First synthesis and pharmacological evaluation of benzoindolizidine and benzoquinolizidine analogues of α- and β-peltatin

Article abstract of DOI:10.1016/S0968-0896(00)00130-9

The benzoindolizidine and-quinolizidine analogues of α- and β-peltatin were designed and synthesized by two different synthetic routes involving as the key step the Bischler-Napieralski cyclization of suitably substituted N-acyl-2-arylmethylpyrrolidine and -piperidine derivatives. The in vitro biological activity of these analogues as well as some of their derivatives was subsequently evaluated. Copyright (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(00)00130-9

Bioorganic & Medicinal Chemistry 8 (2000) 2113±2125
First Synthesis and Pharmacological Evaluation of
Benzoindolizidine and Benzoquinolizidine Analogues of
ꢀ- and ꢁ-Peltatin
Axel Couture,a,* Eric Deniau,a Pierre Grandclaudon,a Stephane Lebrun,a
Stephane Leonce,b Pierre Renardc and Bruno Pfei€erc
a
Â
Laboratoire de Chimie Organique Physique, ESA CNRS 8009, Universite des Sciences et Technologies de Lille,
F-59655 Villeneuve d'Ascq Cedex, France
b
Â
Â
Institut de Recherches Servier, Division de Cancerologie Experimentale, 11 rue des Moulineaux, F-92415 Suresnes, France
c
Â
ADIR, 1 rue Carle Hebert, F-92415 Courbevoie Cedex, France
Received 20 March 2000; accepted 19 May 2000
AbstractÐThe benzoindolizidine and-quinolizidine analogues of a- and b-peltatin were designed and synthesized by two di€erent
synthetic routes involving as the key step the Bischler±Napieralski cyclization of suitably substituted N-acyl-2-arylmethylpyrroli-
dine and -piperidine derivatives. The in vitro biological activity of these analogues as well as some of their derivatives was subse-
quently evaluated. # 2000 Elsevier Science Ltd. All rights reserved.
Introduction
by several groups which have notably designed and syn-
thesized a variety of analogues in which the sp3 carbon is
replaced by a sp2 nitrogen, anticipating con®gurational
integrity and avoiding any problems of epimerization.18 24
Paradoxically, despite the fact that they retain anti-tumour
activities, to our knowledge, such modi®cations have
been mainly con®ned to the aza-analogues of podo-
phyllotoxin 4±61,18 26 (Fig. 1) and the study of the a and
b-peltatin congeners appears to be an unexplored area.
We wish therefore to report in this paper the ®rst synthesis
and the in vitro biological evaluation of hexahydro-
pyrrolo[1,2-b] and hexahydropyrido[1,2-b]isoquinoline
analogues of a and b-peltatin 7±10.
Podophyllotoxin 1 and related compounds, including a
and b-peltatin 2 and 3 (Fig. 1) are a class of cyclolignan
type compounds characterized by several vicinal oxygen
functions on the di€erently substituted aromatic moieties
and/or in the central carbocyclic unit.1 They have been
obtained from the rhizoma resin of Podophyllum peltatum
L2 and have long been known to display anti-tumour3
and mitotoxic activities.4,5 However due to toxic side-
e€ects they have in most cases failed to give satisfactory
clinical trial results.6 9 In fact, the clinical utility of these
compounds is compromised by the highly reactive nature
of the fused g-lactone moiety present in these molecules
and by epimerization at C2 under physiological condi-
tions.10 This has been shown to be deleterious to the
therapeutic action by giving rise to products devoid of
anti-tumour activity.11 Consequently, a number of
groups have carried out modi®cations at this stereogenic
centre and new analogues which are incapable of loss of
con®gurational integrity at the C2 centre, which encom-
pass a broad spectrum of antineoplastic activities and thus
overcome clinical limitations, have been explored.12 17
In particular, ingenious variations have been proposed
Results and Discussion
Chemistry
Two complementary and undoubtedly transposable
methods were adopted for the synthesis of the benzoin-
dolizidine and -quinolizidine analogues of a and b-peltatin
7, 8 and 9, 10 respectively (Fig. 1). The ®rst one which is
depicted in the retrosynthetic Scheme 1 involved as the
key step the formation of the enamides 13, 14 which
could be derived from the Horner reaction between the
appropriate O-benzyl protected aromatic carboxal-
dehyde 15 and the phosphorylated N-acyl pyrrolidine
*Corresponding author. Tel.: +33-(0)3-20-43-44-32; fax: +33-(0)3-
20-33-63-09; e-mail: axel.couture@univ-lille1.fr
0968-0896/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(00)00130-9

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A. Couture et al. / Bioorg. Med. Chem. 8 (2000) 2113±2125
Figure 1.
derivatives 16, 17 respectively. Reduction and subsequent
Bischler±Napieralski cyclization then should complete
the synthesis of the target compounds 7, 8.
The synthesis started with the preparation of the benzyl
protected 4-hydroxybenzo[d][1,3]dioxole-5-carbaldehyde
15. To reach this protected phenol derivative, the
metallation and subsequent hydroxylation of piperonal
was investigated. Critical to the success of this strategy
was the ability to identify a masked carbonyl synthon that
was capable not only of retaining the formyl functionality
but also of directing subsequent lithiation at the `in
between' ring position. The metallation conditions
described by Comins for various aldehydes by using
lithium N,N,N0-trimethylethylenediamine27 were tested on
piperonal but failed. After considerable experimentation
we found that adding 2 equiv of t-butyllithium in pentane
to 1 equiv of imidazolidine 1828 in Et2O at 78 ꢀC and
then quenching with B(OMe)3 at 0 ꢀC followed by
It was obvious that this synthetic strategy would be
fraught with diculties associated with the presence of
several hydroxy phenolic functions at di€erent sites on
the environmentally di€erent aromatic moieties of the
®nal compounds 7, 8. Simple retrosynthetic analysis
indicates that the hydroxy phenolic function at the
western part of the molecule should be incorporated via
the trialkoxybenzaldehyde derivative 15 whereas the
corresponding function on the pendant aromatic unit of
7 should be integrated into the models via the protected
1-aroyl-2-diphenylphosphinoylpyrrolidine derivative 16.

A. Couture et al. / Bioorg. Med. Chem. 8 (2000) 2113±2125
2115
hydrogen peroxide led to the target compound after
regeneration of the formyl functionality and ultimate
benzyl protection (Scheme 2). The second partners, 16 and
17, involved in the Horner reaction were easily obtained
by coupling the phosphorylated amine 20 with the aro-
matic carboxylic acids 22, 23, the former being obtained
by sequential protection-oxidation of syringaldehyde
(Scheme 3). Initially the phosphorylated pyrrolidine 20
was readily obtained by addition of diphenylphosphine
oxide 26 to the triazine 24.29,30 This protocol delivered the
phosphorylated carboxamides 16, 17 in 84 and 78% yield
respectively. Horner reaction between compounds 16, 17
Scheme 1.
Scheme 2.

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A. Couture et al. / Bioorg. Med. Chem. 8 (2000) 2113±2125
Scheme 3.
Scheme 4.

