56.3,
71.2, 102.4, 107.7, 111.7, 112.6, 121.4, 127.7, 127.9,
128.6, 131.6, 134.3, 139.2, 153.6; IR(KBr) 3371, 2241
cmꢀ1
;
CIMS m/z 344 (MH)+. Anal. calcd for
C22H21N3O.0.7 H2O: C, 74.22; H, 6.34; N, 11.80.
Found: C, 74.27; H, 6.15; N, 11.62.
Compound 23a (from 20a) (23 mg, 61%): mp >250 ꢁC;
(1R,11bR)- and (1S, 1 1 Sb)-1,2,3,5,6,11b-Hexahydro-11-
N-methylindolizino[8,7-b]indole-1-carbonitrile [(ꢃ)-21a],
(1S,11bR)- and (1R,11bS)-1,2,3,5,6,11b-hexahydro-11-
N-methylindolizino[8,7-b]indole-1-carbonitrile [(ꢃ)-21b].
Following the same procedure as for the preparation of
compounds 19a–d, compound 16 (300 mg, 1.1 mmol)
was reacted with acrylonitrile (58 mg, 1.1 mmol) in the
presence of cesium fluoride (0.61 g, 4 mmol) in acetoni-
trile to afford compounds 21a,b (200 mg, 70%) whose
Rf’s on silica gel (ethyl acetate/heptane 3:1) were 0.50
(21a) and 0.18 (21b). Compound 21b was isolated by
crystallization of the crude product in ethyl acetate and
recrystallization in ethyl acetate 95% ethanol (85 mg,
1H NMR(250 MHz, CDCl ) d 2.03–2.17 (m, 1H), 2.27–
3
2.41 (m, 1H), 2.44–3.26 (m, 7H), 4.37 (d, J=5.1 Hz,
1H), 6.67 (d, J=8.6 Hz, 1H), 6.83 (s, 1H), 7.18 (d,
J=8.6 Hz, 1H), 7.66 (br s, 1H), 9.79 (br s, 1H); IR
(KBr) 3344, 2244 cmꢀ1; CIMS m/z 254 (MH)+. Anal.
calcd for C15H15N3O. 0.25 H2O: C, 69.88; H, 6.06; N,
16.30. Found: C, 69.75; H, 6.21; N, 16.06.
Compound 23b (from 20b) (22 mg, 60%): mp 205–
208 ꢁC (decomp); 1H NMR(250 MHz, acetone- d6) d
2.04–2.35 (m, 2H), 2.62–2.84 (m, 4H), 3.17–3.27 (m,
2H), 3.51–3.57 (m, 1H), 3.77 (d, J=6.3 Hz, 1H), 6.67
(dd, J=8.3 Hz and 2.4 Hz, 1H), 6.85 (d, J=2.4 Hz,
1H), 7.14 (d, J=8.3 Hz, 1H), 9.78 (br s, 1H); IR(KBr)
3386, 2239 cmꢀ1; CIMS m/z 254 (MH)+.
ꢁ
1
30%): mp 185–187 C; H NMR(250 MHz, CDCl ) d
3
2.23–2.45 (m, 2H), 2.80–2.99 (m, 4H), 3.15–3.31 (m,
2H), 3.33–3.40 (m, 1H), 3.69 (s, 3H), 4.04 (d, J=7.1 Hz,
1H), 7.07 (t, J=7.1 Hz, 1H), 7.21 (t, J=7.1 Hz, 1H),
7.29 (d, J=7.9 Hz, 1H), 7.51 (d, J=7.1 Hz, 1H); 13C
Compound 23c (from 20c) (21 mg, 55%): mp 235–
240 ꢁC (decomp); 1H NMR(250 MHz, acetone- d6) d
2.02–2.35 (m, 1H), 2.36–3.29 (m, 8H), 4.30 (d, J=6.0
Hz, 1H), 6.65 (d, J=8.6 Hz, 1H), 6.83 (s, 1H), 7.12 (d,
J=8.6 Hz, 1H), 7.59 (br s, 1H), 9.67 (br s, 1H); IR
(KBr) 3381, 2240 cmꢀ1; CIMS m/z 254 (MH)+
NMR(62.5 MHz, CDCl ) d 21.5, 29.6, 30.1, 32.2, 47.5,
3
52.5, 60.7, 109.1, 109.9, 118.5, 119.1, 120.7, 121.6,
126.4, 131.7, 137.5; IR(KBr) 2235 cm ꢀ1; CIMS m/z
252 (MH)+. Anal. calcd for C16H17N3.0.1 H2O: C,
75.92; H, 6.85; N, 16.60. Found: C, 75.85; H, 6.96; N,
16.81.
