D-ring substituted rhazinilam analogues: Semisynthesis and evaluation of antitubulin activity

Article abstract of DOI:10.1016/S0968-0896(99)00241-2

Novel (-)- and (+)-rhazinilam derivatives substituted on the D-ring (compounds 3, 4, 5 and 6) have been prepared from (+)-vincadifformine 7 and (-)-tabersonine and evaluated against the disassembly of microtubules into tubulin. Along with this study, a re

Full text of DOI:10.1016/S0968-0896(99)00241-2

Bioorganic & Medicinal Chemistry 7 (1999) 2961±2969
D-Ring Substituted Rhazinilam Analogues: Semisynthesis and
Evaluation of Antitubulin Activity
Christophe Dupont, Daniel Guenard, Luba Tchertanov, Sylviane Thoret
and Francoise Gueritte *
Institut de Chimie des Substances Naturelles, Centre National de la Recherche Scienti®que, avenue de la Terrasse,
91198 Gif-sur-Yvette Cedex, France
Received 20 April 1999; accepted 2 August 1999
AbstractÐNovel ( )- and (+)-rhazinilam derivatives substituted on the D-ring (compounds 3, 4, 5 and 6) have been prepared from
(+)-vincadi€ormine 7 and ( )-tabersonine and evaluated against the disassembly of microtubules into tubulin. Along with this
study, a reproducible `one pot' semisynthesis of ( )-rhazinilam 1 from (+)-1,2-didehydroaspidospermidine 2 was performed
allowing the easy preparation of these new compounds. # 1999 Elsevier Science Ltd. All rights reserved.
Introduction
been synthesized. Most of the chemical modi®cations
realised in these previous studies concerned the phenyl±
pyrrole part and the amide function of the molecule. In
this study, we focused our attention on chemical mod-
i®cations of the D-ring, relatively unknown regarding
interaction with the receptor. We thus report here the
synthesis as well as the biological activity evaluation of
new D-ring substituted rhazinilam analogues 3, 4, 5
and 6.
Among the di€erent classes of natural antimitotic com-
pounds, ( )-rhazinilam 1 is a unique molecule due to its
activity on tubulin. This compound induces spiraliza-
tion of tubulin such as vinblastine and inhibits the cold-
induced disassembly of microtubules (paclitaxel like
activity).1 Despite the apparent similarity between the
e€ects of ( )-rhazinilam and paclitaxel on the micro-
tubule skeleton, these two compounds possess two dis-
tinct mechanisms of action.1 First isolated by Linde
from Melodinus australis, in 1965,2 the structure of rha-
zinilam 1 was established in 1972.3,4 A total synthesis of
(‹)-rhazinilam was performed by Smith's group a
year later along with a semisynthesis of ( )-1 from (+)-
1,2-didehydroaspidospermidine 2.5 The antitubulin
activity of rhazinilam 1 was discovered in our labora-
tory during a tubulin assay based systematic screening
of the Malaysian Flora.6,7 Bioassay-guided puri®cation
of a crude extract of Kopsia singapurensis Ridl. led to
the isolation of ( )-rhazinilam 1 as the main active
product. Structure±activity relationships then have been
studied from analogues obtained by chemical modi®ca-
tions of ( )-rhazinilam 1, and by semisynthesis from
(+)-1,2-didehydroaspidospermidine analogues.8,9 Other
phenyl±pyrrole10,11 and biphenyl compounds.12,13 showing
a similar activity as natural ( )-rhazinilam 1 also have
Results and Discussion
D-Ring substituted rhazinilam analogues can be pre-
pared rapidly by two di€erent ways. The ®rst method is
the direct functionalization of rhazinilam 1. The second
one is the semisynthesis of ( )-rhazinilam analogues
from (+)-1,2-didehydroaspidospermidine derivatives
according to Smith's methodology.5 The former
method was not readily adapted to our purpose due to
the instability of rhazinilam 1 under acid and basic
conditions.
The synthetic strategy, using the semisynthetic pathway,
starts from (+)-vincadi€ormine 7 a€ording easily (+)-
1,2-didehydroaspidospermidine 2 after acid treatment.
Our investigations in the preparation of ( )-rhazinilam
analogues led us ®rst to improve the semisynthesis of
( )-rhazinilam 1 from (+)-1,2-didehydroaspidospermi-
dine 2 (Scheme 1). Indeed, the reactions previously
described5 were not always reproducible and poor
yields were obtained due to work up diculties. Two
Key words: Natural products; rhazinilam; tubulin; cytotoxicity; struc-
ture±activity relationships.
* Corresponding author. Tel.:+33-1-6982-4580; fax:+33-1-6907-
7247; e-mail: gueritte@icsn.cnrs±gif.fr
0968-0896/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(99)00241-2

2962
C. Dupont et al. / Bioorg. Med. Chem. 7 (1999) 2961±2969
observations were made concerning the starting point of
the synthetic modi®cations. On one hand, the reaction
between 2 and meta-chloroperoxybenzoic acid led to a
reaction mixture containing 5,21-dihydrorhazinilam N-
oxide 8 as it was previously described.8 Submitted to the
action of Fe(II) salt, compound 8 a€orded rhazinilam 1
(45% yield) along with 5,21-dihydrorhazinilam 9 (5%
yield), in approximately 30 min (Scheme 1, pathway b).
On the other hand, compound 9 led in 1 to 2 days to
( )-rhazinilam 1 by autoxidation. These two con-
siderations were not consistent with the hypothesis of
the only action of Fe(II) as reductive agent of the N-
oxide function of 8 leading to 9 which gave rhazinilam
1 after autoxidation.8 We thus considered that rhazini-
lam 1 could be formed directly from the N-oxide 8
through a Polonovski reaction involving the Fe(II)/
Fe(III) redox reaction of iron. In order to avoid the
work up worries due to the presence of iron ions, we
submitted the N-oxide 8 to more classical Polonovski
conditions (Ac2O, Et3N) to form a pyrrole group from
an a,a0-dihydropyrrole N-oxide moiety.14 Under these
conditions compound 8 a€orded directly ( )-rhazinilam
1 in 81% yield (Scheme 1, pathway c). Starting from
(+)-1,2-didehydroaspidospermidine 2, the `one pot'
reaction yielded as well ( )-rhazinilam 1 in reproducible
yield (50%) (see Experimental). Recently, seco-rhazini-
lams were also prepared from seco-didehydroaspido-
spermidine derivatives by meta-chloroperoxybenzoic
acid oxidation and thermolysis.15
Scheme 1. (a) mCPBA, CH2Cl2, 20ꢀC (8 (65%)); (b) FeSO4, H2O, rt
(1 (45%), 9 (5%)); (c) Ac2O, Et3N, CH2Cl2, 0ꢀC (1 (81%)).
in tetrahydrofuran at 78ꢀC. The reaction of the anion
generated in situ with ethyl iodide and benzyl bromide
a€orded the kinetically favored compounds 13 and 14,
respectively, whereas phenylselenenyl chloride17 led to
the two epimers 15a and 15b due to the acidity of the
hydrogen adjacent to the selenium. The b stereoselec-
tivity observed in the case of 13 and 14 as well as the
con®guration of each product (a:b=2:1) in the case of
15a and 15b were deduced from NMR spectra analysis
(1H, NOESY). Moreover, the b-position of the ethyl
group at C-14 of compound 13 was con®rmed by X-ray
analysis (Fig. 1).
The selective reduction of 13 and 14 at position 3 was
realized with borane±tetrahydrofuran complex in tetra-
hydrofuran at 0ꢀC.18 Under these conditions, no reac-
tivity of the carbonate and ester groups was observed
and compounds 16 and 17 were both obtained in quan-
titative yield. Concerning the 14,15 unsaturated series,
compounds 15a and 15b were treated with meta-chloro-
peroxybenzoic acid in methylene chloride at 78ꢀC to
yield the corresponding selenoxide products. Elimina-
tion of the selenoxide group occurred smoothly at room
temperature to a€ord 18 in quantitative yield. In the
case of 15a, addition of sodium hydroxide was necessary
to aid elimination. Finally, 1,2 reduction of the a,b-
unsaturated amide 18 was achieved selectively with dii-
sobutylaluminum hydride19 giving the Na-protected
derivative of (+)-tabersonine 19.