A. Couture et al. / Bioorg. Med. Chem. 8 (2000) 2113±2125
2117
and 15 was then carried out by smooth deprotonation at
78 ꢀC with n-BuLi in THF and subsequent treatment
with the suitably substituted aldehyde 15. Warming the
reaction mixture to room temperature ensured the
completion of the reaction and the N-acylenamides 13,
14 were isolated in high yields (71% and 69% respec-
tively) as a mixture of Z- and E-isomers with E-isomers
predominating (40:60 for 13, 45:55 for 14) (Scheme 4). The
stereochemistry was unambiguously assigned from the 1H
NMR spectra and further con®rmed by a one-dimen-
sional di€erence nuclear Overhauser experiment. Thus, in
contrast to the E-isomer, irradiation of the vinylic proton
of the Z-form led to an increased intensity of the allylic
protons of the pyrrolidine ring.
the reduced di- and monophenolic compounds 11 and
12 with excellent yields (87 and 90% respectively)
(Scheme 4). Compounds 11, 12 were then subjected to
Bischler±Napieralski conditions by standard treatment
with phosphorus oxichloride (POCl3) in toluene at re¯ux
and subsequent reduction with sodium borohydride
(NaBH4) in methanol. Unfortunately all attempts to
obtain the cyclocondensed products by this protocol, even
under mild conditions,23,33 were unrewarding probably
due to the presence of the hydroxy phenolic groups in the
opened compounds, a precedented complicating factor in
related systems.17 Assuming that a reprotection step
might be a prerequisite to the carboannulation reaction
to give the target molecules 7 and 8, compounds 11 and
12 were then fully benzyl protected prior to e€ecting the
Bischler±Napieralski reaction (Scheme 5). Gratifyingly,
this protocol a€orded the expected cyclized products 29
and 30, in 73 and 70% yield respectively, as a mixture of
diastereomers in the ratio 10:1. The structure of the
We then focused on the hydrogenation of the poly-
substituted enamides 13 and 14. The rarely employed
method making use of Pd on C and ammonium for-
mate31,32 could o€er, if feasible, a double advantage by
e€ecting simultaneously the reduction of the enamine
function with concomitant removal of the benzyloxy
protecting group thus giving straightforward access to
direct candidates for the Bischler±Napieralski reaction.
As anticipated, treatment of enamides 13, 14 delivered
1
major diastereoisomer was deduced from the H NMR
spectrum, namely by the presence of long range coupling
between the mono and dibenzylic protons attributable
to their axial relationship.34,35 Final removal of the
benzyl protecting group of the major isomers delivered
Scheme 5.

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A. Couture et al. / Bioorg. Med. Chem. 8 (2000) 2113±2125
the benzoindolizidine analogues of a- and b-peltatin 7 and
8 with very acceptable yields (46 and 45% respectively
over the last three steps).
mixture of Z- and E-isomer (5:95). N-Deprotection of the
enecarbamate 32 with tri¯uoroacetic acid and subsequent
reduction of the transient iminium ion was performed as a
single, one-pot reaction and this procedure a€orded
straightforwardly the 2-arylmethylpiperidine 33 in fairly
good yield. Schotten±Baumann reaction between amine
33 and the appropriate carboxylic acid chlorides 34, 35
furnished the amides 36, 37 candidates for the Bischler±
Napieralski reaction. The O-benzyl protections in 36, 37
survived the cyclization conditions since treatment with
POCl3 followed by NaBH4 delivered the cyclocondensed
products 38 and 39 as a mixture of diastereoisomers
(90:10 for 38 and 95:5 for 39). Final removal of the
benzyl protection in 38, 39 completed the synthesis of
the target products 9, 10.
For the synthesis of the benzoquinolizidine analogues of
a- and b-peltatin 9 and 10 (Fig. 1) we decided to adopt an
alternative strategy which hinges upon the same synthetic
principles but precludes the rather unsatisfactory and
tedious deprotection-reprotection steps. This alternative
approach which is depicted in the Scheme 6 involves as
the key step the formation of the 2-arylmethylpiperidine
derivative 33 readily obtained after deprotection and
subsequent reduction of enecarbamate 32. This N-pro-
tected enamine 32 was derived from the Horner reaction
between the aromatic carboxaldehyde 15 and the phos-
phorylated cyclic carbamate 31. Initially compound 31 was
prepared by treatment of the corresponding phosphory-
lated cyclic amine 21 (Scheme 2) with di-tert-butyldicarbo-
nate.29,30 Horner reaction proceeded uneventfully to a€ord
the N-tert-butoxycarbonyl-2-arylmethylpiperidine 32 as a
Pharmacological evaluation
Eleven analogues of a and b-peltatin and particularly
their urethane derivatives (Scheme 7) were prepared in
Scheme 6.

A. Couture et al. / Bioorg. Med. Chem. 8 (2000) 2113±2125
2119
Scheme 7.
both the hexahydropyrrolo[1,2-b] and in the hexahy-
dropyrido[1,2-b]isoquinoline series and evaluated in vitro
on L1210 leukemia cell line. The results expressed as IC50
(concentration reducing by 50% the cell proliferation) are
reported in Table 1. The perturbation of the cell cycle
induced by the most active compounds were also inves-
tigated on the same cell line (Figure 2 and Table 2).
By contrast, introduction of a para-¯uorophenylcarbamate
group on the b-peltatin analogues has no in¯uence in both
the pyrido[1,2-b]isoquinoline (IC50 (L1210)=9.9 mM ver-
sus 9.5 mM for compound 42 and 10) and the pyrrolo[1,2-
b]isoquinoline (IC50 (L1210)=0.86mM versus 0.6 mM for
compound 41 and 8) series. This is the same thing for
the disubstituted analogue of a-peltatin 40.
Surprisingly, the strict analogue of a-peltatin is more
active in the pyrido[1,2-b]isoquinoline series (IC50
(L1210)=2.3 mM for compound 9 versus 5.4 mM for
compound 7) whilst the strict analogue of b-peltatin is
15 times more potent in the pyrrolo[1,2-b]isoquinoline
series (IC50 (L1210)=0.6 mM for compound 8 versus 9.5
mM for compound 10).
Due to their good cytotoxicity, compounds 8 and 41
were selected for investigation of their interaction with
the cell cycle of the L1210 cells.
Compared to untreated control, both induced a partial
accumulation of the cells on the G2+M and >G2+M
(probably 8N) phases of the cell cycle at 2.5 mM as
reported on Figure 2 and Table 2. Between 65% and
69% of the cells are in the G2M phase of the cell cycle
compared to 24% for untreated control. About 20% are
in the 8N phase (tetraploid cells) compared to 1% for
untreated control. The DNA histogram of compounds 8
and 41 is comparable to that obtained with podo-
phyllotoxin at 0.025 mM. Accumulation of the cell in the
tetraploid state could be compatible with antimitotic
properties.
Without any exception, all the mono or di O-benzylated
intermediates were found inactive or signi®cantly less
active than their deprotected analogues (IC50 between
9.7 and 19.3 mM for compounds 29, 30, 38, 39).
Table 1. Antiproliferative activity of the target compounds 7±10 and
some of their derivatives
Compound
IC50 (mM)a
Podophyllotoxin 1
7
8
0.025
5.4
0.6
Table 2. Typical DNA histogram and distribution into the di€erent
phases of the cell cycle of untreated (light) or treated (heavy) L1210
cells
9
2.3
9.5
9.7
17.3
18.4
19.3
4.1
0.86
9.9
10
29
30
38
39
40
41
42
% of L1210 cells in cell cycle phases
Compound
G2+M (mM)
>G2+M (8N) (mM)
Untreated control
Podophyllotoxin
8
41
24%
1%
55% (0.025)
65% (2.5)
69% (2.5)
26% (0.025)
21% (2.5)
20% (2.5)
aInhibition of L1210 cell proliferation measured by the MTT assay
(mean of at least 2 values obtained in separate experiments).