Compound 23d (from 20d) (24 mg, 64%): mp >250 ꢁC;
1H NMR(250 MHz, CD 3OD) d 2.12–2.34 (m, 2H),
2.68–3.32 (m, 7H), 4.15 (d, J=6.6 Hz, 1H), 6.68–6.72
(m, 1H), 6.86–6.90 (m, 1H), 7.13–7.17 (m, 1H), 7.13 (s,
1H); IR(KBr) 3387, 2234 cm ꢀ1; CIMS m/z 254 (MH)+.
The mother liquor was purified by preparative TLC on
silica gel (ethyl acetate/heptane 3:1) to provide com-
pound 21a which was crystallized in ethyl acetate/95%
ethanol (85 mg, 30%): mp 140–141 ꢁC; 1H NMR
(250 MHz, CDCl3) d 2.23–2.39 (m, 1H), 2.41–2.48 (m,
1H), 2.71–3.18 (m, 7H), 3.78 (s, 3H), 4.56 (d, J=6.4 Hz,
1H), 7.11 (t, J=6.9 Hz, 1H), 7.23 (t, J=6.9 Hz, 1H),
7.30 (d, J=7.9 Hz, 1H), 7.51 (d, J=7.9 Hz, 1H); IR
(KBr) 2237 cmꢀ1; CIMS m/z 252 (MH)+. Anal. calcd
for C16H17N3: C, 76.46; H, 6.82; N, 16.72. Found: C,
76.54; H, 6.86; N, 16.65.
General procedure for the reduction of the nitrile deriva-
tives 18, 20, 22 to the aminomethyl derivatives 24, 25,
26. A stirring suspension of lithium aluminum hydride
(41 mg, 1.1 mmol) in anhydrous THF (2 mL) was trea-
ted dropwise under argon with aluminum chloride (122
mg, 0.92 mmol) in THF (2 mL). The reaction mixture
was maintained at rt during the addition by means of a
water bath. After addition was complete, the reaction
mixture was stirred vigorously for 20 min and then
cooled to 0 ꢁC before adding dropwise a solution of the
nitrile (1 mmol) in THF (2 mL). After complete addi-
tion, stirring was continued for an additional hour and
methanol followed by water were slowly added to the
reaction mixture. The mixture was then concentrated
under reduced pressure, the residue was taken up in di-
chloromethane (20 mL) and the mixture was washed
with water (20 mL). The organic phase was extracted
with aqueous HCl (2ꢂ20 mL of a 1 M solution) and the
aqueous phases were combined, washed with dichloro-
(1R,11bR)- and (1S, 1 1Sb)-1,2,3,5,6,11b-Hexahydro-8-
hydroxyindolizino[8,7-b]indole-1-carbonitrile [(ꢃ)-23a],
(1S,11bR)- and (1R,11bS)-1,2,3,5,6,11b-hexahydro-8-hy-
droxyindolizino[8,7-b]indole-1-carbonitrile
[(ꢃ)-23b],
(2S,11bR)- and (2R,11bS)-1,2,3,5,6,11b-hexahydro-8-hy-
droxyindolizino[8,7-b]indole-2-carbonitrile [(ꢃ)-23c] and
(2R,11bR)- and (2S,11bS)-1,2,3,5,6,11b-hexahydro-8-hy-
droxyindolizino[8,7-b]indole-2-carbonitrile [(ꢃ)-23d]. A
solution of the 8-benzyloxy derivative 20a, 20b, 20c or
20d (50 mg, 0.15 mmol) and thioanisole (0.30 mL, 2.5
mmol) in triflic acid (0.70 mL) was stirred at rt for 20

2162
M. Bertrand et al. / Bioorg. Med. Chem. 9 (2001) 2155–2164
1
methane (20 mL) and neutralized by addition of aqu-
eous sodium hydroxide (10 M). The mixture was
extracted with dichloromethane (2ꢂ20 mL), the organic
phases were combined, washed with water (20 mL),
dried over sodium sulfate and evaporated under
reduced pressure. The residue was filtered through a pad
of silica gel, the filtrate was evaporated and the crude
amine was crystallized in the solvent indicated and/or
was characterized as the l-(+)-tartrate salt, crystallized
in 95% ethanol. The following compounds were
prepared in this manner.
