Elaboration of simplified vinca alkaloids and phomopsin hybrids

Article abstract of DOI:10.1111/j.1747-0285.2009.00922.x

Nine simplified vinca alkaloids and phomospin A hybrids, in which vindoline moiety has been replaced by a simpler scaffold, have been elaborated to evaluate their activity on the inhibition of tubulin polymerization. This article deals with the synthesis of various simplified vinca alkaloids, using a stereoselective coupling of catharantine with reactive aromatic compounds and methanol as well as their subsequent condensation with a large peptide chain mimicking those of phomopsin A. Biological evaluation and molecular modeling studies are also reported.

Full text of DOI:10.1111/j.1747-0285.2009.00922.x

ª 2010 John Wiley & Sons A/S
doi: 10.1111/j.1747-0285.2009.00922.x
Chem Biol Drug Des 2010; 75: 284–294
Research Article
Elaboration of Simplified Vinca Alkaloids and
Phomopsin Hybrids
opposite directions (Figure 1). In particular, phomopsin A possess
extensive contacts with Tyr b224, one of the residues sandwiching
the GDP–GTP nucleotide exchange site. Microtubule instability
depends on GTP hydrolysis so that, this activity is crucial to the
regulation of microtubule assembly.
Quoc Anh Ngo, Fanny Roussi*, Sylviane
´
Thoret and Franc¸oise Gueritte
Centre de recherche de Gif, Institut de Chimie des Substances
Naturelles, CNRS, Avenue de la Terrasse, 91198 Gif-sur-Yvette
Cedex, France
*Corresponding author: Fanny Roussi, fanny.roussi@icsn.cnrs-gif.fr
Since the publication of these important results, we have been
involved in the elaboration of original compounds that could inter-
fere with both binding sites (that of phomopsin and that of vinblas-
tine) as these compounds may have increased biological activities
(see for example 10) and should permit a better understanding of
the interactions of drugs with tubulin.
Nine simplified vinca alkaloids and phomospin A
hybrids, in which vindoline moiety has been
replaced by a simpler scaffold, have been elabo-
rated to evaluate their activity on the inhibition of
tubulin polymerization. This article deals with the
synthesis of various simplified vinca alkaloids,
using a stereoselective coupling of catharantine
with reactive aromatic compounds and methanol
as well as their subsequent condensation with a
large peptide chain mimicking those of phomopsin
A. Biological evaluation and molecular modeling
studies are also reported.
In that way, in a previous article (11), we have reported the elabo-
ration of ten original vinca alkaloids-phomopsin A hybrids bridged
via an ammonium salt linking anhydrovinblastine (12,13) or vinorel-
bine (14) to the octahydrophomopsin lateral chain (Figure 1). Some
of these, among which, compounds 1 and 2 (in spite of their large
size), are very active on the inhibition of tubulin polymerization and
present a strong cytotoxicity. These results are very promising
because, among the many SAR studies that have been carried out
on vinblastine derivatives, most have shown that even a slight mod-
ification, particularly on the cleavamine moiety, dramatically
decreases the activity on tubulin. Nevertheless, NMR, molecular
modeling studies and GDP–GTP exchange experiments showed that
the orientation of the added peptide chains of these hybrids is not
the same as that of phomopsin A. In particular, these peptide
chains do not interact with Tyr b224 near the GDP–GTP nucleotide
exchange site. The excellent biological activities of these hybrids on
tubulin was explained by the fact that even if the peptide side
chain of all these compounds is not oriented in the same direction
as that of phomopsin, they may still be accommodated at the inter-
face of tubulin a and b, in a pocket distinct from that of the pho-
mopsin A peptide.
Key words: phomopsin A, tubulin, vinca alkaloids
Received 27 March 2009, revised 12 November 2009 and accepted for
publication 14 November 2009
Tubulin-binding molecules are one of the most important classes of
anticancer agents with major drugs on the market and many prom-
ising compounds in clinical trials (1). Most of these are derived
from complex natural products. They interfere with microtubule
assembly and functions, thus resulting in mitotic arrest of eukary-
otic cells. On-going efforts are still made to elaborate potent new
derivatives and to better understand their interaction with tubulin.