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A. Couture et al. / Bioorg. Med. Chem. 8 (2000) 2113±2125
Starting materials
Triazines 24,33 2529 and diphenylphosphine oxide 2636
were prepared according to the literature methods. The
phosphorylated amines 20,30 21,29 amides 16, 1729 and
carbamate 3129,30 were synthesized according to already
reported procedures. The benzyl protected aromatic
carboxylic acid 2237 was obtained by oxidation with
Jones reagent of the corresponding aldehyde38 deriving
from syringaldehyde. The carboxylic acid chloride 3439
was easily obtained by treatment of the corresponding
acid 22 with thionyl chloride and was used directly
without further puri®cation.
Phosphorylated amide 16. Prepared in 84% yield as
described;29 mp 153±154 ꢀC; IR (KBr) n: 1646 (CO) and
1
1
1161 cm (PO); H NMR: d 1.79±1.94 (m, 1H, CH2),
1.96±2.17 (m, 1H, CH2), 2.18±2.33 (m, 1H, CH2), 2.47±
2.63 (m, 1H, CH2), 3.33±3.45 (m, 2H, NCH2), 3.72 (s, 6H,
OCH3), 4.95 (s, 2H, OCH2Ph), 5.43±5.52 (m, 1H, CH-P),
6.40 (s, 2H, Harom), 7.21±7.43 (m, 7H, Harom), 7.48±7.58
(m, 4H, Harom), 7.79±7.91 (m, 2H, Harom), 8.04±8.16 (m,
2H, Harom); 13C NMR: d C 170.0 (CO), 153.1, 137.4,
132.2, 131.8, 131.1, 130.5 (d, JCP=92.5 Hz), CH 131.4 (d,
JCP=9Hz), 128.8 (d, JCP=11.5 Hz), 128.7, 128.4, 128.1,
128.0 (d, JCP=8Hz), 104.6, 56.1 (d, JCP=71.5 Hz), CH2
74.9, 50.7, 25.6, 24.9, CH3 56.1; 31P NMR: d 34.6. Anal.
calcd for C32H32NO5P: C, 70.97; H, 5.96; N, 2.59. Found:
C, 71.12; H, 5.88; N, 2.63.
Phosphorylated amide 17. Prepared in 78% yield as
described;29 mp 148±149 ꢀC; IR (KBr) n: 1638 (CO) and
1
1
1168 cm (PO); H NMR: d 1.82±1.94 (m, 1H, CH2),
1.97±2.15 (m, 1H, CH2), 2.19±2.30 (m, 1H, CH2), 2.45±
2.60 (m, 1H, CH2), 3.31±3.59 (m, 2H, NCH2), 3.73 (s,
3H, OCH3), 3.76 (s, 3H, OCH3), 3.82 (s, 3H, OCH3),
5.53±5.56 (m, 1H, CH-P), 6.28 (s, 2H, Harom), 7.31±7.45
(m, 3H, Harom), 7.47±7.55 (m, 3H, Harom), 7.82±7.90 (m,
2H, Harom), 8.05±8.11 (m, 2H, Harom); 13C NMR: d C
170.1 (CO), 152.8, 146.3, 131.5 (d, JCP=94 Hz), 130.6,
CH 131.8 (d, JCP=8 Hz), 131.4 (d, JCP=9 Hz), 128.9 (d,
JCP=9 Hz), 128.2 (d, JCP=10 Hz), 104.6, 56.0 (d,
JCP=79 Hz), CH2 50.6, 25.5, 24.9, CH3 60.8, 55.9; 31P
NMR: d 34.6. Anal. calcd for C26H28NO5P: C, 67.09;
H, 6.02; N, 3.01. Found: C, 67.18; H, 5.93; N, 2.88.
Figure 2. Typical DNA histogram and distribution into the di€erent
phases of the cell cycle of untreated (light) or treated (heavy) L1210 cells.
Experimental
General information
Synthesis of the benzaldehyde derivative 15. The synth-
esis of benzaldehyde 15 was carried out as follows. A
solution of piperonal (5 g, 33.3 mmol) and N,N0-dime-
thylethylenediamine (3.22 g, 36.6 mmol) in toluene
(100 mL) was re¯uxed for 5 h. The resulting mixture was
washed with H2O. The organic layer was dried over
MgSO4 and the solvent was removed in vacuo to leave an
oil which was puri®ed by distillation under reduced pres-
sure to a€ord the imidazolidine 18 (5.8 g, 79%, bp 108 ꢀC/
5 10 3 torr).
Methanol was distilled from magnesium turnings. Tetra-
hydrofuran (THF) and ether (Et2O) were pre-dried with
anhydrous Na2SO4 and distilled over sodium benzophe-
none ketyl under Ar before use. CH2Cl2, NEt3, toluene
were distilled from CaH2. Dry glassware was obtained by
oven-drying and assembly under dry Ar. The glassware
was equipped with rubber septa and reagent transfer
was performed by syringe techniques. For ¯ash chro-
matography, Merck silica gel 60 (230-400 mesh ASTM)
was used. The melting points were taken on a Reichert-
Thermopan apparatus and are not corrected. Unless
otherwise stated, proton, carbon and phosphorus NMR
spectra were taken with CDCl3 as solvent at 300, 75 and
121 MHz respectively on a Bruker AM 300 spectro-
meter. Microanalyses were performed by the CNRS
microanalysis centre.
Imidazolidine 18. 1H NMR: d 2.15 (s, 6H, NCH3), 2.46±
2.53 (m, 2H, NCH2), 3.14 (s, 1H, NCH), 3.32±3.38 (m,
2H, NCH2), 5.92 (s, 2H, OCH2O), 6.73 (d, J=7.8 Hz,
1H, Harom), 6.81 (dd, J=7.8, 1.6 Hz, 1H, Harom), 6.99
(d, J=1.6 Hz, 1H, Harom); 13C NMR: d C 147.9, 147.8,
133.8, CH 122.5, 108.4, 107.5, 92.1, CH2 100.9, 53.1,