116 ꢁC; H NMR(250 MHz, CDCl ) d 1.36–1.41 (m,
3
1H), 1.59 (br s, 2H), 1.93–2.04 (m, 2H), 2.50–3.09 (m,
7H), 3.21–3.27 (m, 1H), 3.42 (d, J=6.7 Hz, 1H), 5.07 (s,
2H), 6.82 (dd, J=8.6 Hz and 2.3 Hz, 1H), 7.01 (d,
J=2.3 Hz, 1H), 7.26 (d, J=7.1 Hz, 1H), 7.28–7.38 (m,
4H), 7.45 (d, J=7.3 Hz, 1H), 10.41 (br s, 1H); 13C
NMR(62.5 MHz, CDCl ) d 20.2, 28.2, 45.9, 46.4, 48.1,
3
51.1, 65.4, 70.9, 102.0, 106.2, 111.1, 111.5, 127.5, 127.6,
127.8, 128.4, 130.8, 136.8, 137.9, 152.7; IR(KBr) 3148,
1587 cmꢀ1; CIMS m/z 348 (MH)+. Anal. calcd for
C22H25N3O. 2.3 C4H6O6. 0.5 C2H5OH: C, 54.04; H,
5.89; N, 5.87. Found: C, 54.09; H, 5.99; N, 5.95.
(1R,11bR)- and (1S, 1 1 Sb)-1-Aminomethyl-1,2,3,5,6,11b-
hexahydroindolizino[8,7-b]indole (24a). Prepared from
18a11 in 75% yield as described above and was crystal-
lized in ethyl acetate : mp 132–133 ꢁC (tartrate: 170–
180 ꢁC, decomp); 1H NMR(250 MHz, DMSO- d6) d
1.39–1.47 (m, 1H), 1.84–2.14 (m, 4H), 2.50–2.92 (m,
5H), 3.14–3.29 (m, 1H), 3.29–3.50 (br s, 2H), 3.69 (d,
J=5.5 Hz, 1H), 6.88–7.02 (m, 2H), 7.28–7.36 (m, 2H),
(1S,11bR)- and (1R, 1 1 Sb)-1-Aminomethyl-8-benzyloxy-
1,2,3,5,6,11b-hexahydro-indolizino[8,7-b]indole (25b).
Prepared from 20b in 69% yield as described above
and was characterized as the tartrate salt: mp 140 ꢁC
1
(decomp); H NMR(250 MHz, CDCl 3) d 1.25 (br s,
2H), 1.61–1.89 (m, 1H), 1.89–2.29 (m, 1H), 2.34–3.29
(m, 9H), 4.32 (d, J=5.8 Hz, 1H), 5.07 (s, 2H), 6.84 (t,
J=7.5 Hz, 1H), 7.00–7.11 (m, 2H), 7.19 (d, J=8.7 Hz,
1H), 7.28–7.38 (m, 3H), 7.45 (d, J=6.5 Hz, 1H), 8.36
10.85 (br s, 1H); 13C NMR(62.5 MHz, CDCl ) d 18.7,
3
28.4, 45.2, 46.7, 47.1, 65.6, 106.4, 111.2, 118.1, 119.0,
122.3, 127.3, 136.1; IR(film) 3342, 1576 cm ꢀ1; CIMS
m/z 242 (MH)+. Anal. calcd for C15H19N3: C, 74.65;
H, 7.94; N, 17.41. Found: C, 74.83; H, 7.94; N,
17.22.