Bisindole alkaloids like vinblastine (for a general review see 2)
known since the end of the 1950s (3,4) are one of these antimitotic
drugs, inhibiting tubulin polymerization into microtubules and are
commonly used in cancer chemotherapy (5–7). Knossow and collea-
gues published the 4.1 ꢀ X-ray structures of vinblastine (8) and
phomopsin A (9) (another inhibitor of tubulin polymerization that
binds in the vinca domain) bound to a tubulin-colchicine ⁄ RB3-SLD
complex ((Tc)2R). Superimposition of both binding sites showed that
they significantly overlap: the cleavamine moiety of vinblastine and
the macrocycle of phomopsin A occupy the same area and define
the core of the vinca domain. However, the vindoline moiety of
vinblastine and the lateral chain of phomopsin A are oriented in
We are now willing to determine the real impact of the added
peptide chains on the biological activity of these ammonium
salts. If the peptide chains have a real influence on activity, a
simplification of the vinca part (particularly on the vindoline moi-
ety) should maintain the activity. On the contrary, supposing that
the added peptide chains have no impact on the biological activ-
ity of these ammonium salts, a simplification of the vinblastine
moiety should decrease it. To address this question, we have
elaborated a set of new compounds in which vindoline moiety
was replaced by a simpler scaffold i.e. a methoxy group or
simple aromatics.
284

Elaboration of Simplified Vinca Alkaloids and Phomopsin Hybrids
Figure 1: Previous synthezised anhydrovinblastine- and vinorelbine-phomopsine A hybrids.
(1H, s, 17¢-H), 6.35 (1H, s, 14¢-H), 7.13 – 7.23 (3H, m, 12-H,13-H,14-
H), 7.73 (1H, d, J 7.7 Hz, 11-H), 8.25 (1H, s, 16-H); dC(75 MHz;
CDCl3) 8.5 (C-21¢), 11.7 (C-21), 19.6 (C-8), 20.9 (C-27¢), 27.2 (C-20),
30.7 (C-2), 30.9 (C-20¢), 33.7 (C-1), 38.0 (C-23¢), 42.3 (C-5¢), 45.9 (C-
11¢), 50.2 (C-8¢); 50.3 (C-10¢); 52.0 (C-25¢); 52.5 (C-26); 53.3 (C-12¢);
53.4 (C-7), 53.5 (C-23); 54.1 (C-18); 55.4 (C-22¢), 56.8 (C-5); 62.2 (C-
24); 63.9 (C-19), 65.2 (C-19¢), 76.4 (C-4¢), 79.8 (C-3¢); 83.0 (C-2¢),
94.5 (C-17¢), 111.0 (C-14), 111.1 (C-9), 118.0 (C-11), 118.3 (C-15¢),
120.8 (C-12), 121.6 (C-3), 122.4 (C-14¢), 123.7 (C-13), 124.4 (C-13¢),
125.3 (C-7¢), 129.3 (C-10), 129.8 (C-6¢); 132.7 (C-17); 133.0 (C-4),
134.5 (C-15), 154.1 (C-18¢), 158.3 (C-16¢), 170.7 (C-24¢); 171.0 (C-
Experimental Section
General information
Dichloromethane was distilled over P2O5. N,N-Dimethylformamide
was distilled over CaH2. Flash column chromatography was performed
on silica gel (60 ꢀ, 230–400 mesh). All the commercially available
chemicals were used as purchased. H and 13C NMR spectra were
1
recorded on a Brꢁker AC 300 or ARX 500 spectrometer. Chemical
shifts (d) are reported in p.p.m. relative to tetramethylsilane as an
internal standard. The spectra were fully assigned using correlation
spectroscopy (COSY), heteronuclear multiple-quantum coherence
(HMQC) and heteronuclear multiple-bond correlation (HMBC). High
resolution mass spectra were performed on a AQA Navigator Thermo-
Quest. IR spectra were recorded on a Perkin–Elmer Spectrum BX
spectrometer. The [a]D were measured using a JASCO P-1010 polar-
imeter. The circular dichroꢂsm spectrum was recorded on a JASCO
J-810 spectrometer. Catharanthine was a gift from Jacques Fahy and
Philippe Maillas (Institut de Recherche Pierre Fabre,Toulouse, France).