A. Couture et al. / Bioorg. Med. Chem. 8 (2000) 2113±2125
2121
CH3 39.4. Anal. calcd for C12H16N2O2: C, 65.43; H,
7.32; N, 12.72. Found: C, 65.56; H, 7.31; N, 12.61.
138.1, 137.5, 136.1, 130.9, 130.5, 123.3, CH 128.6, 128.4,
128.3, 128.2 127.8, 127.7, 123.3, 108.0, 104.5, 102.7,
CH2 100.7, 74.9, 73.0, 48.6, 32.2, 20.7, CH3 55.6. Anal.
calcd for C35H33NO7: C, 72.54; H, 5.70; N, 2.42. Found:
C, 72.36; H, 5.58; N, 2.63.
To a stirred solution of imidazolidine 18 (2 g, 9.1 mmol)
in Et2O (50 mL) was added t-BuLi (10.7 mL, 1.7 M in
pentane, 18.2mmol) with stirring under Ar at 78ꢀC. The
solution was stirred at 78ꢀC for 15min, then warmed to
rt over a period of 30min and re-cooled to 78ꢀC. A
solution of trimethyl borate (0.62 g, 10.9 mmol) was added
dropwise by syringe and the mixture was warmed to 0 ꢀC
over a period of 1 h. Glacial AcOH (0.5 mL) was added
followed by hydrogen peroxide (30%, 1.2 mL) and the
reaction mixture was stirred at room temperature for 16h.
The crude product was then poured onto an aqueous 6N
HCl solution (50 mL) and the mixture was stirred for an
additional 30 min. The aqueous layer was extracted
twice with CH2Cl2 (2Â30 mL) and the organic extract
subsequently washed with aqueous NaOH (10%,
2Â40 mL). Acidi®cation with dilute HCl was followed
by extraction with CH2Cl2 (2Â50 mL). The organic
layer was then washed with water and brine and dried
(MgSO4). The dried extract was concentrated in vacuo
to a€ord 19 (1.08 g, 70%) as a yellow solid, mp 115±
116 ꢀC (lit.:40 115±116 ꢀC). Benzylation of 19 under
standard procedure furnished quantitatively the benzyl
protected benzaldehyde derivative 15.41
N-Acyl enamine 14. (69%); (E)-Isomer, mp 116±117 ꢀC;
1H NMR: d 1.79±1.88 (m, 2H, CH2), 2.72 (td, J=7.3,
2.2 Hz, 2H,=CCH2), 3.69 (t, J=6.8 Hz, 2H, NCH2), 3.82
(s, 6H, OCH3), 3.84 (s, 3H, OCH3), 5.18 (s, 2H, OCH2Ph),
5.92 (s, 2H, OCH2O), 6.49 (d, J=8.1 Hz, 1H, Harom), 6.65
(d, J=8.1 Hz, 1H, Harom), 6.74 (s, 2H, Harom), 7.32±7.46
(m, 6H, 5Harom+Hvinyl); 13C NMR: d C 169.8 (CO),
153.1, 147.7, 139.9, 139.8, 137.2, 133.6, 130.5, 128.6, 124.8,
CH 128.3, 128.0, 127.9, 121.8, 108.9, 104.5, 102.7, CH2
101.0, 73.6, 50.9, 30.2, 22.4, CH3 60.9, 56.2; (Z)-isomer,
mp 96±97 ꢀC; 1H NMR: d 1.92±2.13 (m, 2H, CH2), 2.64 (t,
J=7.0 Hz, 2H,=CCH2), 3.63 (s, 6H, OCH3), 3.82 (s, 3H,
OCH3), 3.84 (t, J=7.3 Hz, 2H, NCH2), 4.95 (s, 2H,
OCH2Ph), 5.67 (s, 1H, Hvinyl), 5.83 (s, 2H, OCH2O), 6.33
(s, 2H, Harom), 6.37 (d, J=8.0 Hz, 1H, Harom), 6.53 (d,
J=8.0 Hz, 1H, Harom), 7.32±7.41 (m, 5H, Harom); 13C
NMR: d C 169.8 (CO), 152.8, 147.8, 145.4, 141.8, 136.1,
133.7, 131.0, 130.1, 129.5, CH 127.5, 121.1, 112.9, 108.0,
104.7, 102.6, CH2 100.6, 73.7, 49.8, 31.9, 21.1, CH3 60.8,
56.3. Anal. calcd for C29H29NO7: C, 69.17; H, 5.80; N,
2.78. Found: C, 68.91; H, 5.89; N, 2.60.
Synthesis of the N-acyl enamines 13, 14. A solution of n-
BuLi (1.6 M in hexanes, 1.7 mL, 2.75 mmol) was added
dropwise to a solution of the phosphorylated amide 16,
17 (2.5 mmol) in THF (30 mL) at 78 ꢀC with stirring
under Ar. The orange solution was stirred for an addi-
tional 15 min and a solution of the appropriate aldehyde
15 (2.5 mmol) in THF (5 mL) was then added slowly.
After being stirred at 78 ꢀC for 15 min the reaction
mixture was allowed to come to room temperature over
2 h. Aqueous NH4Cl was added and the organic layer was
separated, rinsed with brine, dried (MgSO4) and con-
centrated to dryness. The crude products were analyzed
by 1H NMR in order to determine the E/Z-isomers ratio
and compounds 13, 14 (E- and Z-isomers) were sepa-
rated by ¯ash column chromatography using AcOEt:
hexane (60:40) as eluent and ®nally recrystallized from
hexane-toluene.
Synthesis of the N-acyl pyrrolidine derivatives 11, 12. A
suspension of compounds 13, 14 (2 mmol) in methanol
(30 mL) was stirred with activated Pd/C (10%, 20 mg)
and a solution of HCOONH4 (640 mg, 10 mmol) in dis-
tilled water (5 mL) was slowly added. The reaction mix-
ture was re¯uxed for 2 h, ®ltered on Celite1 and water
was added. Extraction with CH2Cl2 (3Â20 mL), drying
over MgSO4 and concentration in vacuo left an oily
product which was puri®ed by ¯ash chromatography
using AcOEt:hexane (1:1) as eluent.
N-Acyl pyrrolidine 11. (87%); mp 168±169 ꢀC; 1H NMR:
d 1.75±1.87 (m, 2H, CH2), 1.93±2.17 (m, 2H, CH2), 2.36
(dd, J=13.9, 9.6 Hz, 1H, CH2Ar), 3.44 (d, J=13.9 Hz,
1H, CH2Ar), 3.55±3.66 (m, 2H, NCH2), 3.87 (s, 6H,
OCH3), 3.96±4.05 (m, 1H, NCH), 5.93 (s, 2H, OCH2O),
6.31 (d, J=7.8 Hz, 1H, Harom), 6.48 (d, J=7.8 Hz, 1H,
Harom), 6.84 (s, 2H, Harom); 13C NMR: d C 170.4 (CO),
148.0, 146.7, 140.8, 137.1, 134.9, 126.5, 99.8, CH 121.9,
120.0, 105.1, 59.5, CH2 101.2, 51.0, 36.8, 31.3, 25.0, CH3
56.5. Anal. calcd for C27H23NO7: C, 62.84; H, 5.73; N,
3.49. Found: C, 62.92; H, 5.79; N, 3.39.
N-Acyl enamine 13. (71%); (E)-Isomer, mp 125±126 ꢀC;
1H NMR: d 1.78±1.91 (m, 2H, CH2), 2.73 (td, J=7.6,
1.9 Hz, 2H,=CCH2), 3.70 (t, J=7.0 Hz, 2H, NCH2), 3.79
(s, 6H, OCH3), 5.02 (s, 2H, OCH2Ph), 5.17 (s, 2H,
OCH2Ph), 5.92 (s, 2H, OCH2O), 6.52 (d, J=8.1 Hz, 1H,
Harom), 6.67 (d, J=8.1 Hz, 1H, Harom), 6.77 (s, 2H,
Harom), 7.25±7.51 (m, 11H, 10Harom+Hvinyl); 13C NMR: d
C 169.1 (CO), 153.4, 147.7, 139.9, 139.7, 138.1, 137.5,
137.2, 133.0, 130.6, 124.9, CH 128.4, 128.3, 128.1, 128.0,
127.9, 127.8, 121.8, 108.9, 104.6, 102.1, CH2 101.0, 75.0,
73.6, 50.4, 30.1, 22.4, CH3 56.2; (Z)-Isomer, mp 117±
N-Acyl pyrrolidine 12. (90%); mp 185±186 ꢀC; 1H NMR:
d 1.68±1.79 (m, 2H, CH2), 1.83±2.01 (m, 2H, CH2), 2.33
(dd, J=14.1, 9.5 Hz, 1H, CH2Ar), 3.35 (d, J=14.1 Hz,
1H, CH2Ar), 3.42±3.51 (m, 2H, NCH2), 3.79 (s, 6H,
OCH3), 3.85 (s, 3H, OCH3), 3.75±3.83 (m, 1H, NCH),
5.89 (s, 2H, OCH2O), 6.25 (d, J=8.0 Hz, 1H, Harom),
6.41 (d, J=8.0 Hz, 1H, Harom), 6.80 (s, 2H, Harom); 13C
NMR: d C 170.0 (CO), 147.8, 146.2, 140.4, 137.9, 134.2,
127.1, 100.0, CH 122.3, 121.6, 104.9, 58.9, CH2 100.9,
50.1, 37.5, 29.6, 23.2, CH3 56.8. Anal. calcd for
C22H25NO7: C, 63.61; H, 6.07; N, 3.37. Found: C,
63.50; H, 6.18; N, 3.31.
118 ꢀC; H NMR: d 1.92±2.20 (m, 2H, CH2), 2.63 (t,
1
J=7.4 Hz, 2H,=CCH2), 3.61 (s, 6H, OCH3), 3.78 (t,
J=7.1 Hz, 2H, NCH2), 4.94 (s, 2H, OCH2Ph), 4.98 (s,
2H, OCH2Ph), 5.69 (s, 1H, Hvinyl), 5.78 (s, 2H, OCH2O),
6.35 (s, 2H, Harom), 6.38 (d, J=8.1 Hz, 1H, Harom), 6.57
(d, J=8.1 Hz, 1H, Harom), 7.28±7.49 (m, 10H, Harom);
13C NMR: d C 168.6 (CO), 152.4, 147.7, 139.1, 138.6,