(br s, 1H); 13C NMR(62.5 MHz, CDCl ) d 17.4, 28.6,
3
43.4, 44.0, 46.7, 48.2, 60.7, 71.2, 101.9, 108.4, 111.5,
111.7, 127.6, 127.7, 128.5, 131.1, 134.0, 137.9, 152.8; IR
(KBr) 3402, 1588 cmꢀ1; FABMS m/z 348 (MH)+. Anal.
calcd for C22H25N3O. 2.3 C4H6O6. 0.5 C2H5OH: C,
54.04; H, 5.89; N, 5.87. Found: C, 54.12; H, 6.01; N,
6.05.
(1S,11bR)- and (1R, 1 1 Sb)-1-Aminomethyl-1,2,3,5,6,11b-
hexahydroindolizino[8,7-b]indole (24b). Prepared from
18b11 in 68% yield as described above and was char-
acterized as the tartrate salt: mp 170–180 ꢁC (decomp);
(2S,11bR)- and (2R, 1 1 Sb)-2-Aminomethyl-8-benzyloxy-
1,2,3,5,6,11b-hexahydro-indolizino[8,7-b]indole (25c).
Prepared from 20c in 76% yield as described above
1H NMR(250 MHz, CDCl ) d 1.25–1.38 (m, 1H), 1.82
3
(br s, 2H), 2.01–2.20 (m, 1H), 2.54–3.29 (m, 9H), 4.41
(d, J=5.5 Hz, 1H), 7.04–7.14 (m, 2H), 7.32 (d, J=7.9
H, 1H), 7.49 (d, J=7.1 Hz, 1H), 10.85 (br s, 1H); 13C
and was characterized as the tartrate salt: mp 128 ꢁC
1
(decomp); H NMR(250 MHz, CDCl 3) d 1.25 (br s,
NMR(62.5 MHz, CDCl ) d 17.2, 28.7, 43.6, 44.1, 46.6,
2H), 1.80–2.12 (m, 4H), 2.87–3.22 (m, 7H), 4.05–4.15
(m, 1H), 5.06 (s, 2H), 6.82 (d, J=8.4 Hz, 1H), 7.00–7.37
(m, 6H), 7.44 (d, J=6.8 Hz, 1H), 8.61 (br s, 1H); 13C
3
48.0, 60.5, 108.6, 111.0, 117.8, 118.6, 120.9, 128.0, 133.1,
136.0; IR(film) 3407, 1544 cm ꢀ1; FABMS m/z 242
(MH)+. Anal. calcd for C15H19N3. 1.9 C4H6O6: C,
51.56; H, 5.78; N, 7.98. Found: C, 51.62; H, 5.61; N,
7.91.
NMR(62.5 MHz, CDCl ) d 17.8, 33.8, 40.0, 46.0, 46.7,
3
53.3, 57.5, 71.1, 102.1, 107.6, 111.5, 111.6, 111.7, 127.5,
127.7, 128.4, 131.4, 136.0, 137.8, 153.1; IR(KBr) 3403,
1589 cmꢀ1; FABMS m/z 348 (MH)+. Anal. calcd for
C22H25N3O. 2.5 C4H6O6. 0.5 C2H5OH: C, 54.13; H,
5.81; N, 5.63. Found: C, 54.33; H, 6.06; N, 5.75.
(2S,11bR)- and (2R, 1 1 Sb)-2-Aminomethyl-1,2,3,5,6,11b-
hexahydroindolizino[8,7-b]indole (24c). Prepared fom
18c11 in 72% yield as described above and was char-
acterized as the tartrate salt: mp 170–180 ꢁC (decomp);
1H NMR(200 MHz, CDCl 3) d 1.91–3.26 (m, 13H), 4.19
(m, 1H), 7.03–7.14 (m, 2H), 7.26 (d, J=7.0 Hz, 1H),
7.46 (d, J=6.3 Hz, 1H), 8.51 (br s, 1H); IR(KBr) 3410,
1580 cmꢀ1; CIMS m/z 242 (MH)+. Anal. calcd for
C15H19N3. 2C4H6O6. 0.5 H2O: C, 50.18; H, 5.86; N,
7.63. Found: C, 50.11; H, 5.89; N, 7.53.
(2R,11bR)- and (2S, 1 1 Sb)-2-Aminomethyl-8-benzyloxy-
1,2,3,5,6,11b-hexahydro-indolizino[8,7-b]indole (25d).