Compounds 16, 17 and 19 were then submitted to de-
carboxylation and Boc-deprotection with hydrochloric
acid to a€ord 1,2-didehydroaspidospermidine deriva-
tives 20, 21 and 22 in high yields. No puri®cation was
used at this time since these compounds showed a great
tendency to autooxidise. The `one pot' semisynthesis
was ®nally performed as described above to give ( )-
14b-ethylrhazinilam 3, ( )-14b-benzylrhazinilam 4 and
( )-14,15-didehydrorhazinilam 5 in 50% yield each
from compounds 20, 21 and 22, respectively. In order to
compare the e€ects of the enantiomer series ((+)-form)
to that of natural ( )-rhazinilam series, we also prepared
(+)-14,15-didehydrorhazinilam 6 from ( )-1,2,14,15-
In order to prepare ( )-rhazinilam analogues sub-
stituted on the D-ring, we aimed to obtain a lactam
function at position 3 (Scheme 2).
(+)-Vincadi€ormine 7 was ®rst protected by a Boc
group to yield 10. The oxidation of 10 was best
performed with bromine in tetrahydrofuran:water
according to Picot and Lusinchi.16 No regioselec-
tivity was observed and 10 a€orded 11 (45%) along
with 12 (43%). Compound 11 was then functiona-
lized at position 14 using lithium diisopropylamide

C. Dupont et al. / Bioorg. Med. Chem. 7 (1999) 2961±2969
2963
Scheme 2. (a) Boc2O, DMAP, DMF, 100%; (b) Br2, NaHCo3, THF/H2O, 95%; (c) LDA, RX, THF, 78ꢀC ±rt, (RX=Etl 9 60%; RX=BnBr 12
83%); (d) BH3-THF, THF, 0ꢀC, 100%; (e) LDA 2 equiv, PhSeCl 1 equiv, THF, 78ꢀC ±rt, 80%; (f) mCPBA, CH2Cl2, 78ꢀC ±rt, 100%;
(g) DIBAH, THF, 20ꢀC, 95%.
tetradehydroaspidospermidine (23)20,21 (Scheme 3). The
e€ects of ( )-14b-ethylrhazinilam 3, ( )-14b-benzyl-
rhazinilam 4, ( )- and (+)-14,15-didehydrorhazinilam
5 and 6 on microtubules disassembly and their cyto-
toxicity are summarized in Table 1. Inhibition of
microtubules disassembly was evaluated using tubulin
from bovine brain according to the previously described
methodology.8 Cytotoxicity was examined on the KB
human cancer line.22
The substitution at position 14 with the hydrophobic
ethyl and benzyl groups, results in a clear decrease of
the activity inhibiting the disassembly of microtubules
into tubulin. ( )-14b-Ethylrhazinilam 3 was found 50-
fold less active than ( )-rhazinilam 1 and ( )-14b-ben-
zylrhazinilam 4 was found inactive. The unsaturated D-
ring derivative, ( )-14,15-didehydrorhazinilam 5 is only
2 times less active than rhazinilam 1. Unexpectedly, its
enantiomer (+)-14,15-didehydrorhazinilam 6 retains
some interaction with microtubules. This result is at
variance with the fact that the binding interaction with
microtubules was shown to be stereoselective in the
biphenyl series mimicking the activity of rhazinilam.13
Regarding the cytotoxicity of the compounds, one
Table 1. Cytotoxicity and antitubulin activity of rhazinilam 1 and
analogues 3, 4, 5 and 6
Compound
Cytotoxicity
(KB cell line)
IC50 (mM)a,b
Inhibition of
microtubules disassembly
IC50 (mM)c
Rhazinilam 1
2
100
44
4
300
3
150
Inactive
6
130
3
4
5
6
a
The IC50 for cytotoxicity and inhibition of microtubules disassembly
was evaluated from measurements run in triplicate.
b
The cytotoxicity IC50 values refer to the concentration of com-
pounds corresponding to 50% growth inhibition after 72 h incubation.
c
IC50 is the concentration of test compound required to inhibit 50%
of the rate of microtubules disassembly.
Figure 1. X-ray crytallographic structure of compound 13 (crystal-
lographic numerotation of atoms was used).

2964
C. Dupont et al. / Bioorg. Med. Chem. 7 (1999) 2961±2969
by spraying with a solution of molybdatophosphoric
acid in EtOH. Melting points (mp) were measured on a
Ko¯er apparatus. Optical rotations have been measured
on a Perkin±Elmer 141 MC polarimeter. Infrared spec-
tra were recorded on a Nicolet FT±IR 205 and UV
spectra on a Elmer-Lambda 5 spectrometer. 1H and 13
C
NMR spectra were recorded on Bruker AC-200, AC-
250 or AC-300 spectrometers using tetramethylsilane
as internal standard. Chemical shifts are expressed in
part per million (ppm). s, bs, d, bd, t, dd, q and m
indicate singlet, broad singlet, doublet, broad doublet,
triplet, doublet of doublet, quartet and multiplet,
respectively. Mass spectra were measured on a AEI
MS-50 spectrometer. All physicochemical measure-
ments and elemental analyses were performed at the
ICSN, CNRS, Gif-sur-Yvette, France.
Preparation of 5,21-dihydrorhazinilam N-oxide (8). To a
solution of 2 (100 mg, 0.36 mmol) in methylene chloride
(10 mL) at 20ꢀC was added sodium hydrogen carbonate
(36 mg, 0.43 mmol) followed by a solution of meta-
chloroperoxybenzoic acid (62 mg, 0.36 mmol) in meth-
ylene chloride (1 mL). The formation of the N-oxide
compound was observed by TLC on alumina (eluent
CH2Cl2:MeOH: 90:10; Rf ꢁ0.8). An additional solution
of meta-chloroperoxybenzoic acid (0.25 g, 1.44 mmol) in
methylene chloride (4 mL) was then added. The orange
solution was then stirred at 20ꢀC until N-oxide con-
sumption (4±8 h). The solvent was then evaporated in
vacuo and water was added. The mixture was extracted
with butan-1-ol (5Â). After evaporation of the solvent,
the crude product was puri®ed by chromatography on
alumina (eluent CH2Cl2:MeOH, 90:10) to a€ord 8 as a
yellowish amorphous solid (72 mg, 65%); 1H NMR
(250 MHz, CDCl3) d 0.65 (3H, t, J=7.3 Hz, H-18); 1.20
(1H, m, H-19); 1.29 (1H, m, H-19); 1.44 (2H, m, H-
15+H-17); 1.74 (4H, m, 2H-14+H-15+H-17); 1.97
(1H, m, H-16); 2.16 (1H, m, H-16); 3.51 (1H, m, H-3);
3.84 (1H, m, H-3); 4.10 (1H, m, H-5); 4.37 (1H, s, H-
21); 4.59 (1H, m, H-5); 5.85 (1H, s, H-6); 7.16 (1H, m,
H-12); 7.25 (2H, m, H-10+H-11); 7.50 (1H, m, H-9);
13C NMR (62.5 MHz, CDCl3) d 7.4 (C-18); 19.1 (C-14);
27.5 (C-17); 27.7 (C-16); 32.5 (C-19); 34.7 (C-15); 44.9
(C-20); 66.8 (C-3); 73.2 (C-5); 92.2 (C-20); 125.8 (C-6);
126.3 (C-12); 127.2+129.5 (C-10+C-11); 129.0 (C-9);
133.3 (C-7); 136.0 (C-8); 138.0 (C-13); 178.3 (C-2); IR
(CHCl3) n 1666 cm 1; MS (CI+) m/z 295 ([M±
H2O+H]+).
Scheme 3. (a) HCl 12 N, Á, 5 mn; (b) mCPBA, Ac2O, NEt3, CH2Cl2,
20ꢀC, 50%.
observes a rather good correlation with the micro-
tubules disassembly assay. However, a discrepancy is
observed for compound 4 which is 20 times less cyto-
toxic than rhazinilam 1 on KB cells but does not show
any interaction with microtubules. In this case, the
cytotoxicity may be related to a mode of action di€erent
from a direct interaction with microtubules.
Conclusion
In summary, this work provides new information on the
structure±activity relationships in the rhazinilam series.
The presence of hydrophobic groups at C-14 on the D-
ring led to a large decrease in the `antitubulin' activity
indicating that this part of the molecule may be in
interaction with the binding site. In order to con®rm
this hypothesis, future studies will concern substitutions
at position 14 with polar groups as well as chemical
modi®cations at positions 3 and 15.
Preparation of rhazinilam (1) from 5,21-dihydrorhazini-
lam N-oxide (8). Acetic anhydride (5.23 mL, 1 mmol) was
added to a suspension of 8 (20 mg, 0.064 mmol) in
methylene chloride at 0ꢀC. The mixture was allowed to
warm to room temperature and was stirred for 30 min.