2122
A. Couture et al. / Bioorg. Med. Chem. 8 (2000) 2113±2125
Synthesis of the benzylated compounds 27, 28. Com-
pounds 27, 28 were prepared by benzylation of the par-
ent phenolic compounds 11, 12 under standard
conditions (BnBr, KOH, MeOH).
acetone:petroleum ether (1:1) as eluent and by recrys-
tallization from Et2O:hexane.
Compound 29 (major isomer). Mp 122±123 ꢀC; 1H NMR:
d 1.59±1.87 (m, 3H, CH2), 2.03±2.16 (m, 2H, CH2), 2.34±
2.56 (m, 2H, 1H NCH2+1H CH2Ar), 2.87 (td, J=9.4,
2.4 Hz, 1H, NCH2), 3.12 (dd, J=15.9, 2.4 Hz, 1H, NCH),
3.78 (s, 6H, OCH3), 4.05 (s, 1H, NCHAr), 5.03 (s, 2H,
OCH2Ph), 5.29 (dd, J=16.8, 11.7Hz, 1H, OCH2Ph), 5.85
(dd, J=7.6, 1.3 Hz, 2H, OCH2O), 5.96 (s, 1H, Harom), 6.52
(s, 2H, Harom), 7.27±7.51 (m, 10H, Harom); 13C NMR: d C
153.4, 147.0, 139.7, 139.0, 138.0, 137.7, 136.0, 134.4, 133.5,
121.1, CH 128.5, 128.4, 128.0, 127.9, 127.8, 127.3, 106.2,
102.3, 72.3, 60.8, CH2 100.6, 74.9, 73.2, 53.8, 31.2, 30.7,
21.3, CH3 56.2. Anal. calcd for C35H35NO6: C, 74.34;
H, 6.19; N, 2.48. Found: C, 74.18; H, 6.03; N, 2.55.
1
Compound 27. (74%); oil; H NMR: d (mixture of two
rotational isomers A and B, 70:30) 1.51±1.98 (m, 4H,
CH2), 2.31±2.46 (m, 3H, CH2Ar+1H NCH2, B), 2.59±
2.66 (m, 1H, NCH2, B), 2.88 (dd, J=13.2, 7.9 Hz, 1H,
CH2Ar, A), 3.15 (dd, J=13.2, 3.7 Hz, 1H, CH2Ar, A),
3.26±3.38 (m, 2H, NCH2, A), 3.67 (s, 6H, OCH3, B),
3.74 (s, 6H, OCH3, A), 4.32±4.39 (m, 1H, NCH, B),
4.46±4.57 (m, 1H, NCH, A), 5.01 (dd, J=15.9, 10.8 Hz,
4H, OCH2Ph, A), 5.26 (dd, J=14.9, 11.3 Hz, 4H,
OCH2Ph, B), 5.86 (s, 2H, OCH2O, B), 5.92 (s, 2H,
OCH2O, A), 6.14 (d, J=7.7 Hz, 1H, Harom, B), 6.36 (d,
J=7.7 Hz, 1H, Harom, B), 6.41±6.53 (m, 2H, Harom, A),
6.73 (s, 2H, Harom, A), 6.75 (s, 2H, Harom, B), 7.18±7.49
(m, 10H, Harom); 13C NMR: d (mixture of two rota-
tional isomers A and B, 70:30) C 169.6 (CO), 153.2,
148.2, 147.7, 141.6, 137.6, 137.4, 132.9, 132.8, 129.4, CH
128.9, 128.1, 128.0, 127.9, 127.4, 124.5, 123.9, 123.2,
104.8, 104.2, 102.9, 102.6, 58.3 (A), 57.1 (B), CH2 100.9
(A), 100.2 (B), 75.0, 73.6, 50.4 (A), 45.6 (B), 34.9 (B),
32.1 (A), 29.3 (B), 29.2 (A), 24.8 (A), 22.7 (B), CH3 56.2
(A), 55.2 (B). Anal. calcd for C35H35NO7: C, 71.79; H,
5.98; N, 2.39. Found: C, 71.66; H, 6.09; N, 2.51.
Compound 30 (major isomer). Mp 172±173 ꢀC; 1H
NMR: d 1.51±1.89 (m, 3H, CH2), 2.01±2.17 (m, 2H,
CH2), 2.33±2.56 (m, 2H), 2.86 (t, J=7.9 Hz, 1H), 3.09 (d,
J=14.9 Hz, 1H, NCH), 3.82 (s, 6H, OCH3), 3.85 (s, 3H,
OCH3), 4.04 (s, 1H, NCHAr), 5.28 (dd, J=16.8, 11.2 Hz,
2H, OCH2Ph), 5.84 (d, J=5.6 Hz, 2H, OCH2O), 5.94 (s,
1H, Harom), 6.52 (s, 2H, Harom), 7.32±7.48 (m, 5H,
Harom); 13C NMR: d C 153.1, 147.0, 139.6, 139.0, 137.6,
137.1, 134.4, 133.4, 121.2, CH 128.4, 128.0, 127.8, 106.0,
102.2, 72.2, 60.8, CH2 100.7, 73.1, 53.8, 31.2, 30.7, 21.2,
CH3 60.9, 56.2. Anal. calcd for C29H31NO6: C, 71.16; H,
6.34; N, 2.86. Found: C, 71.39; H, 6.51; N, 2.73.
ꢀ
1
Compound 28. (80%); mp 61±62 C; H NMR: d (mix-
ture of two rotational isomers A and B, 77:23) 1.46±2.05
(m, 4H, CH2), 2.28±2.43 (m, 3H, CH2Ar+1H NCH2,
B), 2.52±2.64 (m, 1H, NCH2, B), 2.84 (dd, J=13.2,
8.0 Hz, 1H, CH2Ar, A), 3.09 (dd, J=13.2, 3.4 Hz, 1H,
CH2Ar, A), 3.17±3.35 (m, 2H, NCH2, A), 3.56±3.61 (m,
1H, NCH, B), 3.71 (s, 3H, OCH3), 3.78 (s, 6H, OCH3),
4.34±4.52 (m, 1H, NCH, A), 4.81 (dd, J=11.9 Hz, 2H,
OCH2Ph, A), 5.58 (dd, J=15.9, 11.6 Hz, 2H, OCH2Ph,
B), 5.78 (s, 2H, OCH2O, B), 5.81 (s, 2H, OCH2O, A),
6.13 (d, J=7.6 Hz, 1H, Harom, B), 6.29 (d, J=7.6 Hz,
1H, Harom, B), 6.33±6.47 (m, 2H, Harom, A), 6.66 (s, 2H,
Harom, B), 6.68 (s, 2H, Harom, A), 7.13±7.45 (m, 5H,
Harom); 13C NMR: d (mixture of two rotational isomers
A and B, 77:23) C 168.9 (CO), 152.4, 147.5, 140.6,
136.9, 136.7, 134.8, 132.4, 132.3, CH 128.4, 127.9, 127.3,
123.8 (A), 123.4 (B), 107.7 (A), 107.6 (B), 102.9 (A),
102.2 (B), 59.1 (B), 58.2 (A), CH2 101.6 (B), 100.9 (A),
73.6 (A), 72.6 (B), 50.4 (A), 45.2 (B), 35.3 (B), 32.1 (A),
29.7 (B), 29.1 (A), 24.8 (A), 22.1 (B), CH3 60.8, 56.2.
Anal. calcd for C29H31NO7: C, 68.91; H, 6.14; N, 2.77.
Found: C, 68.78; H, 6.18; N, 2.90.
Synthesis of the benzoindolizidine analogues of ꢀ- and
ꢁ-peltatin 7, 8. A suspension of compounds 29, 30
(major isomer, 0.5 mmol) and Pd/C (10%, 15mg) in dry
MeOH (20 mL) was treated with a solution of ammonium
formate (320mg, 5 mmol) in distilled water (3 mL). The
mixture was re¯uxed for 2 h, ®ltered on Celite1 and con-
centrated in vacuo. CH2Cl2 (30 mL) and water (30 mL)
were added, the organic layer was rinsed with brine and
dried (Na2SO4). The crude residue was puri®ed by recrys-
tallization from EtOH to a€ord compounds 7, 8 as pale
yellow crystals.
Compound 7. (85%); mp 270±271 ꢀC; 1H NMR (d6-
DMSO): d 1.51±1.85 (m, 3H, CH2), 2.00±2.21 (m, 2H,
CH2), 2.31±2.75 (m, 3H, NCH2+1H CH2Ar), 3.17 (d,
J=15.8 Hz, 1H, NCH), 3.77 (s, 6H, OCH3), 4.04 (s, 1H,
NCHAr), 5.88 (s, 2H, OCH2O), 6.01 (s, 1H, Harom),
6.50 (s, 2H, Harom); 13C NMR: d C 153.3, 146.7, 139.0,
138.2, 135.4, 134.2, 132.8, 99.3, CH 118.1, 105.8, 72.0,
60.3, CH2 100.8, 53.4, 30.9, 30.5, 21.0, CH3 56.1. Anal.
calcd for C21H23NO6: C, 65.45; H, 5.97; N, 3.64. Found:
C, 65.56; H, 5.89; N, 3.80.
Synthesis of the cyclocondensed products 29, 30. A mix-
ture of compound 27, 28 (2 mmol), POCl3 (1.53 g,
10mmol) in dry toluene (30 mL) was re¯uxed for 6 h under
Ar with stirring. The solvent and excess reagent were
removed under vacuum and the residue was dissolved in
dry methanol (20 mL). Sodium borohydride (0.38 g,
10 mmol) was then added portionwise until pH 9,
AcOEt (50 mL) was added and the organic layer washed
with aqueous NaOH (10%) and dried (MgSO4). Eva-
poration of the solvent left an oily residue which was
Compound 8. (80%); mp 257±258 ꢀC; 1H NMR (d6-
DMSO): d 1.51±1.86 (m, 3H, CH2), 2.05±2.19 (m, 2H,
CH2), 2.28±2.50 (m, 2H), 2.68±281 (m, 1H), 2.97±3.02
(m, 1H, NCH), 3.81 (s, 6H, OCH3), 3.84 (s, 3H, OCH3),
4.07 (s, 1H, NCHAr), 5.68 (s, 1H, Harom), 5.86 (s, 1H,
OCH2O), 6.56 (s, 2H, Harom); 13C NMR: d C 152.7,
145.9, 139.6, 137.2, 136.1, 132.2, 99.0, CH 117.9, 105.7,
70.9, 60.1, CH2 100.4, 53.1, 30.8, 30.1, 20.8, CH3 60.0,
55.8. Anal. calcd for C22H25NO6: C, 66.16; H, 6.27; N,
3.51. Found: C, 66.01; H, 6.36; N, 3.62.
1
analyzed by H NMR spectroscopy. Major isomer was
®nally puri®ed by ¯ash column chromatography using