Prepared from 20d in 66% yield as described above
and was characterized as the tartrate salt: mp 137 ꢁC
1
(decomp); H NMR(250 MHz, CDCl ) d 1.17–1.25 (m,
3
1H), 1.44–1.51 (m, 1H), 1.66 (br s, 2H), 2.26–3.29 (m,
9H), 4.12–4.22 (m, 1H), 5.07 (s, 2H), 6.83 (d, J=8.5 Hz,
1H), 7.01 (s, 1H), 7.10 (d, J=8.6 Hz, 1H), 7.28–7.38 (m,
4H), 7.44 (d, J=7.1 Hz, 1H), 8.40 (br s, 1H); 13C NMR
(62.5 MHz, CDCl3) d 17.6, 33.4, 41.2, 46.0, 46.6, 53.4,
56.9, 71.1, 102.2, 107.0, 111.5, 111.7, 127.6, 127.7, 128.5,
(2R,11bR)- and (2S, 1 1 Sb)-2-Aminomethyl-1,2,3,5,6,11b-
hexahydroindolizino[8,7-b]indole (24d). Prepared from
18d11 in 65% yield as described above and was char-
acterized as the tartrate salt: mp 185 ꢁC (decomp); IR
(KBr) 3330, 1576 cmꢀ1; FABMS m/z 242 (MH)+.
ꢀ1
131.5, 136.7, 137.8, 153.2; IR(KBr) 3402, 1589 cm
;
FABMS m/z 348 (MH)+. Anal. calcd for C22H25N3O.
2.2 C4H6O6. 0.5 C2H5OH: C, 54.51; H, 5.93; N, 6.00.
Found: C, 54.63; H, 5.84; N, 6.13.
(1R,11bR)- and (1S, 1 1 Sb)-1-Aminomethyl-8-benzyloxy-
1,2,3,5,6,11b-hexahydro-indolizino[8,7-b]indole (25a).
Prepared from 20a in 74% yield as described above
and was characterized as the tartrate salt: mp 114–
(1R,11bR)- and (1S, 1 1 Sb)-1-Aminomethyl-11-N-benzyl-
1,2,3,5,6,11b-hexahydro-indolizino[8,7-b]indole
(26a).

M. Bertrand et al. / Bioorg. Med. Chem. 9 (2001) 2155–2164
2163
Prepared from 22a11 in 71% yield as described above
solution of compound 25a (150 mg, 0.43 mmol) in
methanol (2 mL) was hydrogenated at atmospheric
pressure for 4 h in the presence of 10% palladium on
carbon (15 mg). The reaction mixture was filtered
through Celite, the filter pad was washed with methanol
and the collected filtrates were evaporated under
reduced pressure, affording compound 27a (90 mg,
82%). The latter was transformed into its tartrate salt
and crystallized in 95% ethanol: mp 112 ꢁC (decomp);
1H NMR(250 MHz, CD 3OD) d 1.41–1.57 (m, 1H),
1.91–3.25 (m, 9H), 3.45–3.51 (m, 2H), 6.60 (dd, J=8.4
Hz and 2.2 Hz, 1H), 6.79 (d, J=2.2 Hz, 1H), 7.10 (d,
J=8.4 Hz, 1H); IR(KBr) 3330, 1590 cm ꢀ1; L-SIMS m/z
258 (MH)+. Anal. calcd for C15H19N3O. 2.5 C4H6O6.
H2O: C, 46.16; H, 5.58; N, 6.46. Found: C, 46.22; H,
5.64; N, 6.76.