Water was then added, the aqueous layer was extracted
with methylene chloride (2Â). The combined organic
layers were dried over sodium sulfate and the solvent
was removed in vacuo to yield pure 1 as a white amor-
phous solid (15.2 mg, 81%); 1H NMR (300 MHz,
CDCl3) d 0.72 (3H, t, J=7.3 Hz, H-18); 1.25 (1H, dq,
J=14.6, 7.3 Hz, H-19); 1.47 (1H, dq, J=14.6, 7.3 Hz,
H-19); 1.49 (1H, m, H-17b); 1.96 (1H, m, H-16a); 2.24 (1H,
m, H-14a); 2.38 (1H, m, H-16b); 2.46 (1H, m, H-17a);
Experimental
All reactions were followed by TLC on glass plates
coated with Si gel 60 F254 silical gel and spots revealed

C. Dupont et al. / Bioorg. Med. Chem. 7 (1999) 2961±2969
2965
3.79 (1H, m, H-3b); 4.01 (1H, m, H-3a); 5.76 (1H, m, H-
6); 6.51 (1H, m, H-5); 6.69 (1H, bs, NH); 7.21 (1H, m,
H-12); 7.30+7.35 (2H, m, H-10+H-11); 7.43 (1H, m,
H-9); 13C NMR (75 MHz, CDCl3) d 8.2 (C-18); 19.5 (C-
14); 28.2 (C-16); 30.2 (C-19); 33.1 (C-15); 36.7 (C-17);
38.9 (C-20); 46.1 (C-3); 109.9 (C-6); 117.4 (C-7); 119.1
(C-5); 126.9 (C-12); 127.2+128.0 (C-10+C-11); 130.6
(C-20); 131.5 (C-9); 138.2 (C-8); 140.4 (C-13); 177.4 (C-
2); MS (CI+) m/z 295 ([M+H]+).
(C-3+C-5); 53.2 (C-7); 71.1 (C-21); 82.0 (C(CH3)3);
112.4 (C-16); 116.1 (C-12); 120.5 (C-9); 123.5 (C-10);
127.3 (C-11); 138.5 (C-8); 140.7 (C-13); 149.6 (C-2);
151.3 (CO2C(CH3)3); 168.3 (CO2CH3); IR (CHCl3) n
1717 cm 1; MS (EI) m/z 438 (M+); 338. Anal. calcd
(found) for C26H34N2O4: C, 71.21 (70.94); H, 7.81
(7.88); N, 6.39 (6.22); O, 14.59 (14.78).
Oxidation of compound 10. A solution of bromine
(0.3 mL, 5.86 mmol) in tetrahydrofuran (40 mL) was
added dropwise at 20ꢀC to a stirred solution of 10 (1 g,
2.28 mmol) and sodium carbonate (0.5 g, 4.72 mmol) in
tetrahydrofuran:water 1:1 (200 mL). The red solution
was decolorized by treatment with a saturated solution
of sodium thiosulfate. Sodium chloride was added until
the layers separated. The aqueous layer was then
extracted with ethyl acetate. The combined organic lay-
ers were dried over sodium sulfate and the solvent was
evaporated under vacuo to yield 1.3 g of crude product
as a slight yellow oil. The residue was chromatographed
on silica gel (eluent heptane:acetone: 70:30) to a€ord 11
(0.47 g, 45%) and 12 (0.42 g, 43%) as colorless oils.
Compound 11 was further crystallized from ethyl ace-
tate for analysis.
`One pot' preparation of ( )-rhazinilam 1 from 2. To a
solution of 2 (0.50 mmol) in methylene chloride (10 mL)
at
20ꢀC was added sodium hydrogen carbonate
(50 mg, 0.60 mmol) followed by a solution of meta-
chloroperoxybenzoic acid (86 mg, 0.50 mmol) in meth-
ylene chloride (1 mL). The formation of the Nb-oxide
compound was observed by TLC on alumina (eluent
CH2Cl2:MeOH: 90:10; Rf ꢁ0.8). An additionnal solution
of meta-chloroperoxybenzoic acid (0.35 g, 2.00 mmol) in
methylene chloride (4 mL) was then added. The orange
solution was then stirred at 20ꢀC until Nb-oxide con-
sumption (4±8 h). The formation of the 5,21-dihy-
drorhazinilam Nb-oxide was observed by TLC on
alumina (eluent CH2Cl2:MeOH: 90:10; Rfꢁ0.5). Triethyl-
amine (0.7 mL, 5.00 mmol) was then added, followed by
acetic anhydride (74 mL, 1.00 mmol). The mixture was
allowed to warm to room temperature and was stirred
for 30 min. Water was then added, the aqueous layer
was extracted with methylene chloride (2Â). The com-
bined organic layers were dried over sodium sulfate and
the solvent was removed in vacuo. Excess triethylamine
was removed by azeotropic distillation with ethanol.
The crude product (brown oil) was chromatographed
on silica gel to yield 1 (50%).
3-oxo-(Na-tert-Butoxycarbonyl)vincadi€ormine (11). Mp
193±4ꢀC (AcOEt); [a]d25 +27.4 ꢀ (c 1.43, CHCl3); 1H
NMR (300 MHz, CDCl3) d 0.67 (3H, t, J=7.4 Hz, H-
18); 1.04 (1H, dq, J=14.8, 7.4 Hz, H-19); 1.16 (1H, dq,
J=14.8, 7.4 Hz, H-19); 1.44 (1H, m, H-15b); 1.58 (9H,
s, C(CH3)3); 1.91 (1H, m, H-6b); 1.96 (1H, m, H-15a);
2.05 (1H, m, H-6a); 2.16 (1H, m, H-17a); 2.30 (1H, m,
H-14); 2.39 (1H, m, H-14); 2.46 (1H, m, H-17b); 3.32
(1H, m, H-5b); 3.51 (1H, s, H-21); 3.77 (3H, s,
CO2CH3); 4.11 (1H, m, H-5a); 7.08 (1H, m, H-10); 7.15
(1H, m, H-9); 7.28 (1H, m, H-11); 7.67 (1H, m, H-12);
13C NMR (75 MHz, CDCl3) d 7.5 (C-18); 27.8 (C-19);
28.1 (C(CH3)3); 29.9 (C-15); 30.6 (C-17+C-14); 39.1 (C-
6+C-20); 42.4 (C-5); 51.6 (CO2CH3); 54.4 (C-7); 66.8
(C-21); 83.0 (C(CH3)3); 110.4 (C-16); 116.2 (C-12); 121.2
(C-9); 124.0 (C-10); 128.3 (C-11); 136.5 (C-8); 140.6 (C-
13); 148.7 (C-2); 150.6 (CO2C(CH3)3); 167.6 (CO2CH3);
170.9 (C-3); IR (CHCl3) n 1724; 1625 cm 1; MS (CI+)
m/z 453 ([M+H]+); 353. Anal. calcd (found) for
C26H32N2O5: C, 69.00 (68.82); H, 7.13 (7.18); N, 6.19
(6.08); O, 17.68 (17.53).
(Na-tert-Butoxycarbonyl)vincadi€ormine (10). tert-Butoxy-
carbonic anhydride (4.6 g, 28 mmol), 4-(N,N-dimethyl-
amino)pyridine (0.33g, 2.8mmol) and (+)-vincadi€ormine
7 (4.6 g, 14 mmol) were dissolved in minimum di-
methylformamide. A strong gas evolution occurred. The
mixture was stirred at room temperature overnight. tert-
Butoxycarbonic anhydride (2.3 g, 14 mmol) and 4-(N,N-
dimethylamino)pyridine (0.165 g, 1.4 mmol) were then
added again. After stirring 1 h at room temperature, a
white precipitate appeared. The solvent was evaporated.