A. Couture et al. / Bioorg. Med. Chem. 8 (2000) 2113±2125
2123
Synthesis of the enecarbamate 32. Enecarbamate 32 was
prepared by metallation of the N-protected phosphory-
lated piperidine 3130 and subsequent treatment with aro-
matic aldehyde 15 as already described for enamides 13,
14 and obtained in 80% yield as a mixture of (E)- and (Z)-
isomers (95:5). (E)-Isomer; mp 101±102 ꢀC; 1H NMR
(C6D6): d 1.27±1.38 (m, 4H, CH2), 1.42 (s, 9H, CH3),
2.21±2.27 (m, 2H, CH2), 3.51±3.58 (m, 2H, CH2), 5.17 (s,
2H, OCH2Ph), 5.37 (s, 2H, OCH2O), 6.44 (d, J=8.0Hz,
1H, Harom), 6.60 (s, 1H, Hvinyl), 6.67 (d, J=8.0Hz, 1H,
Harom), 7.02±7.18 (m, 3H, Harom), 7.33±7.37 (m, 2H,
Harom); 13C NMR (C6D6): d C 154.3, 148.9, 141.0, 139.7,
138.2, 124.1, 79.2, CH 128.6, 128.3, 123.5, 120.5, 103.1,
CH2 101.1, 73.8, 47.1, 28.3, 26.0, 25.6, CH3 28.5. Anal.
calcd for C25H29NO5: C, 70.90; H, 6.90; N, 3.31. Found:
C, 70.96; H, 6.78; N, 3.26.
3.79 (s, 6H, OCH3), 3.82 (s, 3H, OCH3), 5.51 (s, 2H,
OCH2Ph), 5.91 (s, 2H, OCH2O), 6.18 (s, 1H, Harom),
6.67 (s, 2H, Harom), 7.12±7.39 (m, 5H, Harom); 13C
NMR: d C 169.1 (CO), 152.1, 147.8, 140.7, 137.1, 134.5,
132.1, 131.7, CH 128.5, 128.0, 127.5, 123.9, 107.5, 55.1,
CH2 100.9, 73.1, 45.2, 35.3, 30.9, 25.3, 19.2, CH3 60.8,
56.3. Anal. calcd for C30H33NO7: C, 69.35; H, 6.40; N,
2.70. Found: C, 69.13; H, 6.36; N, 2.64.
Synthesis of the cyclocondensed products 38, 39. The
Bischler±Napieralski cyclization reaction of amides 36,
37 was performed as previously described for the corre-
sponding pyrrolidine derivatives 27, 28 to give 38, 39 in
76 and 71% yield respectively.
ꢀ
1
Compound 38 (major isomer). Mp 149±150 C; H NMR:
d 1.23±1.88 (m, 7H, CH2), 2.24 (br. t, J=9.7 Hz, 1H,
CH2), 2.47 (dd, J=16.4, 11.0 Hz, 1H, CH2), 2.79±2.88
(m, 2H, CH2), 3.77 (s, 6H, OCH3), 3.96 (s, 1H, NCH),
5.00 (s, 2H, OCH2Ph), 5.27 (dd, J=16.0, 11.7 Hz, 2H,
OCH2Ph), 5.82 (dd, J=5.2, 1.2 Hz, 2H, OCH2O) 5.87
(s, 1H, Harom), 6.50 (s, 2H, Harom), 7.25±7.49 (m, 10H,
Harom); 13C NMR: d C 153.4, 146.8, 140.5, 138.3, 138.0,
137.6, 134.0, 132.3, 127.7, 119.9, CH 128.5, 128.4, 128.0,
127.8, 106.4, 102.3, 72.6, 57.8, CH2 100.5, 74.9, 73.1,
54.1, 34.0, 31.8, 26.0, 24.4, CH3 56.2. Anal. calcd for
C36H37NO6: C, 74.59; H, 6.43; N, 2.42. Found: C,
74.65; H, 6.32; N, 2.30.
Synthesis of the 2-arylmethylpiperidine derivative 33.
Tri¯uoroacetic acid (1.8 mL, 23 mmol) was added
under Ar with stirring to a solution of the enecarbamate
32 (0.973 g, 2.3 mmol) in anhydrous CH2Cl2 (5 mL) and
stirring was maintained overnight. Evaporation of the
solvent and excess reagent under vacuum left a residue
which was dissolved in anhydrous MeOH (50 mL).
Sodium borohydride (885 mg, 23.3 mmol) was then
added portionwise at 0 ꢀC and the reaction mixture was
stirred at this temperature for an additional 1 h. EtOAc
(60 mL) was then added and the organic solution was
washed twice with aqueous NH4OH (2Â50mL) then
with water and brine and ®nally dried over Na2SO4. The
solvent was removed under vacuum to leave an oily resi-
due of the compound 33 (0.56 g, 75%) which was used
Compound 39 (major isomer). Mp 173±174 ꢀC; H NMR:
1
d 1.26±1.85 (m, 7H, CH2), 2.26 (t, J=10.5 Hz, 1H,
CH2), 2.45 (dd, J=16.1, 10.5 Hz, 1H, CH2), 2.75±2.92
(m, 2H, CH2), 3.82 (s, 6H, OCH3), 3.84 (s, 3H, OCH3),
3.96 (s, 1H, NCH), 5.27 (dd, J=16.2, 11.6 Hz, 2H,
OCH2Ph), 5.80 (s, 2H, OCH2O), 5.91 (s, 1H, Harom),
6.52 (s, 2H, Harom), 7.26±7.44 (m, 5H, Harom); 13C
NMR: d C 153.0, 146.8, 140.5, 138.3, 137.6, 136.3,
134.0, 132.2, 119.9, CH 128.4, 128.0, 127.8, 106.2, 102.3,
72.5, 57.7, CH2 100.5, 73.1, 54.1, 34.0, 31.7, 26.0, 24.4,
CH3 60.8, 56.1. Anal. calcd for C30H33NO6: C, 71.55;
H, 6.61; N, 2.78. Found: C, 71.61; H, 6.69; N, 3.73.
1
without further puri®cation; H NMR (C6D6): d 1.61±
1.80 (m, 7H, CH2+NH), 2.63±2.81 (m, 2H, CH2), 2.92±
2.98 (m, 1H, CH2), 3.21 (br. t, J=6.7 Hz, 1H, CH2), 3.83
(dd, J=8.9, 3.1 Hz, 1H, CH), 5.23 (s, 2H, OCH2Ph), 5.89
(s, 2H, OCH2O), 6.46 (d, J=7.9 Hz, 1H, Harom), 6.62 (d,
J=7.9Hz, 1H, Harom), 7.21±7.48 (m, 5H, Harom); 13C
NMR (C6D6): d C 147.8, 140.5, 137.5, 128.6, 125.9, CH
128.3, 128.0, 127.8, 123.0, 102.8, 59.5, CH2 100.8, 73.4,
45.9, 36.1, 31.1, 25.5, 24.7. Anal. calcd for C20H23NO3: C,
73.82; H, 7.12; N, 4.30. Found: C, 73.63; H, 6.98; N, 4.44.
Synthesis of the benzoquinolizidine analogues of ꢀ- and
ꢁ-peltatin 9, 10. The ®nal deprotection of the annu-
lated compounds 38, 39 (major isomer) was carried out
as described previously for the indolizidine analogues.
Synthesis of the N-acyl piperidines 36,37. The Schotten±
Baumann reaction between amine 33 and the appro-
priate carboxylic acid chlorides 34, 35 was carried out
under standard conditions.
Compound 9. (87%); mp 290±291 ꢀC; H NMR: d 1.50±
1
1
N-Acyl piperidine 36. (78%); yellow oil; H NMR: d
2.10 (m, 7H, CH2), 2.85±3.18 (m, 3H, CH2), 3.46±3.52
(m, 1H, CH2), 3.75 (s, 6H, OCH3), 3.83 (s, 1H, NCH),
5.50 (s, 1H, Harom), 5.91 (s, 2H, OCH2O), 6.95 (s, 2H,
Harom); 13C NMR: d C 146.5, 136.7, 136.1, 133.4, 126.8,
124.8, 98.0, CH 115.5, 106.1, 70.8, 60.8, CH2 100.9,
52.0, 30.4, 28.2, 22.4, 21.7, CH3 56.1. Anal. calcd for
C23H27NO6: C, 66.15; H, 6.31; N, 3.51. Found: C,
66.21; H, 6.36; N, 3.40.
1.30±1.96 (m, 6H, CH2), 2.43±2.57 (m, 2H, CH2), 2.94
(dd, J=13.5, 8.0 Hz, 1H, CH2), 3.16±3.31 (m, 2H, CH2),
3.86 (s, 6H, OCH3), 5.45 (s, 2H, OCH2Ph), 5.50 (s, 2H,
OCH2Ph), 5.90 (s, 2H, OCH2O), 6.21 (s, 1H, Harom), 6.72
(s, 2H, Harom), 7.15±7.43 (m, 10H, Harom); 13C NMR: d C
169.2 (CO), 153.4, 148.1, 147.6, 140.3, 138.0, 137.1, 134.2,
132.1, 131.8, 129.5, CH 129.0, 128.2, 128.1, 128.0, 127.5,
124.5, 107.7, 55.2, CH2 100.8, 74.1, 73.2, 45.4, 36.0, 31.1,
25.4, 19.3, CH3 56.2. Anal. calcd for C36H37 NO7: C,
72.59; H, 6.26; N, 2.35. Found: C, 72.71; H, 6.46; N, 2.17.
Compound 10. (90%); mp 308±309 ꢀC; 1H NMR: d
1.56±2.18 (m, 7H, CH2), 2.78±3.09 (m, 3H, CH2), 3.35±
3.54 (m, 1H, CH2), 3.69 (s, 3H, OCH3), 3.77 (s, 6H,
OCH3), 3.81 (s, 1H, NCH), 5.49 (s, 1H, Harom), 5.92 (s,
2H, OCH2O), 7.02 (s, 2H, Harom); 13C NMR: d C 146.4,
136.8, 136.1, 133.6, 126.6, 125.0, 98.2, CH 115.6, 106.2,
N-Acyl piperidine 37. (75%); yellow oil; 1H NMR: d
1.34±1.98 (m, 6H, CH2), 2.40±2.56 (m, 2H, CH2), 2.96
(dd, J=13.4, 8.1 Hz, 1H, CH2), 3.12±3.26 (m, 2H, CH2),