22
25¢); 171.6 (C-25); 173.3 (C-22); [a]D 52 (c 0.8 in CHCl3); m ⁄ z (ESI)
865.4341 (M+. C49H61N4O10 requires 865.4388), 865.1 (100%).
N-Methyl-5, 7-dimethoxyindoline (8)
To a stirred solution of 4,6-dimethoxyindole (114 mg, 0.64 mmol) in
anhydrous DMF (3 mL) at room temperature, under argon was
added NaH (52 mg, 1.30 mmol) and iodomethane (60 lL,
0.96 mmol). The resulting suspension was stirred at room tempera-
ture for 4 h and concentrated under reduced pressure. The residue
was dissolved in ethyl acetate and the crude mixture was washed
with brine. The organic layer was dried over magnesium sulfate
and concentrated under reduced pressure, to afford N-methyl-4,6-
dimethoxyindole 7 (134 mg, 100%) that was used without further
purification. To a solution of N-methyl-4,6-dimethoxyindole (134 mg,
0.64 mmol) in glacial acetic acid (3 mL) was added slowly, under
argon at 10 ꢀC, sodium cyanoborohydryde (132 mg, 2.10 mmol). The
resulting solution was stirred for 1 h at the same temperature.
Water was then added and the reaction mixture was cooled to
0 ꢀC and neutralized carefully with sodium hydroxyde pellets. The
aqueous layer was extracted with ethyl acetate (x3). The combined
organic layer was washed with brine, dried over magnesium sulfate
and concentrated in vacuo to give pure N-methyl-4,6-dimethoxyindo-
line (111 mg, 90%): dH(500 MHz; CDCl3) 2.67 (3H, s, CH3N), 2.77
(2H, t, J 8.4, NCH2CH2), 3.25 (2H, t, J 8.4, NCH2CH2), 3.72 (6H, s, 2
OCH3), 5.73 (1H, d, J 1.3, CHar), 5.82 (1H, d, J 1.3, CHar), dC(75
MHz; CDCl3) 25.2 (C-3), 36.2 (C-10), 55.4 (C-11), 55.5 (C-12), 56.6 (C-
2), 87.6 (C-6), 88.6 (C-8), 108.3 (C-4), 155.4 (C-5), 156.0 (C-7), 161.5
Compound 4
Anhydrovinblastine (22 mg, 0.05 mmol) and methyl bromoacetate
(8 mg, 0.05 mmol) were stirred at room temperature in acetonitrile
for 2 h. The solvent was removed under reduced pressure and the
crude mixture was purified by column chromatography on silica
(97:3 dichloromethane ⁄ methanol then a gradient of methanol) to
give compound 4 (36 mg, 77%). mmax(neat) ⁄ cm 1741, 1233; dH(500
MHz; CDCl3) 0.77 (3H, t, J 7.3 Hz, 21¢-H), 0.98 (3H, t, J 7.3 Hz, 21-
H), 1.28 (1H, m, 20¢-H), 1.68 (1H, m, 20¢-H), 1.94 (1H, m, 2-H), 1.97
(1H, m, 11¢-H), 1.98 (2H, m, 20-H), 2.03 (3H, s, 27¢-H), 2.15 (1H, m,
11¢-H), 2.51 (1H, m, 1-H), 2.66 (1H, m, 10¢-H), 2.67 (3H, s, 23¢-H),
2.70 (1H, s, 19¢-H), 2.84 (1H, d, J 15.5 Hz, 8¢-H), 3.01 (1H, m, 1-H),
3.19 (1H, m, 8-H), 3.25 (1H, m, 10¢-H), 3.27 (1H, d, J 15.5 Hz, 8¢-H),
3.60 (3H, s, 23-H), 3.71 (1H, s, 2¢-H), 3.72 (3H, s, 26-H), 3.77 (3H, s,
25¢-H), 3.82 (3H, s, 22¢-H), 3.91 (1H, m, 7-H), 3.92 (1H, m, 8-H), 4.21
(1H, d, J 16.8 Hz, 19-H), 4.37 (2H, s, 5-H), 4.43 (1H, m, 7-H), 4.58
(1H, d, J 16.8 Hz, 24-H), 4.90 (1H, d, J 16.8 Hz, 24-H), 4.91 (1H, d,
J 16.8Hz, 19-H), 5.26 (1H, d, J 11.5 Hz, 6¢-H), 5.32 (1H, s, 4¢-H),
5.51 (1H, s, 3-H), 5.82 (1H, dd, J 11.5 Hz and 4.5 Hz, 7¢-H), 6.04
Chem Biol Drug Des 2010; 75: 284–294
285

Ngo et al.