The crude product was puri®ed by ¯ash chromato-
graphy (eluent heptane:ethyl acetate, 80:20) to provide
10 (5.66 g) in 95% yield as a yellowish solid. Compound
10 was further recrystallized from ethyl acetate as yel-
lowish needles; mp 149±150ꢀC (AcOEt); [a]d25+115.4 ꢀ
5-oxo-(Na-tert-Butoxycarbonyl)vincadi€ormine (12). [a]d25
+54.5 ꢀ (c 0.94, CHCl3); 1H NMR (300 MHz, CDCl3) d
0.61 (3H, t, J=7.4 z, H-18); 0.85 (1H, dq, J=14.8,
7.4 Hz, H-19); 1.24 (1H, dq, J=14.8, 7.4 Hz, H-19); 1.54
(9H, s, C(CH3)3); 1.55 (1H, m, H-15b); 1.63 (2H, m, H-
14); 1.91 (1H, m, H-15a); 2.10 (1H, m, H-17b); 2.48
(1H, m, H-6b); 2.58 (1H, m, H-17a); 2.77 (1H, m, H-
3b); 2.88 (1H, m, H-6a); 3.77 (3H, s, CO2CH3); 3.78
(1H, s, H-21); 4.34 (1H, m, H-3a); 7.09 (1H, m, H-10);
7.15 (1H, m, H-9); 7.26 (1H, m, H-11); 7.67 (1H, m, H-
12); 13C NMR (75 MHz, CDCl3) d 6.8 (C-18); 19.8 (C-
14); 26.7 (C-17); 28.1 (C(CH3)3); 28.9 (C-19); 33.0 (C-
15); 37.0 (C-20); 40.0 (C-3); 46.6 (C-6); 51.7 (C-
7+CO2CH3); 66.1 (C-21); 82.8 (C(CH3)3); 110.8 (C-16);
116.2 (C-12); 120.6 (C-9); 124.3 (C-10); 128.2 (C-11);
136.4 (C-8); 140.8 (C-13); 148.5 (C-2); 150.8
1
(c 1.20, CHCl3); H NMR (250 MHz, CDCl3) d 0.56
(3H, t, J=7.4 Hz, H-18); 0.70 (1H, qd, J=14.8,
7.4 Hz, H-19); 1.13 (1H, qd, J=14.8, 7.4 Hz, H-19); 1.24
(1H, m, H-15b); 1.53 (9H, s, C(CH3)3); 1.59 (1H, m, H-
14b); 1.64 (1H, m, H-6b); 1.77 (1H, m, H-15a); 1.86
(1H, m, H-14a); 1.98 (1H, m, H-17b); 2.16 (1H, m, H-
6a); 2.26 (1H, m, H-3b); 2.34 (1H, s, H-21); 2.37 (1H, m,
H-5b); 2.91 (1H, m, H-5a); 3.01 (1H, m, H-17a); 3.12
(1H, m, H-3a); 3.75 (3H, s, CO2CH3); 7.01 (1H, m, H-
10); 7.15 (1H, m, H-9); 7.19 (1H, m, H-11); 7.65 (1H, m,
H-12); 13C NMR (75 MHz, CDCl3) d 7.2 (C-18); 22.4
(C-14); 28.0 (C-17); 28.3 (C(CH3)3); 28.9 (C-19); 32.9
(C-15); 38.0 (C-20); 42.7 (C-6); 51.6 (CO2CH3); 51.8

2966
C. Dupont et al. / Bioorg. Med. Chem. 7 (1999) 2961±2969
(CO2C(CH3)3); 167.4 (CO2CH3); 170.0 (C-5); IR
(CHCl3) n 1723; 1678 cm 1; MS (CI+) m/z 453
([M+H]+); 353; HRMS (CI+) m/z calcd (found) for
C26H33N2O5 (MH+): 453.2390 (453.2397).
2.24 mmol), and benzyl bromide (0.27 mL, 2.27 mmol).
The crude product (yellow oil, 1.2 g) was chromato-
graphed on silica gel (eluent heptane:ethyl acetate,
70:30) to yield 1.0 g of the title compound as a colorless
oil (83%); [a]d25 +114.1 ꢀ (c 1.07, CHCl3); 1H NMR
(300 MHz, CDCl3) d 0.52 (3H, t, J=7.4 Hz, H-18); 0.82
(1H, dq, J=14.8, 7.4 Hz, H-19); 1.21 (1H, dq, J=14.8,
7.4 Hz, H-19); 1.54 (1H, m, H-15b); 1.54 (9H, s,
C(CH3)3); 1.79 (1H, m, H-15a); 1.84 (1H, m, H-6b);
1.95 (1H, m, H-6a); 2.14 (1H, m, H-17b); 2.34 (1H, m,
H-17a); 2.72 (1H, m, H-14a); 2.75 (1H, m, CH2-È); 3.15
(1H, m, H-5b); 3.47 (1H, m, CH2-È); 3.75 (3H, s,
CO2CH3); 3.75 (1H, s, H-21); 4.43 (1H, m, H-5a); 7.07
(1H, m, H-10); 7.17 (1H, m, H-9); 7.22 (1H, m, H-11);
7.24 (3H, m, H-Èo+H-Èp); 7.31 (2H, m, H-Èm); 7.67
(1H, m, H-12); 13C NMR (62.5 MHz, CDCl3) d 7.2 (C-
18); 27.1 (C-17); 28.2 (C(CH3)3); 28.8 (C-19); 35.0 (C-
15); 37.0 (C-20); 38.4 (CH2-È); 39.6 (C-14); 41.4 (C-6);
43.1 (C-5); 51.8 (CO2CH3); 54.9 (C-7); 66.3 (C-21); 82.9
(C(CH3)3); 110.7 (C-16); 116.4 (C-12); 120.6 (C-9); 123.9
(C-10); 126.4 (C-Èp); 128.3 (C-11); 128.5 (C-Èm); 129.3
(C-Èo); 136.4 (C-8); 139.4 (Cq-È); 140.6 (C-13); 149.6
(C-2); 150.8 (CO2C(CH3)3); 167.4 (CO2CH3); 170.0 (C-
3); IR (CHCl3) n 1722; 1625 cm 1; MS (CI+) m/z 543
([M+H]+); 453; 443; HRMS (CI+) m/z calcd (found)
for C33H39N2O5 (MH+): 543.2859 (543.2870).
14ꢀ-Ethyl-3-oxo-(Na-tert-butoxycarbonyl)vincadi€ormine
(13). To a stirred solution of diisopropylamine (0.62 mL,
4.42 mmol) in anhydrous tetrahydrofuran (5 mL) at
78ꢀC was added n-butyl lithium 1.6 M in hexane
(2.8 mL, 4.42 mmol). After 15 min at 78ꢀC, a solution
of compound 11 (1 g, 2.21 mmol) in anhydrous tetra-
hydrofuran (10 mL) was added, followed after 30 min at
78ꢀC by ethyl iodide (0.36 mL, 4.42 mmol). Then the
mixture was allowed to warm to room temperature over
2 h. The yellow solution was quenched carefully with
water. The aqueous layer was extracted with ethyl ace-
tate (2Â). The combined organic layers were dried over
sodium sulfate and the solvent was evaporated in vacuo.
The crude product (yellow oil, 1 g) was chromato-
graphed on silica gel (eluent heptane:acetone: 70:30) to
yield 0.64 g of the title compound as a colorless oil
(60%). Compound 13 was crystallized from cyclohexane
for analysis: mp 192±193ꢀC (cyclohexane); [a]d25+82.1 ꢀ
1
(c 1.23, CHCl3); H NMR (300 MHz, CDCl3) d 0.65
(3H, t, J=7.4 Hz, H-18); 0.89 (1H, dq, J=14.8, 7.4 Hz,
H-19); 0.99 (3H, t, J=7.4 Hz, CHCH2CH3); 1.27 (1H,
dq, J=14.8, 7.4 Hz, H-19); 1.55 (9H, s, C(CH3)3); 1.59
(1H, m, H-15b); 1.60 (1H, m, CHCH2CH3); 1.84 (1H,
m, H-6b); 1.92 (1H, m, H-6a); 1.97 (1H, m, H-15aa);
1.99 (1H, m, CHCH2CH3); 2.26 (1H, m, H-17b); 2.34
(1H, m, H-17a); 2.38 (1H, m, H-14a); 3.16 (1H, m, H-
5b); 3.77 (3H, s, CO2CH3); 3.79 (1H, s, H-21); 4.36 (1H,
m, H-5a); 7.08 (1H, m, H-10); 7.20 (1H, m, H-9); 7.27
(1H, m, H-11); 7.68 (1H, m, H-12); 13C NMR (75 MHz,
Compounds 15a and 15b. Compounds 15a and 15b were
obtained from 11 (3.89 g, 8.61 mmol) by the above pro-
cedure using diisopropylamine (2.41 mL, 17.2 mmol), n-
butyllithium (10.8 mL, 17.3 mmol), and a solution of
phenylselenenyl chloride (1.65 g, 8.62 mmol) in anhy-
drous tetrahydrofuran (10 mL). The crude product (yel-
low oil, 5.5 g) was chromatographed on silica gel (eluent
heptane:ethyl acetate, 50:50) to a€ord 15b (1.39 g, 26%)
as white needles and 15a (2.80 g, 54%) as a colorless oil.
CDCl3)
d
7.4 (C-18); 11.4 (CHCH2CH3); 25.3
(CHCH2CH3); 27.7 (C-17); 28.3 (C(CH3)3); 29.1 (C-19);
34.9 (C-15); 37.4 (C-20); 39.7 (C-14); 41.0 (C-6); 43.1 (C-
5); 51.8 (CO2CH3); 55.0 (C-7); 66.4 (C-21); 83.0
(C(CH3)3); 110.8 (C-16); 116.5 (C-12); 120.8 (C-9); 124.0
(C-10); 128.4 (C-11); 136.5 (C-8); 140.8 (C-13); 149.6 (C-
2); 150.9 (CO2C(CH3)3); 167.6 (CO2CH3); 171.2 (C-3);
IR (CHCl3) n 1724; 1625 cm 1; MS (CI+) m/z 481
([M+H]+); 381. Anal. calcd (found) for C28H36N2O5:
C, 69.98 (70.02); H, 7.55 (7.34); N, 5.83 (5.67); O, 16.64
(16.59).