2124
A. Couture et al. / Bioorg. Med. Chem. 8 (2000) 2113±2125
70.8, 60.8, CH2 100.9, 52.0, 34.0, 28.2, 22.4, 21.7, CH3
56.1. Anal. calcd for C23H27NO6: C, 66.81; H, 6.58; N,
3.39. Found: C, 66.74; H, 6.65; N, 3.36.
the compounds, then ®xed 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 ¯ow cytometer (Bruker, France) using an
argon laser (Spectra-Physics) emitting 400 mW at
488 nM. The ¯uorescence of propidium iodide was
collected through a 615 nM long-pass ®lter. Data are
displayed as linear histograms and results are expressed
as the percentage of cells found in the G2+M phase of
the cell cycle.
Synthesis of the urethanes 40±42. Urethanes 40±42 were
prepared according to the literature methods.42
Compound 40. (75%); mp 115±116 ꢀC; 1H NMR: d
1.51±1.82 (m, 5H, CH2), 1.96±2.12 (m, 2H, CH2), 2.33±
2.59 (m, 1H, CH2), 2.75±2.97 (m, 1H, CH), 3.81 (s, 6H,
OCH3), 4.11 (s, 1H, NCH), 5.90 (d, J=5.6 Hz, 2H,
OCH2O), 6.15 (s, 1H, Harom), 6.61 (s, 2H, Harom), 6.96±
7.43 (m, 10H, 8Harom+2NH); 13C NMR: d C 156.2 (d,
JCF=249 Hz, CF), 154.1 (d, JCF=249 Hz, CF), 152.7,
150.6, 147.1, 142.2, 137.7, 133.9, 133.2, 133.0, 130.5,
122.5, 120.7, 120.5, CH 116.0, 115.8, 115.7, 115.5, 106.0,
105.7, 72.1, 60.3, CH2 101.9, 53.7, 31.1, 30.2, 21.2, CH3
56.3. Anal. calcd for C35H31F2N3O8: C, 63.73; H, 4.74;
N, 6.37. Found: C, 63.87; H, 4.58; N, 6.15.
References
1. Ramos, A. C.; Pelaez-Lamamie de Clairac, R.; Medarde,
M. Heterocycles 1999, 51, 1443.
2. (a) Kotchetkov, N. K.; Kharlin, A. Y.; Ghizhov, O. G.;
Sheichenko, V. L. Tetrahedron Lett. 1961, 730. (b) Hartwell,
J. L.; Schrecker, A. N. Fortschr. Chem. Org. Naturst. 1958, 15,
83.
3. Horwitz, S. B.; Loike, J. D. Lloydia 1977, 40, 82.
4. Weiss, S. G.; Tin-Wa, M.; Perdue, R. E.; Farnsworth, N.
R. J. Pharm. Sci. 1975, 64, 95.
5. Keller-Juslen, C.; Kuhn, M.; von Wartburg, A.; Stehelin,
H. J. Med. Chem. 1971, 14, 936.
6. Cortese, F.; Bhattacharyya, B.; Wol€, J. J. Biol. Chem.
1977, 252, 1134.
7. King, L. S.; Sullivan, M. S. Science 1946, 104, 244.
8. Jardine, I. In Anticancer Agents Based on Natural Products;
Cassady, J. M.; Douros, J. D., Eds.; Academic: New York,
1980, pp 319±351.
9. Ayres, D. C.; Loike, J. D. Lignans, Chemical, Biological and
Clinical Properties. Cambridge University Press: Cambridge,
1990.
10. Emmenegger, H.; Stahelin, H.; Rutschmann, J.; Renz, J.;
von Wartburg, A. Arzneim. Forsch 1961, 327, 459.
11. Evans, W. E.; Sinkule, J. A.; Horwath, A.; Crom, W. R.;
Dow, L. W.; Riviera, G. Proc. Am. Assoc. Cancer Res. 1981,
690.
Compound 41. (70%); mp 157±158 ꢀC; 1H NMR: d 1.48±
1.79 (m, 5H, CH2), 1.98±2.11 (m, 2H, CH2), 2.36±2.65 (m,
1H, CH2), 2.80±3.03 (m, 1H, CH), 3.82 (s, 6H, OCH3),
3.85 (s, 3H, OCH3), 4.06 (s, 1H, NCH), 5.92 (s, 2H,
OCH2O), 6.12 (s, 1H, Harom), 6.54 (s, 2H, Harom), 6.97±
7.04 (m, 2H, Harom), 7.27±7.43 (m, 3H, 2Harom+NH); 13
C
NMR: d C 155.9 (d, JCF=240Hz, CF), 153.1, 150.5,
147.0, 139.2, 133.4, 130.5, 122.5, 120.7, 120.6, CH 115.9,
115.7, 106.1, 105.9, 72.1, 60.4, CH2 101.8, 53.7, 31.2, 30.2,
21.1, CH3 60.9, 56.2. Anal. calcd for C29H29FN2O7: C,
64.92; H, 5.45; N, 5.22. Found: C, 67.17; H, 5.65; N, 5.06.
Compound 42. (78%); mp 121±122 ꢀC; 1H NMR: d
1.28±1.57 (m, 4H, CH2), 1.71±1.86 (m, 3H, CH2), 2.30±
2.28 (m, 1H, CH2), 2.51±2.60 (m, 1H, CH2), 2.67 (dd,
J=14.3, 3.5 Hz, 1H), 2.79±2.83 (m, 1H, CH), 3.82 (s,
6H, OCH3), 3.85 (s, 3H, OCH3), 3.98 (s, 1H, NCH),
5.86 (dd, J=6.1, 1.4 Hz, 2H, OCH2O), 6.08 (s, 1H,
Harom), 6.55 (s, 2H, Harom), 6.95±7.03 (m, 2H, Harom),
7.36±7.44 (m, 3H, 2Harom+NH); 13C NMR: d C 157.7
(d, J=245 Hz, CF), 153.2, 150.6, 146.8, 140.0, 137.2,
133.3, 132.2, 129.7, 121.3, 120.6, CH 115.9, 115.6, 106.2,
106.0, 72.4, 57.5, CH2 101.6, 54.0, 34.0, 31.2, 25.9, 24.3,
CH3 60.9, 56.2. Anal. calcd for C30H31FN2O7: C, 65.45;
H, 5.68; N, 5.09. Found: C, 65.71; H, 5.60; N, 5.30.
12. Ward, R. S. Chem. Soc. Rev. 1982, 11, 75.
13. Ward, R. S.. Synthesis, 719.
14. Ward, R. S. Nat. Prod. Rep. 1993, 10, 1.
15. Ward, R. S. Nat. Prod. Rep. 1995, 12, 183.
16. Ward, R. S. Nat. Prod. Rep. 1997, 14, 43.
17. Ward, R. S. Nat. Prod. Rep. 1999, 16, 75.
18. Tomioka, K.; Kubota, Y.; Koga, K. Tetrahedron 1993, 49,
1891.
19. Lienard, P.; Quirion, J.-C.; Husson, H.-P. Tetrahedron
1993, 49, 3995.
20. Arai, Y.; Enomoto, K. Yakugaku Zasshi 1968, 88, 1197.
Chem. Abstr. 1969, 70, 27530.
21. Pearce, H. L.; Bach, N. J.; Cramer, T. L. Tetrahedron
Lett. 1989, 30, 907.
22. Kadow, J. F.; Vyas, D. M.; Doyle, T. W. Tetrahedron
Lett. 1989, 30, 3299.
23. van der Eycken, J.; Bosmans, J. P.; van Haver, D.; Van-
dewalle, M.; Hulkenberg, A.; Veerman, W.; Nieuwenhuisen,
R. Tetrahedron Lett. 1989, 30, 3873.
24. Bosmans, J. P.; van der Eycken, J.; Vandewalle, M.;
Hulkenberg, A.; van Hes, R.; Veerman, W. Tetrahedron Lett.
1989, 30, 3877.
25. Hitotsuyanagi, Y.; Yamagami, K.; Fujii, A.; Naka, Y.;
Ito, Y.; Tahana, T. Bioorg. Med. Lett. 1995, 5, 1039.
26. Hitotsuyanagi, Y.; Kobayashi, M.; Morita, H.; Itokawa,
H.; Takeya, K. Tetrahedron Lett. 1999, 40, 9107.
27. Comins, D. L.; Hong, H.; Saha, J. K.; Jianhua, G. J. Org.
Chem. 1994, 59, 5120.
Biological materials: cell culture and cytotoxicity
L1210 cells (Murine Leukemia) provide by the NCI,
Frederik, USA were cultivated in RPMI 1640 medium
(Gibco) supplemented with 10% foetal calf serum,
2 mM l-glutamine, 100 units/mL penicillin, 100 mg/mL
streptomycin, and 10mM HEPES bu€er (pH=7.4).
Cytotoxicity was measured by the microculture tetra-
zolium assay as described.43 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).
For the cell cycle analysis, L1210 cells (2.5 105 cells/mL)
were incubated for 21 h with various concentrations of