and was characterized as the tartrate salt: mp 80 ꢁC
1
(decomp); H NMR(250 MHz, CDCl ) d 1.16 (s, 2H),
3
1.80–1.95 (m, 2H), 2.14–2.22 (m, 1H), 2.60–3.07 (m,
7H), 3.27–3.40 (m, 1H), 4.20 (d, J=1.6 Hz, 1H), 5.26–
5.47 (m, 2H), 6.92 (d, J=6.6 Hz, 1H), 7.07–7.26 (m,
7H), 7.50–7.54 (m, 1H); 13C NMR(62.5 MHz, CDCl 3) d
16.3, 28.7, 45.3, 46.2, 47.1, 47.4, 48.2, 59.8, 108.0, 109.5,
117.9, 119.2, 121.3, 125.5, 127.0, 127.1, 128.7, 136.3,
137.3, 137.9; IR(KBr) 3369, 1605 cm ꢀ1; CIMS m/z 332
(MH)+. Anal. calcd for C22H25N3.2.2 C4H6O6. 0.8
C2H5OH: C, 55.71; H, 6.05; N, 6.02. Found: C, 56.12;
H, 6.16; N, 5.77.
(1S,11bR)- and (1R, 1 1 Sb)-1-Aminomethyl-11-N-benzyl-
(26b).
1,2,3,5,6,11b-hexahydro-indolizino[8,7-b]indole
Prepared from 22b11 in 67% yield as described above
and was characterized as the tartrate salt: mp 101 ꢁC
(1S,11bR)- and (1R, 1 1 Sb)-1-Aminomethyl-1,2,3,5,6,11b-
hexahydro-8-hydroxy-indolizino[8,7-b]indole (27b). Fol-
lowing the same procedure as for the preparation of
27a, hydrogenation of 25b (150 mg, 0.43 mmol) affor-
ded compound 27b (85 mg, 77%) which was character-
1
(decomp); H NMR(250 MHz, CDCl 3) d 1.00 (br s,
2H), 1.79–1.86 (m, 1H), 1.94–1.99 (m, 1H), 2.30–3.16
(m, 9H), 3.62 (d, J=4.9 Hz, 1H), 5.16–5.37 (m, 2H),
6.93 (d, J=6.2 Hz, 1H), 7.05–7.25 (m, 7H), 7.49–7.53
(m, 1H); 13C NMR(62.5 MHz, CDCl 3) d 22.1, 27.5,
43.6, 44.7, 47.4, 48.2, 51.9, 61.3, 109.4, 109.8, 118.1,
119.1, 121.2, 126.0, 127.1, 127.2, 128.6, 134.7, 137.0,
137.6; IR(KBr) 3368, 1609 cm ꢀ1; CIMS m/z 332
(MH)+. Anal. calcd for C22H25N3. 3.1 C4H6O6.
C2H5OH: C, 51.88; H, 5.89; N, 4.98. Found: C, 51.86;
H, 5.86; N, 4.68.
ꢁ
1
ized as the tartrate salt: mp 135 C (decomp); H NMR
(250 MHz, CD3OD) d 1.50–1.78 (m, 1H), 1.98–2.23 (m,
1H), 2.44–3.16 (m, 9H), 3.89 (d, J=5.6 Hz, 1H), 6.65
(dd, J=8.5 Hz and 2.3 Hz, 1H), 6.79 (s, 1H), 7.10 (m,
1H); IR(KBr) 3349, 1592 cm ꢀ1; L-SIMS m/z 258
(MH)+. Anal. calcd for C15H19N3O. 1.8 C4H6O6. 0.5
C2H5OH: C, 50.62; H, 6.01; N, 7.63. Found: C, 50.63;
H, 6.28; N, 7.74.
(2S,11bR)- and (2R, 1 1 Sb)-2-Aminomethyl-11-N-benzyl-
(26c).
1,2,3,5,6,11b-hexahydro-indolizino[8,7-b]indole
Prepared from 22c11 in 73% yield as described above
(2S,11bR)- and (2R, 1 1 Sb)-2-Aminomethyl-1,2,3,5,6,11b-
hexahydro-8-hydroxy-indolizino[8,7-b]indole (27c). Fol-
lowing the same procedure as for the preparation of
27a, hydrogenation of 25c (150 mg, 0.43 mmol) afforded
compound 27c (92 mg, 83%) which was characterized
as the tartrate salt: mp 140 ꢁC (decomp); 1H NMR
(250 MHz, CD3OD) d 1.85–1.92 (m, 1H), 2.13–2.22 (m,
1H), 2.49–3.36 (m, 9H), 4.12–4.18 (m, 1H), 6.63 (dd,
J=8.5 Hz and 2.1 Hz, 1H), 6.80 (d, J=2.1 Hz, 1H),
7.11 (d, J=8.5 Hz, 1H); IR(KBr) 3389, 1629 cm ꢀ1; L-
SIMS m/z 258 (MH)+. Anal. calcd for C15H19N3O. 1.7
C4H6O6 0.7 C2H5OH: C, 51.16; H, 6.14; N, 7.72.