14ꢁ-Phenylselenenyl-3-oxo-(Na-tert-butoxycarbonyl)-vin-
cadi€ormine (15a). [a]2d5 46.5 ꢀ (c 1.14, CHCl3); 1H
NMR (300 MHz, CDCl3) d 0.62 (3H, t, J=7.4 Hz, H-
18); 1.19 (1H, dq, J=14.8, 7.4 Hz, H-19); 1.21 (1H, dq,
J=14.8, 7.4 Hz, H-19); 1.57 (9H, s, C(CH3)3); 1.90 (1H,
m, H-6b); 1.97 (1H, m, H-15b); 2.02 (1H, m, H-6a); 2.15
(1H, m, H-17a); 2.26 (1H, m, H-15a); 2.40 (1H, m, H-
17b); 3.33 (1H, m, H-5b); 3.76 (3H, s, CO2CH3); 3.90
(1H, s, H-21); 4.04 (1H, m, H-14b); 4.13 (1H, m, H-5a);
7.10 (1H, m, H-10); 7.14 (1H, m, H-9); 7.29 (1H, m, H-
11); 7.34 (3H, m, H-Èp+H-Èm); 7.67 (1H, m, H-12);
7.70 (2H, m, H-Èo); 13C NMR (75 MHz, CDCl3) d 7.3
(C-18); 28.1 (C(CH3)3); 28.8 (C-19); 30.5 (C-17); 36.3
(C-15); 39.1 (C-20); 39.6 (C-6); 40.3 (C-14); 43.3 (C-5);
51.7 (CO2CH3); 54.9 (C-7); 65.8 (C-21); 83.1 (C(CH3)3);
110.3 (C-16); 116.3 (C-12); 121.2 (C-9); 124.1 (C-10);
128.2+128.4 (C-Èp+C-11); 128.9 (Cq-È); 129.3 (C-
Èm); 134.5 (C-Èo); 136.2 (C-8); 140.7 (C-13); 149.1 (C-
2); 150.7 (CO2C(CH3)3); 167.7 (CO2CH3); 168.8 (C-3);
IR (CHCl3) n 1727; 1648 cm 1; MS (CI+) m/z 609
([M+H]+); 509; 453; 353. Anal. calcd (found) for
C32H36N2O5Se: C, 63.26 (63.31); H, 5.97 (6.07); N, 4.61
(4.55); O, 13.17 (13.24).
X-ray determination of compound 13:23 Mr=478.57,
orthorhombic, space group P212121, a=7.7849(3),
b=11.932(1), c=28.486(1) A, V=2646.0(3) A3, Z=4,
r=1.201 g cm 1. Of the 7229 re¯ections collected, 4351
re¯ections (I>2s(I)) were used for the re®nement. The
®nal residuals were R1=0.0506, wR2=0.1398, and
GOF=1.031. Structure was solved by direct methods
with SHELX8624 and re®ned by the full-matrix least
squares approximation based on F2 with SHELXL9325
programs. Re®nement was anisotropic for all non-H
atoms. Hydrogen atoms were located from a di€erence
map and re®ned isotropically.
14ꢀ-Benzyl-3-oxo-(Na-tert-butoxycarbonyl)vincadi€ormine
(14). Compound 14 was obtained from 11 (1 g,
2.21 mmol) by the above procedure using diisopropyla-
mine (0.31 mL, 2.21 mmol), n-butyllithium (1.4 mL,
14ꢀ-Phenylselenenyl-3-oxo-(Na-tert-butoxycarbonyl)-vin-
cadi€ormine (15b). Mp 185±186ꢀC (heptane±ethyl acetate);

C. Dupont et al. / Bioorg. Med. Chem. 7 (1999) 2961±2969
2967
[a]2d5 19.5 ꢀ (c 1.13, CHCl3); 1H NMR (300 MHz,
CDCl3) d 0.45 (3H, t, J=7.3H z, H-18); 0.93 (1H, dq,
J=14.6, 7.3 Hz, H-19); 1.05 (1H, dq, J=14.6, 7.3 Hz,
H-19); 1.52 (1H, m, H-15b); 1.57 (9H, s, C(CH3)3); 1.92
(1H, m, H-6b); 2.08 (1H, m, H-17a); 2.09 (1H, m, H-
6a); 2.17 (1H, m, H-15a); 2.48 (1H, m, H-17b); 3.43
(1H, m, H-5b); 3.46 (1H, s, H-21); 3.74 (3H, s,
CO2CH3); 4.07 (1H, m, H-14a); 4.08 (1H, m, H-5a);
7.08 (1H, m, H-10); 7.13 (1H, m, H-9); 7.28 (4H, m, H-
11+H-Èp+H-Èm); 7.62 (2H, m, H-Èo); 7.65 (1H, m,
H-12); 13C NMR (75 MHz, CDCl3) d 7.5 (C-18); 27.7
(C-19); 28.2 (C(CH3)3); 32.1 (C-17); 38.4 (C-6); 39.0 (C-
15); 41.1 (C-20); 43.1 (C-5); 43.5 (C-14); 51.8 (CO2CH3);
54.5 (C-7); 67.0 (C-21); 83.4 (C(CH3)3); 110.3 (C-16);
116.4 (C-12); 121.5 (C-9); 124.2 (C-10); 128.0+128.6
(C-Èp+C-11); 129.2 (C-Èm+Cq-È); 135.1 (C-Èo); 136.6
(C-8); 141.9 (C-13); 148.5 (C-2); 150.8 (CO2C(CH3)3);
167.6 (CO2CH3); 169.6 (C-3); IR (CHCl3) n 1728; 1656
cm 1; MS (CI+) m/z 609 ([M+H]+); 509; 453; 353.
Anal. calcd (found) for C32H36N2O5Se: C, 63.26 (63.01);
H, 5.97 (6.18); N, 4.61 (4.54); O, 13.17 (13.25).
NMR (300 MHz, CDCl3) d 0.55 (3H, t, J=7.4 Hz, H-
18); 0.71 (1H, qd, J=14.8, 7.4 Hz, H-19); 0.99 (1H, m,
H-15b); 1.13 (1H, qd, J=14.8, 7.4 Hz, H-19); 1.51 (9H,
s, C(CH3)3); 1.63 (1H, m, H-6b); 1.83 (1H, m, H-15a);
1.96 (1H, m, H-3b); 1.98 (1H, m, H-17b); 2.13 (1H, m,
H-6a); 2.20 (1H, m, H-14a); 2.35 (1H, m, H-21+H-5b);
2.46 (1H, m, CH2-È); 2.57 (1H, m, CH2-È); 2.82 (1H,
m, H-5a); 2.95 (1H, m, H-17a); 3.04 (1H, m, H-3a); 3.74
(3H, s, CO2CH3); 7.00 (1H, m, H-10); 7.14 (1H, m, H-
9); 7.19 (4H, m, H-11+H-Èo+H-Èp); 7.30 (2H, m, H-
Èm); 7.65 (1H, m, H-12); 13C NMR (75 MHz, CDCl3) d
7.2 (C-18); 28.3 (C(CH3)3); 28.7 (C-17); 28.9 (C-19); 34.9
(C-14); 38.2 (C-20); 40.6 (C-15); 41.3 (CH2-È); 42.9 (C-
6); 51.5 (C-5); 51.6 (CO2CH3); 52.9 (C-7); 57.3 (C-3);
71.0 (C-21); 82.0 (C(CH3)3); 112.3 (C-16); 116.1 (C-12);
120.5 (C-9); 123.5 (C-10); 126.0 (C-Èp); 127.3 (C-11);
128.4 (C-Èm); 129.0 (C-Èo); 138.4 (C-8); 140.3 (Cq-È);
140.7 (C-13); 149.7 (C-2); 151.3 (CO2C(CH3)3); 168.2
(CO2CH3); IR (CHCl3) n 1717 cm 1; MS (CI+) m/z
529 ([M+H]+); 429; HRMS (CI+) m/z calcd (found)
for C33H41N2O4 (MH+): 529.3067 (529.3076).