A. Couture et al. / Bioorg. Med. Chem. 8 (2000) 2113±2125
2125
28. Posner, G. H.; Canella, K. A. J. Am. Chem. Soc. 1985,
107, 2571.
29. Couture, A.; Deniau, E.; Grandclaudon, P.; Woisel, P.
Tetrahedron 1996, 52, 4433.
30. Lebrun, S.; Couture, A.; Deniau, E.; Grandclaudon, P.
Tetrahedron 1999, 55, 2659.
31. Sonesson, C.; Larhed, M.; Nyqvist, C.; Hallberg, A. J.
Org. Chem. 1996, 61, 4756.
36. Miller, R. C. J. Org. Chem. 1959, 24, 2013.
37. Jogia, M. K.; Vakamoce, V.; Weavers, R. T. Aust. J.
Chem. 1985, 38, 1009.
38. Heiser, B.; Broger, E. A.; Crameri, Y. Tetrahedron:
Asymmetry 1991, 2, 51.
39. Bradley, R. J. Chem. Soc., 1928, 1555.
40. Loriot, M.; Robin, P.; Brown, E. Tetrahedron 1984, 40,
2529.
32. Sonesson, C.; Wikstroem, H.; Smith, M. W.; Svensson, K.;
Carlsson, A.; Waters, N. Bioorg. Med. Chem. Lett. 1997, 7, 241.
33. Itoh, N.; Sugasawa, S. Tetrahedron 1959, 6, 16.
34. Kametani, T.; Fukumoto, K. Heterocycles 1975, 3, 311.
35. Overman, L. E.; Kakimoto, M.; Okazaki, M. E.; Meier,
G. P. J. Am. Chem. Soc. 1983, 105, 6622.
41. Brown, E.; Loriot, M.; Robin, J.-P. Tetrahedron Lett.
1982, 23, 949.
42. Musser, J. H.; Chakraborty, U.; Bailey, K.; Sciortino, S.;
Whyzmuzis, C. J. Med. Chem. 1987, 30, 62.
43. Leonce, S.; Perez, V.; Casabianca-Pignede, M. R.; Anslett,
M.; Bisagni, E.; Atassi, G. Invest. New Drugs 1996, 14, 169.