(C9); m ⁄ z(ESI) 194.1178 (M + H+. C11H16NO2 requires 194.118),
194.1(100%).
J 7.4, 21-H), 1.68 (1H, m, 2-H), 1.87 (2H, q, J 7.4, 20-H), 2.00 (1H,
d, J 14.9, 1-H), 2.50 (1H, d, J 12.8, 19-H), 2.81 (1H, dd, J 14.5 and
4.6, 8-H), 3.00 (1H, dd, J 13.6 and 4.6, 7-H), 3.13 (1H, m, 1-H), 3.15
(1H, m, 7-H), 3.30 (2H, m, 8-H, 5-H), 3.40 (1H, m, 5-H), 3.54 (1H, m,
19-H), 3.66 (3H, s, 23-H), 3.72 (6H, s, 7¢-H and 8¢-H), 5.46 (1H, d, J
7.2, 3-H), 5.88 (2H, s, 6¢-H), 6.93 – 7.05 (2H, m, 12-H and 13-H),
7.07 (1H, d, J 7.1, 11-H), 7.38 (1H, d, J 7.1, 14-H), 8.00 (1H, s, 16-
H); dC(75 MHz; CDCl3) 12.4 (C-21), 25.3 (C-8), 28.3 (C-20), 33.4 (C-2),
36.2 (C-1), 47.5 (C-19), 51.9 (C-5), 53.5 (C18), 54.4 (C-7), 55.3 (C-23),
55.9 (C-7¢, C-8¢), 92.7 (C-2¢, C-6¢), 106.5 (C-4¢), 109.8 (C-14), 113.1
(C-9), 118.2 (C-11), 118.4 (C-12), 121.3 (C-13), 125.7 (C-3), 129.4 (C-
10), 132.7 (C-17), 135.2 (C-15), 140.0 (C-4), 147.8 (C-1¢), 165.4 (C-3¢,
C-5¢), 175.7 (C-22); [a]D22+29 (c 0.85 in CHCl3); m ⁄ z (ESI)
490.2682 (M + H+. C29H36N3O4 requires 490.2706), 490.2 (100%);
k(EtOH) (De) 224 (+20), 256 (+12).
5,7-Dimethoxy-3-methyl-2,3-dihydrobenzofuran (10)
A solution of N-iodosuccinimide (730 mg, 3.24 mmol) in anhydrous di-
chloromethane (10 mL) was added to a solution of 3,5-dimethoxyphe-
nol (500 mg, 3.24 mmol) in anhydrous dichloromethane (100 mL) at
0 ꢀC. After 4 h, the reaction mixture was concentrated under reduced
pressure. The residue was purified by column chromatography on sil-
ica (8:2 heptane ⁄ ethyl acetate) to afford a 90 ⁄ 10 inseparable mixture
of 2-iodo-3,5-dimethoxyphenol and 2,6-diiodo-3,5-dimethoxyphenol
(650 mg, 70%). A solution of bromoallyl (60 lL, 0.69 mmol), sodium
bicarbonate (166 mg, 1.20 mmol) and 2-iodo-3,5-dimethoxyphenol
(containing 10% of 2,6-diiodo-3,5-dimethoxyphenol) (176 mg, 0.53
mmol) in acetonitrile (15 mL) was refluxed for 3 h before being con-
centrated under reduced pressure. The residue was dissolved in ethyl
acetate and the crude mixture was washed with water and brine.