Borane reduction of the amide group on compounds 13
and 14. General procedure. To a solution of 13 or 14
(0.25 mmol) in anhydrous tetrahydrofuran (5 mL) at
0ꢀC was added dropwise a solution of borane±tetra-
hydrofuran complex 1 M in tetrahydrofuran (1.25 mL,
1.25 mmol). The mixture was stirred at 0ꢀC for 15 min
and then quenched carefully with water. After the gas
evolution has ®nished, the solution was treated with
10% hydrochloric acid for 30 min. Sodium hydroxide
was then added until pHꢁ13. Sodium chloride was
added until demixion. The aqueous layer was extracted
with ethyl acetate (2Â). The combined organic layers
were dried over sodium sulfate and the solvent was
removed in vacuo to a€ord 16 or 17, respectively.
3-Oxo-(Na-tert-butoxycarbonyl)tabersonine (18). To a
stirred solution of 15a or 15b (0.8 g, 1.32 mmol) in
methylene chloride (20 mL) at 78ꢀC was added drop-
wise a solution of meta-chloroperoxybenzoic acid
(0.25 g, 1.45 mmol) in methylene chloride (3 mL). The
solution was then allowed to warm to room tempera-
ture. After 1 h at room temperature, the solution turned
red indicating the spontanous elimination of the selen-
oxide. Solid sodium hydroxyde was added to aid elim-
ination in the case of 15b. Water was then added. The
aqueous layer was extracted with methylene chloride
(2Â). The combined organic layers were dried over
sodium sulfate and the solvent was removed in vacuo to
a€ord compound 18 as a white amorphous solid (0.59 g,
100%). It was further crystallized from heptane/acetone
for analysis; mp 196±7ꢀC (heptane±acetone); [a]2d5
14ꢀ-Ethyl-(Na-tert-butoxy-carbonyl)vincadi€ormine
(16). Colorless oil (100%); [a]2d5+83.7 ꢀ (c 1.13, CHCl3);
1H NMR (250 MHz, CDCl3) d 0.57 (3H, t, J=7.4 Hz,
H-18); 0.69 (1H, qd, J=14.8, 7.4 Hz, H-19); 0.86 (1H,
m, H-15b); 0.93 (3H, t, J=7.4 Hz, CH-CH2-CH3); 1.13
(1H, qd, J=14.8, 7.4 Hz, H-19); 1.24 (2H, m, CH-CH2-
CH3); 1.53 (9H, s, C(CH3)3); 1.65 (1H, m, H-6b); 1.82
(1H, m, H-14a); 1.84 (1H, m, H-15a); 1.88 (1H, m, H-
3b); 2.00 (1H, m, H-17b); 2.18 (1H, m, H-6a); 2.33 (1H,
s, H-21); 2.36 (1H, m, H-5b); 2.91 (1H, m, H-5a); 2.97
(1H, m, H-17a); 3.18 (1H, m, H-3a); 3.75 (3H, s,
CO2CH3); 7.01 (1H, m, H-10); 7.15 (1H, m, H-9); 7.19
(1H, m, H-11); 7.65 (1H, m, H-12); 13C NMR (75 MHz,
CDCl3) d 7.2 (C-18); 11.7 (CH-CH2-CH3); 27.4 (CH-
CH2-CH3); 28.3 (C(CH3)3); 28.8 (C-17); 29.0 (C-19);
34.6 (C-14); 38.0 (C-20); 40.4 (C-15); 42.9 (C-6); 51.6 (C-
5+CO2CH3); 53.0 (C-7); 57.4 (C-3); 71.1 (C-21); 82.0
(C(CH3)3); 112.5 (C-16); 116.1 (C-12); 120.5 (C-9); 123.5
(C-10); 127.3 (C-11); 138.5 (C-8); 140.7 (C-13); 149.6 (C-
2); 151.3 (CO2C(CH3)3); 168.2 (CO2CH3); IR (CHCl3) n
1721 cm 1; MS (IC+) m/z 467 ([M+H]+); 367; HRMS
(CI+) m/z calcd (found) for C28H39N2O4 (MH+):
467.2910 (467.2886).
19.1 ꢀ (c 1.10, CHCl3); H NMR (300 MHz, CDCl3) d
1
0.71 (3H, t, J=7.4 Hz, H-18); 1.13 (1H, dq, J=14.8,
7.4 Hz, H-19); 1.25 (1H, dq, J=14.8, 7.4 Hz, H-19); 1.58
(9H, s, C(CH3)3); 1.98 (1H, m, H-6b); 2.09 (1H, m, H-
6a); 2.31 (1H, m, H-17a); 2.43 (1H, m, H-17b); 3.33
(1H, m, H-5b); 3.78 (3H, s, CO2CH3); 4.03 (1H, s, H-
21); 4.20 (1H, m, H-5a); 5.98 (1H, m, H-15); 6.43 (1H,
m, H-14); 7.10 (1H, m, H-10); 7.21 (1H, m, H-9); 7.29
(4H, m, H-11); 7.68 (1H, m, H-12); 13C NMR (75 MHz,
CDCl3) d 7.4 (C-18); 26.6 (C-19); 27.9 (C(CH3)3); 28.6
(C-17); 40.2 (C-20); 42.2 (C-6); 42.3 (C-5); 51.6
(CO2CH3); 54.1 (C-7); 64.5 (C-21); 82.9 (C(CH3)3);
109.4 (C-16); 116.0 (C-12); 120.9 (C-9); 123.2 (C-15);
123.9 (C-10); 128.2 (C-11); 135.9 (C-8); 140.4 (C-13);
145.0 (C-14); 149.9 (C-2); 150.5 (CO2C(CH3)3); 161.2
1
(C-3); 167.1 (CO2CH3); IR (CHCl3) n 1726; 1664 cm
;
MS (CI+) m/z 451 ([M+H]+); 351. Anal. calcd (found)
for C26H30N2O5: C, 69.31 (69.07); H, 6.71 (6.93); N,
6.22 (5.93); O, 17.76 (18.11).
(Na-tert-Butoxycarbonyl)tabersonine (19). To a solution
of 18 (0.4 g, 0.89 mmol) in anhydrous tetrahydrofuran
(20 mL) at 20ꢀC was added dropwise a solution of
diisobutylaluminum hydride 1 M in hexane (4.5 mL,
4.5 mmol). The mixture was stirred at 20ꢀC for 15 min
14ꢀ-Benzyl-(Na-tert-butoxycarbonyl)vincadi€ormine (17).
Colorless oil (100%); [a]2d5 +99.7 ꢀ (c 1.05, CHCl3); H
1

2968
C. Dupont et al. / Bioorg. Med. Chem. 7 (1999) 2961±2969
and then quenched carefully with water. After the gas
evolution has ®nished, sodium hydroxide was added
and the solution was stirred until the aqueous layer
was extracted with ethyl acetate (2Â). The combined
organic layers were dried over sodium sulfate and the
solvent was removed in vacuo to give 0.45 g of a
yellowish oil. The residue was then chromatographed
on silica gel (eluent heptane:acetone, 80:20) to a€ord
19 (0.37 g, 95%): [a]2d5 +34.8 ꢀ (c 1.23, CHCl3); 1H
NMR (300 MHz, CDCl3) d 0.61 (3H, t, J=7.4 Hz, H-
18); 0.97 (1H, qd, J=14.8, 7.4 Hz, H-19); 1.10 (1H, qd,
J=14.8, 7.4 Hz, H-19); 1.55 (9H, s, C(CH3)3); 1.78 (1H,
m, H-6b); 2.16 (1H, m, H-6a); 2.29 (1H, m, H-17b); 2.51
(1H, m, H-5b); 2.65 (1H, s, H-21); 2.73 (1H, m, H-17a);
3.04 (1H, m, H-5a); 3.09 (1H, m, H-3b); 3.50 (1H, m, H-
3a); 3.76 (3H, s, CO2CH3); 5.65 (1H, m, H-15); 5.81
(1H, m, H-14); 7.03 (1H, m, H-10); 7.19 (1H, m, H-9);
7.22 (1H, m, H-11); 7.66 (1H, m, H-12); 13C NMR
(75 MHz, CDCl3) d 7.6 (C-18); 26.5 (C-19); 28.3
(C(CH3)3); 31.4 (C-17); 41.6 (C-20); 43.0 (C-6); 51.0 (C-
5); 51.5 (C-3); 51.6 (CO2CH3); 52.6 (C-7); 68.0 (C-21);
82.3 (C(CH3)3); 111.7 (C-16); 116.1 (C-12); 121.0
(C-9); 123.5 (C-10); 125.2 (C-14); 127.5 (C-11); 132.8 (C-
15); 138.5 (C-8); 140.7 (C-13); 150.1 (C-2); 151.2
(CO2C(CH3)3); 168.2 (CO2CH3); IR (CHCl3) n 1719
cm 1; MS (CI+) m/z 437 ([M+H]+); 337; HRMS
(CI+) m/z calcd (found) for C26H33N2O4 (MH+):
437.2440 (437.2440).