Found: C, 51.12; H, 6.42; N, 7.74.
and was characterized as the tartrate salt: mp 74 ꢁC
1
(decomp); H NMR(250 MHz, CDCl 3) d 1.42 (br s,
2H), 1.77–2.01 (m, 2H), 2.17–2.32 (m, 1H), 2.54–3.21
(m, 8H), 4.01 (t, J=7.1 Hz, 1H), 5.16–5.33 (m, 2H),
6.92 (d, J=7.7 Hz, 1H), 7.06–7.26 (m, 7H), 7.50–7.53
(m, 1H); 13C NMR(62.5 MHz, CDCl 3) d 19.6, 34.3,
40.3, 46.5, 46.9, 47.2, 55.7, 56.9, 107.9, 109.5, 118.1,
119.2, 121.3, 125.6, 125.8, 126.9, 127.2, 128.7, 136.8,
137.8; IR(KBr) 3360, 1602 cm ꢀ1; CIMS m/z 332
(MH)+. Anal. calcd for C22H25N3.2.2 C4H6O6.
C2H5OH: C, 55.67; H, 6.30; N, 5.94. Found: C, 55.58;
H, 6.38; N, 5.91.
(2R,11bR)- and (2S, 1 1 Sb)-2-Aminomethyl-1,2,3,5,6,11b-
hexahydro-8-hydroxy-indolizino[8,7-b]indole (27d). Fol-
lowing the same procedure as for the preparation of
27a, hydrogenation of 25d (150 mg, 0.43 mmol) affor-
ded compound 27d (80 mg, 72%) which was character-
(2R,11bR)- and (2S, 1 1 Sb)-2-Aminomethyl-11-N-benzyl-
1,2,3,5,6,11b-hexahydro-indolizino[8,7-b]indole
(26d).
Prepared from 22d11 in 66% yield as described above
and was characterized as the tartrate salt: mp 82 ꢁC
(decomp); H NMR(250 MHz, CDCl 3) d 1.25 (br s,
ized as the tartrate salt: mp 149 ꢁC (decomp); H NMR
1
1
2H), 1.38–1.44 (m, 2H), 2.26–2.99 (m, 8H), 3.27–3.30
(m, 1H), 4.16 (t, J=7.3 Hz, 1H), 5.18–5.28 (m, 2H), 6.94
(d, J=6.2 Hz, 1H), 7.03–7.28 (m, 7H), 7.51–7.54 (m,
(250 MHz, CD3OD) d 1.97–3.27 (m, 11H), 4.23–4.33
(m, 1H), 6.67–6.72 (m, 1H), 6.83 (s, 1H), 7.12–7.20 (m,
1H); IR(KBr) 3385, 1593 cm ꢀ1; L-SIMS m/z 258
(MH)+. Anal. calcd for C15H19N3O 1.7 C4H6O6
C2H5OH: C, 51.18; H, 6.31; N, 7.53. Found: C, 51.39;
H, 6.35; N, 7.71.
1H); 13C NMR(62.5 MHz, CDCl ) d 18.4, 35.5, 41.5,
3
46.5, 46.7, 47.3, 54.3, 56.4, 106.2, 107.5, 109.5, 118.2,
119.3, 121.3, 125.9, 127.1, 127.3, 128.8, 137.4, 137.8; IR
(KBr) 3362, 1605 cmꢀ1; CIMS m/z 332 (MH)+. Anal.
calcd for C22H25N3.2.2 C4H6O6. C2H5OH: C, 55.67; H,
6.30; N, 5.94. Found: C, 55.86; H, 6.18; N, 6.01.
Bioassays
Determination of cytotoxic activity on L1210 cells.