The organic layer was dried over magnesium sulfate and concen-
trated under reduced pressure to afford a 90 ⁄ 10 inseparable mixture
of 1-(allyloxy)-2-iodo-3,5-dimethoxybenzene and 1-(allyloxy)-2,6-diiod-
o-3,5-dimethoxybenzene (160 mg, 83%) that was used without
further purification. To a solution of the 90 ⁄ 10 inseparable mixture of
1-(allyloxy)-2-iodo-3,5-dimethoxybenzene and 1-(allyloxy)-2,6-diiodo-
3,5-dimethoxybenzene (160 mg, 0.44 mmol) in toluene (10 mL) were
added nBu3SnH (67 lL, 0.25 mmol) and AIBN (8 mg, 0.05 mmol). The
reaction mixture was refluxed under argon. After 2 h, nBu3SnH
(67 lL, 0.25 mmol) and AIBN (8 mg, 0.05 mmol) were added and
reflux was carried on until completion (TLC control). The solvent was
removed under reduced pressure. The crude product was purified by
column chromatography on silica (heptane then 95:5 heptane ⁄ ethyl
acetate) to afford 4,6-dimethoxy-3-methyl-2,3-dihydrobenzofuran 10
as a pale yellow oil (66 mg, 77%): dH(500 MHz; CDCl3) 1.21 (3H, d, J
6.8, CHCH3), 3.44 (1H, m, CHCH3), 3.68 (3H, s, OCH3), 3.72 (3H, s,
OCH3), 4.05 (1H, dd, J 8.5 and 5.4, OCH), 4.55 (1H, t, J 9.1, OCH), 5.97
(1H, d, J 1.9, CHar), 5.93 (1H, d, J 1.9, CHar), dC(75 MHz; CDCl3) 19.4
(C-10), 34.9 (C-3), 55.3 (C-11), 55.5 (C-12), 79.6 (C-2), 88.5 (C-6), 91.3
(C-8), 111.0 (C-4), 157.1 (C-5), 161.6 (C-7 and C-9), m ⁄ z (ESI) 195.1012
(M + H+. C11H15O3 requires 195.1021), 195.1(100%).
Polonovsky–Potier reaction for the preparation
of compounds 13 and 14
To a solution of catharantine N-oxide (180 mg, 0.51 mmol) in anhy-
drous dichloromethane (1.5 mL) was added trifluoroacetic anhydride
(284 lL) at -78 ꢀC in an atmosphere of argon. After 40 min, N-
methyl-4,6-dimethoxyindoline 8 (98 mg, 0.51 mmol) or 5,7-dimeth-
oxy-3-methyl-2,3-dihydrobenzofuran 10 (99 mg, 0.51 mmol) was
added, and the reaction mixture was stirred at the same tempera-
ture for 30 min then at room temperature for thirty additional min.
The solvent was removed under reduced pressure, the residue was
dissolved in methanol and sodium borohydride (5 eq) was added.
The reaction mixture was concentrated under reduced pressure at
room temperature and dissolved in dichloromethane. The organic
layer was washed with a saturated solution of sodium carbonate,
dried over magnesium sulfate, filtered and concentrated in vacuo.
The crude product was dissolved in a 7:3 mixture of heptane and
ethyl acetate and filtered on a pad of celite. After evaporation of
the solvents, the residue was stirred with sulfuric acid (1 eq) for
15 min in ethanol (6 mL) and the solvent was removed. The sulfate
salts were washed in a 2:8 mixture of ethyl acetate and heptane.
The pure free amines 13 (translucent oil, 148 mg, 56%) and 14
(162 mg, 60%) were then regenerated using standard conditions.
For 13, as this compound was prone to decomposition, it was
immediately transformed to compound 21.