(250 MHz, CDCl3) d 0.49 (3H, t J=7.3 Hz, H-18); 0.65
(2H, m, 2H-19); 0.78 (1H, m, H-15b); 1.56 (1H, m, H-
15a); 1.60 (1H, m, H-17); 1.65 (1H, m, H-6b); 1.90 (1H,
m, H-3b); 2.15 (1H, m, H-6a); 2.21 (1H, m, H-14a); 2.42
(2H, s, H-21+ CH2-È); 2.43 (1H, m, H-17); 2.58 (2H,
m, H-5+CH2-È); 2.74 (1H, m, H-16); 3.04 (1H, m, H-
16); 3.10 (2H, m, H-3a+H-5a); 7.18 (4H, m, H-10+H-
Èo+H-Èp); 7.30 (4H, m, H-9+H-11+H-Èm); 7.51
(1H, m, H-12); 13C NMR (75 MHz, CDCl3) d 7.4 (C-
18); 23.9 (C-16); 28.2 (C-17); 29.8 (C-19); 34.7 (C-14);
35.5 (C-6); 36.9 (C-20); 41.1 (C-15); 41.2 (CH2-È); 54.3
(C-5); 57.5 (C-3); 61.1 (C-7); 78.9 (C-21); 120.2 (C-12);
121.1 (C-9); 125.2 (C-10); 126.0 (C-Èp); 127.6 (C-11);
128.4 (C-Èm); 129.0 (C-Èo); 140.3 (Cq-È); 147.1 (C-8);
154.7 (C-13); 192.0 (C-2); IR (CHCl3) n 2967, 1578,
1455, 1243 cm 1; MS (CI+) m/z 371 ([M+H]+);
HRMS (CI+) m/z calcd (found) for C26H31N2 (MH+):
371.2487 (371.2471).
(+)-1,2,14,15-Tetradehydroaspidospermidine (22). Yel-
1
low oil (95%); [a]d25 +135.0 (c 1.24, CHCl3); H NMR
(250 MHz, CDCl3) d 0.57 (3H, t, J=7.3 Hz, H-18); 0.90
(2H, q, J=7.3 Hz, 2H-19); 1.68 (1H, m, H-6b); 1.74
(1H, m, H-17); 2.28 (1H, m, H-6a); 2.55 (1H, m, H-17);
2.73 (1H, s, H-21); 2.83 (1H, m, H-5b); 2.86 (1H, m, H-
16); 2.99 (1H, m, H-16); 3.10 (1H, m, H-3b); 3.31 (1H,
m, H-5a); 3.52 (1H, m, H-3a); 5.52 (1H, m, H-15); 5.69
(1H, m, H-14); 7.16 (1H, m, H-10); 7.29 (1H, m, H-11);
7.35 (1H, m, H-9); 7.52 (1H, m, H-12); 13C NMR
(62.5 MHz, CDCl3) d 8.1 (C-18); 24.6 (C-16); 27.4 (C-
19); 29.8 (C-17); 35.6 (C-6); 40.3 (C-20); 51.4 (C-3); 53.3
(C-5); 60.8 (C-7); 73.1 (C-21); 120.0 (C-12); 121.1 (C-9);
124.5 (C-14); 125.2 (C-10); 127.6 (C-11); 134.2 (C-15);
147.3 (C-8); 154.2 (C-13); 190.1 (C-2); IR (CHCl3) n
2968, 1575, 1456, 1251 cm 1; MS (CI+) m/z 279
([M+H]+); HRMS (CI+) m/z calcd (found) for
C19H23N2 (MH+): 279.1861 (279.1861).
Preparation of 1,2-didehydroaspidospermidine analogues
20, 21, 22. General procedure. A suspension of 16, 17,
19 (2.96 mmol) in hydrochloric acid 12 N (15 mL) was
heated to re¯ux (110ꢀC) for 5 min. The yellow solution
was then cooled to 0ꢀC and a 32% ammonia solution
was slowly added until pHꢁ13. The white suspension
was extracted with methylene chloride (3Â). Then the
combined organic layers were dried over sodium sulfate
and the solvent was evaporated in vacuo to yield 20, 21
or 22, respectively.
`One pot' preparation of ( )-rhazinilam analogues 3, 4,
5 and 6. Compounds 3, 4, 5 and 6 were obtained from
20, 21, 22 and 23, respectively, following the experi-
mental conditions described for the `one pot' prepara-
tion of rhazinilam (1) (see above).
14ꢀ-Ethyl-1,2-didehydro-aspidospermidine (20). Yellow
oil (100%); [a]d25 +214.6 (c 0.49, CHCl3); 1H NMR
(300 MHz, CDCl3) d 0.50 (3H, t, J=7.4 Hz, H-18); 0.65
(3H, m, 2H-19+H-15b); 0.92 (3H, t, J=7.4 Hz, CH-
CH2-CH3); 1.23 (2H, m, CH-CH2-CH3); 1.56 (1H, m,
H-15a); 1.61 (1H, m, H-17); 1.65 (1H, m, H-6b); 1.81
(2H, m, H-3b+H-14a); 2.18 (1H, m, H-6a); 2.39 (1H, s,
H-21); 2.44 (1H, m, H-17); 2.60 (1H, m, H-5b); 2.76
(1H, m, H-16); 3.09 (1H, m, H-16); 3.18 (1H, m, H-5a);
3.22 (1H, m, H-3a); 7.15 (1H, m, H-10); 7.27 (1H, m, H-
11); 7.32 (1H, m, H-9); 7.51 (1H, m, H-12); 13C NMR
(75 MHz, CDCl3) d 7.3 (C-18); 11.6 (CH-CH2-CH3);
23.7 (C-16); 27.2 (CH-CH2-CH3); 27.9 (C-17); 29.7 (C-
19); 34.2 (C-14); 35.4 (C-6); 36.5 (C-20); 40.7 (C-15);
54.3 (C-5); 57.4 (C-3); 60.9 (C-7); 78.9 (C-21); 120.0 (C-
12); 120.9 (C-9); 125.1 (C-10); 127.4 (C-11); 147.0 (C-8);
154.4 (C-13); 192.1 (C-2); IR (CHCl3) n 2964, 1577,
1455, 1216 cm 1; MS (CI+) m/z 309 ([M+H]+);
HRMS (CI+) m/z calcd (found) for C21H29N2 (MH+):
309.2331 (309.2332).
14ꢀ-Ethylrhazinilam (3). Puri®ed by chromatography
(eluent heptane:acetone, 70:30), 3 was obtained as a white
amorphous solid (50%); [a]2d5 316.5 ꢀ (c 1.37, CHCl3); 1H
NMR (250 MHz, CDCl3) d 0.71 (3H, t, J=7.3 Hz, H-
18); 0.99 (3H, t, J=7.3 Hz, CH-CH2-CH3); 1.22 (1H,
dq, J=14.6, 7.3 Hz, H-19); 1.27 (2H, q, J=7.3 Hz, CH-
CH2-CH3); 1.47 (3H, m, H-19+2H-15); 1.50 (1H, m, H-
17b); 1.94 (1H, m, H-16a); 2.23 (1H, m, H-14a); 2.37
(1H, m, H-16b); 2.47 (1H, m, H-17a); 3.34 (1H, m, H-
3b); 4.05 (1H, m, H-3a); 5.76 (1H, m, H-6); 6.49 (1H, m,
H-5); 6.68 (1H, bs, NH); 7.20 (1H, m, H-12); 7.32 (2H,
m, H-10+H-11); 7.43 (1H, m, H-9); 13C NMR
(75 MHz, CDCl3) d 8.3 (C-18); 11.3 (CH-CH2-CH3);
27.2 (CH-CH2-CH3); 28.1 (C-16); 30.1 (C-19); 31.7 (C-
14); 37.3 (C-17); 39.5 (C-20); 40.3 (C-15); 51.8 (C-3);
109.9 (C-6); 117.2 (C-7); 119.1 (C-5); 126.9 (C-12);
127.3+128.1 (C-10+C-11); 130.6 (C-20); 131.6 (C-9);
138.2 (C-8); 140.4 (C-13); 177.5 (C-2); IR (CHCl3) n
1665 cm 1; MS (CI+) m/z 323 ([M+H]+); HRMS
14ꢀ-Benzyl-1,2-didehydroaspidospermidine (21). Yellow
oil (100%); [a]d25 +183.7 ꢀ (c 1.75, CHCl3); H NMR
1

C. Dupont et al. / Bioorg. Med. Chem. 7 (1999) 2961±2969
2969
(CI+) m/z calcd (found) for C21H27N2O (MH+):
323.2124 (323.2116).
cadi€ormine. We also express our appreciation to C.
Gaspard for the cytotoxicity assays and to P. Pineau for
the high scale synthesis of 3-oxo-(Na-tert-butox-
ycarbonyl)-vincadi€ormine.