L1210 cells (murine leukemia) provided by the NCI,
Frederick, USA were cultivated in RPMI 1640 medium
(1R,11bR)- and (1S, 1 1 Sb)-1-Aminomethyl-1,2,3,5,6,11b-
hexahydro-8-hydroxy-indolizino[8,7-b]indole (27a).
A

2164
M. Bertrand et al. / Bioorg. Med. Chem. 9 (2001) 2155–2164
(Gibco) supplemented with 10% fetal calf serum, 2 mM
l-glutamine, 100 units/mL penicillin, 100 mg/mL strep-
tomycin, and 10 mM HEPES buffer (pH=7.4). Cyto-
toxicity was measured by the microculture tetrazolium
(MTT) assay as described.19 Cells were exposed to
graded concentrations of the compounds for 48 h and
results expressed as IC50 (concentration which reduced
by 50% the optical density of treated cells with respect
to untreated controls).
References and Notes
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11. Poissonnet, G.; Theret-Bettiol, M.-H.; Dodd, R. H. J.
Org. Chem. 1996, 61, 2273.
12. Because the benzyloxy group of compound 10 did not
survive the highly acidic medium attained during the typical
work up of a Bischler–Napieralski reaction (ex: ref 21), the
product was precipitated as its hydrochloride salt at the end of
the reaction period, thus protecting it from acid hydrolysis.
This procedure was also extended to the preparation of b-car-
bolines 8 and 9. See Experimental.
Cell cycle analysis. L1210 cells (2.5 105 cells/mL) were
incubated for 21 h with various concentrations of the
compounds, then fixed by 70% ethanol (v/v), washed
and incubated in PBS containing 100 mg/mL RNAse
and 25 mg/mL propidium iodide for 30 min at 20 ꢁC.
For each sample, 104 cells were analyzed on an
ATC3000 flow cytometer (Brucker, France) using an
argon laser (Spectra-Physics) emitting 400 nW at 488
nm. The fluorescence of propidium iodide was collected
through a 615 nm long-pass filter. Results are expressed
as the percentage of cells found in the phases of the cell
cycle.
Determination of cytotoxic activities on K562 cells.
Both K562-S (sensitive to doxorubicin) and K562-R
(resistant to doxorubicin, stock cultures being kept
in the presence of 10ꢀ6 M doxorubicin in order
to maintain selection pressure) erythroleukemia
cells20,21 were grown as described above for L1210
cells except that 40 mg/mL of gentamycin were added
to the culture medium and that bicarbonate was used
as buffer. Cells (25,000) in 1 mL of medium were seeded
in each well of 24-well Nunc microplates and various
concentrations of the test compounds in 10 mL of
medium were added immediately to each well. Cul-
tures were incubated for 3 days at 37 ꢁC in a 5%
CO2ꢀ95% air incubator. Cell viability was determined
by the MTT assay as described above.17,18 Values are
reported as the concentration of test substance inhibit-
ing 50% of cell proliferation compared to untreated
cells (IC50).
13. Andriamialisoa, R. Z.; Langlois, N.; Langlois, Y. J. Org.
Chem. 1985, 50, 961.
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R. Z.; Langlois, N. Tetrahedron 1983, 39, 3755.
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ylides (13,14,17) as well as of the cyclization products 18 and
22 have been reported. See ref 11.
16. For reviews of 1,3-cycloaddition reactions, see: (a) Huis-
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F. G. Chem. Rev. 1986, 86, 941.
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M.; Bisagni, E.; Atassi, G. Invest. New Drugs 1996, 14, 169.
20. K562Rand K562S cells were kindly provided by Dr. H.
Tapiero, Institut de Cancerologie et d’Immunogenetique, Vil-
lejuif, France.
Acknowledgements
21. Lozzio, C. B.; Lozzio, B. B. Blood 1975, 45, 321.
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23. Pachter, I. J.; Mohrbacher, R. J.; Zacharias, D. E. J. Am.
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24. Stauffer, R. Helv. Chim. Acta 1966, 49, 1199.
The authors thank the Ministere de l’Education Natio-
nale, de la Recherche et de la Technologie for fellow-
ships (G.P.; M.-H. T.-B.) and Institut de Recherche
International Servier for a fellowship (M.B.) and finan-
cial support.