18(S)-3¢,5¢-Dimethoxyanilinecleavamine (12)
Catharantine sulfate (43 mg, 0.10 mmol) and ferric chloride hexahy-
drate (135 mg, 0.50 mmol) were suspended in 5 mL of an equivolu-
metric mixture of glycine buffer (7.505 g of glycine and 5.85 g of
sodium chloride in 1 L of water) and 0.1 N hydrochloric acid in an
atmosphere of argon. After 10 min at room temperature, 3,5-dime-
thoxyaniline (16 mg, 0.10 mmol) was added and the reaction mix-
ture was stirred at 35 ꢀC for 3 h until the reaction mixture became
clear. Sodium borohydryde (15 mg, 0.40 mmol) in ammonium hydrox-
yde was added dropwise. The reaction mixture was extracted with
dichloromethane (2 · 10 mL). The combined organic layer was dried
over magnesium sulfate, filtered and concentrated under reduced
pressure. The crude product was dissolved in a 7:3 mixture of hep-
tane and ethyl acetate and filtered on a pad of celite. After evapo-
ration of the solvent, pure compound 12 (42 mg, 85%) was
obtained as an oil: mmax(neat) ⁄ cm 3375, 2931, 1715, 1613, 1459,
1219 and 1127; dH(500 MHz; CDCl3) -NH2 are missing- 0.92 (3H, t,
For 14: mmax(neat) ⁄ cm 2955, 2360, 1730, 1614, 1457, 1208, 1099;
dH(500 MHz; CDCl3) 0.93 (3H, t, J 7.4, 21-H), 1.20 (3H, d, J 7.1,
10¢-H), 1.56 (1H,m, 2-H), 1.88 (2H, q, J 7.4, 20-H), 2.05 (1H, m, 1-H),
2.49 (1H, m, 19-H), 2.84 (1H, m, 8-H), 3.00 (1H, m, 7-H), 3.13 (1H,
m, 1-H), 3.14 (1H, m, 7-H), 3.30 (2H, m, 8-H and 5-H), 3.40 (1H, m,
5-H), 3.43 (1H, m, 3¢-H), 3.57 (1H, m, 19-H), 3.70 (3H, s, 23-H), 3.78
(6H, s, 11¢-H and 12¢-H), 4.06 (1H, m, 2¢-H), 4.53 (1H, m, 2¢-H), 5.45
(1H, s, 3-H), 6.03 (1H, s, 8¢-H), 6.97 – 7.10 (3H, m, 12-H, 13-H and
14-H), 7.40 (1H, d, J 8.3, 11-H), 8.06 (1H, s, NH); dC(75 MHz;
CDCl3) 12.3 (C-21), 19.4 (C-10¢), 25.4 (C-8), 27.9 (C-20), 33.7 (C-2),
35.5 (C-3¢), 36.1 (C-1), 46.9 (C-19), 52.3 (C-5), 53.1 (C-18), 54.0 (C-7),
54.2 (C-23), 55.5 (C-11¢, C-12¢), 79.1 (C-2¢), 89.4 (C-8¢), 106.4 (C-6¢),
110.1 (C-14), 111.4 (C-4¢), 114.2 (C-9), 118.3 (C-11), 118.5 (C-12),
121.6 (C-13), 125.2 (C-3), 129.4 (C-10), 132.7 (C-17), 135.5 (C-15),
140.3 (C-4), 156.0 (C-5¢, C-7¢), 159.3 (C-9¢), 175.1 (C-22); m ⁄ z (ESI)
531.2854 (M + H+. C32H39N2O5 requires 531.2859), 531.2 (100%).