14ꢀ-Benzylrhazinilam (4). Puri®ed by chromatography
(eluent heptane:acetone, 80:20), 4 was obtained as a pale
yellow oil (50%); [a]2d5 292.5 ꢀ (c 1.00, CHCl3); 1H
NMR (300 MHz, CDCl3) d 0.70 (3H, t, J=7.4 Hz, H-
18); 1.21 (1H, qd, J=14.8, 7.4 Hz, H-19); 1.43 (1H, m,
H-17b); 1.46 (1H, qd, J=14.8, 7.4 z, H-19); 1.50 (2H, m,
H-15); 1.92 (1H, m, CH-16a); 2.35 (1H, m, CH-16b);
2.44 (1H, m, H-17a); 2.66 (3H, m, CH-14a+CH2-È);
3.41 (1H, m, H-3b); 3.90 (1H, m, H-3a); 5.74 (1H, m, H-
6); 6.43 (1H, m, H-5); 6.71 (1H, bs, NH); 7.18 (1H, m,
H-12); 7.21 (3H, m, H-Èo+H-Èp); 7.31 (4H, m, CH-
10+CH-11+H-Èm); 7.41 (1H, m, H-9); 13C NMR
(75 MHz, CDCl3) d 8.2 (C-18); 28.0 (C-16); 29.9 (C-19);
31.9 (C-14); 37.4 (C-17); 39.5 (C-20); 40.3 (C-15); 40.9
(CH2-È); 51.7 (C-3); 110.0 (C-6); 117.2 (C-7); 119.1 (C-
5); 126.4 (C-Èp); 126.9 (C-12); 127.2+128.1 (C-10+C-
11); 128.6 (C-Èm); 129.0 (C-Èo); 130.3 (C-21); 131.5 (C-
9); 138.1 (C-8); 139.1 (Cq-È); 140.4 (C-13); 177.5 (C-2);
IR (CHCl3) n 1662 cm 1; MS (CI+) m/z 385
([M+H]+); HRMS (CI+) m/z calcd (found) for
C26H29N2O (MH+): 385.2280 (385.2277).
References and Notes
1. David, B.; Sevenet, T.; Morgat, M.; Guenard, D.; Moisand,
A.; Tollon, Y.; Thoison, O.; Wright, M. Cell Motility and the
Cytoskeleton 1994, 28, 317.
2. Linde, H. H. A. Helv. Chim. Acta 1965, 48, 1822.
3. Abraham, D. J.; Rosenstein, R. D.; Lyon, R. L.; Fong, H.
H. S. Tetrahedron Lett. 1972, 10, 909.
4. De Silva, K. T.; Ratcli€e, A. H.; Smith, G. F.; Smith, G. N.
Tetrahedron Lett. 1972, 10, 913.
5. Ratcli€e, A. H.; Smith, G. F.; Smith, G. N. Tetrahedron
Lett. 1973, 52, 5179.
6. This research work is a continuation of the collaborative
program initiated in 1981 by Dr T. Sevenet (ICSN-CNRS,
Gif-sur-Yvette), Dr K. C. Chan and Dr H. Hamid (University
of Malaya, Kuala Lumpur). This program was devoted to the
search of new active molecules from the Malaysian ¯ora.
7. Thoison, O.; Guenard, D.; Sevenet, T.; Kan-Fan, C.; Quir-
ion, J.-C.; Husson, H.-P.; Deverre, J.-R.; Chan, K.-C.; Potier,
P. C. R. Acad. Sc. Paris 11 1987, 304, 157.
8. David, B.; Sevenet, T.; Thoison, O.; Awang, K.; Paõs, M.;
Wright, M.; Guenard, D. Biorg. Med. Chem. Lett. 1997, 7,
2155.
9. David, B. Ph.D. Dissertation, Universite Rene Descartes de
Paris, France, 1990.
10. Alazard, J.-P.; Millet-Paillusson, C.; Boye, O.; Guenard,
D.; Chiaroni, A.; Riche, C.; Thal, C. Bioorg. Med. Chem. Lett.
1991, 1, 725.
11. Alazard, J.-P.; Millet-Paillusson, C.; Guenard, D.; Thal, C.
Bull. Soc. Chim. Fr. 1996, 133, 251.
12. Pascal, C.; Dubois, J.; Guenard, D.; Gueritte, F. J. Org.
Chem. 1998, 63, 6414.
13. Pascal, C.; Dubois, J.; Guenard, D.; Tchertanov, L.;
Thoret, S.; Gueritte, F. Tetrahedron 1998, 54, 14737.
14. Kreher, R.; Setibert, J. Angew. Chem., Int. Ed. Engl. 1964,
3, 639.
15. Levy, J.; Soufyane, M.; Mirand, C. Doe de Maindreville,
Royer, D. Tetrahedron Asymetry 1997, 8, 4127.
16. Picot, A.; Lusinchi, X. Synthesis 1975, 109.
17. Blowers, J. W.; Saxton, J. E.; Swanson, A. G. Tetrahedron
1986, 42, 6071.
18. Curran, W. V.; Angier, R. B. J. Org. Chem. 1966, 31, 3867.
19. Thielke, D.; Wegener, J.; Winterfeldt, E. Angew. Chem.,
Int. Ed. Engl. 1974, 13, 602.
( )-14,15-Didehydrorhazinilam (5). Puri®ed by chroma-
tography (eluent heptane:acetone, 70:30), 20 was
obtained as a white amorphous solid (50%); [a]d25
293.5 ꢀ (c 1.31, CHCl3); H NMR (300 MHz, CDCl3)
1
d 0.66 (3H, t, J=7.4 Hz, H-18); 1.04 (1H, dq, J=14.8,
7.4 Hz, H-19); 1.61 (1H, dq, J=14.8, 7.4 Hz, H-19); 1.73
(1H, m, H-17); 2.00 (1H, m, H-16a); 2.06 (1H, m, H-17);
2.35 (1H, m, H-16a); 4.47 (2H, m, H-3); 4.56 (1H, m, H-
3); 5.53 (1H, m, H-15); 5.88 (1H, m, H-6); 6.62 (1H, m,
H-5); 6.94 (1H, bs, NH); 7.25 (1H, m, H-12); 7.32+7.36
(2H, m, H-10+H-11); 7.40 (1H, m, H-9); 13C NMR
(75 MHz, CDCl3) d 9.1 (C-18); 28.5 (C-16); 29.8 (C-19);
39.6 (C-17); 41.7 (C-20); 44.4 (C-3); 110.1 (C-6); 116.2
(C-7); 118.5 (C-5); 119.6 (C-14); 127.1 (C-12); 127.4 (C-
11); 127.8 (C-21); 128.1 (C-10); 131.0 (C-9); 134.5 (C-
15); 138.1 (C-8); 139.8 (C-13); 177.0 (C-2); IR (CHCl3) n
1665 cm 1; MS (CI+) m/z 293 ([M+H]+); HRMS
(CI+) m/z calcd (found) for C19H21N2O (MH+):
293.1654 (293.1663).
(+)-14,15-Didehydrorhazinilam (6). Puri®ed by chro-
matography (eluent heptane:acetone, 70:30), 6 was
obtained as a white amorphous solid (50%); [a]d25
+292.9 ꢀ (c 1.28, CHCl3); HRMS (CI+) m/z calcd
(found) for C19H21N2O (MH+): 293.1654 (293.1660).
20. Hoizey, M. J.; Sigaut, C.; Le Men-Olivier, L.; Levy, J.; Le
Men, J. Tetrahedron Lett. 1974, 1601.
21. Henriques, A.; Kan, C.; Chiaroni, A.; Riche, C.; Husson,
H.-P.; Kan, S.-K.; Lonasmaa, M. J. Org. Chem. 1982, 47, 803.
22. Borenfreund, E.; Puerner, J. A. Toxicology Lett. 1985, 24,
119.
23. Atomic coordinates, bond distances and angles, and ther-
mal parameters have been deposited at the Cambridge Crys-
tallographic Data Center with the deposition number CCDC
114221 and can be obtained on request at CCDC, Union
Road, Cambridge C82 IEZ, UK.
24. Sheldrick, G. M. SHELXL86. Program for the Solution of
Crystal Structures, University of Gottingen, Germany, 1986.
25. Sheldrick, G. M. SHELXL93. Program for the Structure
Determination, University of Cambridge, UK, 1993.
Acknowledgements
We are very grateful to Professor P. Potier for his con-
tinuous interest and encouragement and to Dr T. Seve-
net and Dr C. Thal for helpful discussions. We would
like to thank RPR and CNRS for a grant (to C.D.) and
J. Hannart (Omnium Chimique) for a gift of (+)-vin-