286
Chem Biol Drug Des 2010; 75: 284–294

Elaboration of Simplified Vinca Alkaloids and Phomopsin Hybrids
18(S)-Methoxycleavamine (15)
m, 1-H), 3.18 (1H, m, 5-H), 3.38 (1H, m, 3¢-H), 3.64 (3H, s, 23-H),
To a solution of catharantine N-oxide (2.8 g, 8.00 mmol) in anhy-
drous dichloromethane (32 mL) was added trifluoroacetic anhydride
(3.33 mL) at )78 ꢀC in an atmosphere of argon. After 40 min, anhy-
drous methanol (6.5 mL) was added and the reaction mixture was
stirred at the same temperature for 10 min then at room tempera-
ture for 30 additional min. The solvent was removed under reduced
pressure, the residue was dissolved in methanol and sodium boro-
hydride (5 eq) was added. The reaction mixture was concentrated
under reduced pressure at room temperature and dissolved in
dichloromethane. The organic layer was washed with a saturated
solution of sodium carbonate, dried over magnesium sulfate, filtered
and concentrated in vacuo. The crude product was purified by two
columns chromatography on silica (7 ⁄ 3 heptane ⁄ ethyl acetate then
95 ⁄ 5 dichloromethane ⁄ methanol) to give pure S-methoxycleavamine
15 (1.26 g, 43%) as a translucent oil: mmax(neat) ⁄ cm 3401, 2930,
1734, 1458 and 1220; dH(500 MHz; CDCl3) 0.96 (3H, t, J 7.5, 21-H),
1.90 (2H, q, J 7.5, 20-H), 2.09 (2H, m, 2-H and 19-H), 2.19 (1H, dd,
J 5.3 and 14.9, 1-H), 2.38 (1H, d, J 11.0, 19-H), 2.56 (1H, m, 5-H),
2.69 (1H, m, 5-H), 2.84 (2H m, 1-H and 8-H), 3.02 (1H, d, J 15.6,
7-H), 3.11 (1H, d, J 15.6, 7-H), 3.20 (3H, s, 22-H), 3.30 (1H, m, -8H),
3.58 (3H, s, H-24), 5.41 (1H d, J 4.7, H-3), 7.01 (1H, t, J 7.9
and 7.1, Har), 7.11 (1H, m, Har), 7.25 (1H, d, J 7.9, Har.), 7.47 (1H, d,
J 7.9, Har.), 8.50 (1H, s, NH1); dC(75 MHz; CDCl3) 12.8 (C-21),
26.3 (C-8), 28.0 (C-20), 33.7 (C-2), 38.6 (C-1), 51.7 (C-19), 52.3
(C-24), 52.6 (C-22), 53.0 (C-5), 56.6 (C-7), 82.5 (C-18), 111.0 (C-14),
113.8 (C-9), 119.2 (C-11), 119.4 (C-12), 122.6 (C-13), 124.7 (C-3),
128.6 (C-10), 129.5 (C-17), 135.4 (C-15), 140.1 (C-4), 170.7 (C-23);
m ⁄ z (ESI) 369.2181 (M + H+. C22H29N2O3 requires 369.2178), 369.2
(100%).
3.68 (1H, m, 19-H), 3.80 (1H, m, 5-H), 3.80 (6H, s, 11¢-H and 12¢-H),
4.09 (1H, m, 2¢-H), 4.28 (2H, m, 8-H), 4.28 (1H, m, 2¢-H), 5.67 (1H, s,
3-H), 7.03 - 7.21 (3H, m, 12-H, 13-H and 14-H), 7.68 (1H, d, J 7.2 ,
11-H), 8.59 (1H, s, NH); dC(75 MHz; CDCl3) 12.2 (C-21), 19.4 (C-10¢),
27.8 (C-20), 28.0 (C-2), 35.1 (C-1), 35.5 (C-3¢), 44.1 (C-19), 47.6 (C-8),
53.2 (C-5), 53.4 (C-18), 53.5 (C-23), 60.9 (C-11¢, C-12¢), 79.1 (C-2¢),
100.1 (C-8¢), 106.4 (C-6¢), 108.0 (C-9), 110.1 (C-14), 118.6 (C-11),
119.8 (C-12), 121.4 (C-4¢), 122.5 (C-13), 123.5 (C-3), 128.7 (C-10),
134.8 (C-15), 135.2 (C-4), 137.0 (C-17), 154.1 (C-5¢, C-7¢), 159.3 (C-
9¢), 174.5 (C-22); m ⁄ z (ESI) 595.1844 (79Br) (M + H+. C31H36BrN2O5
(79Br) requires 595.1808), 595.2 (100%).
18(S)-Methoxy-norcleavamine (18)
To a solution of 18(S)-methoxycleavamine 15 (400 mg, 1.08 mmol)
in anhydrous dichloromethane (5 mL) was added trifluoroacetic acid
(84 lL, 1.08 mmol) at 0 ꢀC and the reaction mixture was stirred for
15 min before being cooled to )78 ꢀC. A solution of N-bromosuc-
cinimide (210 mg, 1.18 mmol) in anhydrous dichloromethane
(2.5 mL) was then added. After 30 min, the reaction mixture was
neutralized by a 10% sodium bicarbonate solution. The aqueous
layer was extracted with dichloromethane and the combined
organic layer was dried over magnesium sulfate, filtered and con-
centrated under reduced pressure to afford a bromo intermediate
that was used without further purification. To a solution of this
compound (1.08 mmol) in a 1 ⁄ 1 mixture of tetrahydrofuran and
water (50 mL), under argon was added AgBF4 (250 mg, 1.28 mmol).
The reaction mixture was stirred for 3 h at 50 ꢀC and cooled to
room temperature. The solvent was removed under reduced pres-
sure at 25–30 ꢀC, the mixture was diluted by a 10% sodium bicar-
bonate solution and extracted three times with dichloromethane.
The combined organic layer was dried over magnesium sulfate, fil-
tered and concentrated. The crude product was purified by column
chromatography on aluminia (7:3 heptane ⁄ ethyl acetate) to afford
pure methoxy-norcleavamine 18 as a pale yellow oil (128 mg,
30%): mmax(neat) ⁄ cm 1458 and 1243; dH(500 MHz; CDCl3) 0.96 (3H,
t, J 7.4, 21-H), 1.86 (2H, q, J 7.4, 20-H), 2.12 (1H, m, 1-H), 2.43 (1H,
dd, J 10.5 and 16.2, 1-H), 2.60 (2H, m, 19-H and 2-H), 3.06 (1H, d,
J 17.5, 5-H), 3.28 (1H, d, J 15.3, 19-H), 3.37 (3H, s, 24-H), 3.65 (1H,
m, 5-H), 3.66 (3H, s, 22-H), 4.10 (2H, d, J 1.7, 8-H), 5.59 (1H, s, 3-
H), 7.03 (1H, t, J 7.0 and 7.7, Har), 7.11 (1H, t, J 7.0 and 7.8, Har),
7.25 (1H, d, J 8.0, Har), 7.59 (1H, d, J 7.8, Har), 8.70 (1H, s, NH);
dC(75 MHz; CDCl3) 12.1 (C-21), 28.0 (C-20), 29.0 (C-2), 35.1 (C-1),
44.1 (C-19), 46.8 (C-8), 52.0 (C-5), 52.7 (C-22), 55.1 (C-24), 80.9 (C-
18), 106.8 (C-9), 110.8 (C-14), 118.7 (C-11), 119.7 (C-12), 122.4 (C-
13), 122.8 (C-3), 128.0 (C-10), 135.0 (C-4), 137.0 (C-17), 139.2 (C-15),
18(S)-Benzofuran-norcleavamine 17
To a solution of benzofurancleavamine 14 (110 mg, 0.20 mmol) in
anhydrous dichloromethane (1.5 mL) was added trifluoroacetic acid
(16 lL, 0.21 mmol) at 0 ꢀC, and the reaction mixture was stirred
for 15 min before being cooled to -78 ꢀC. A solution of N-bromo-
succinimide (78 mg, 0.44 mmol) in anhydrous dichloromethane
(1.5 mL) was then added. After 30 min, the reaction mixture was
neutralized by a 10% sodium bicarbonate solution. The aqueous
layer was extracted with dichloromethane and the combined
organic layer was dried over magnesium sulfate, filtered and con-
centrated under reduced pressure to afford a bromo intermediate
that was used without further purification. To a solution of this
compound (0.20 mmol) in a 1 ⁄ 1 mixture of tetrahydrofuran and
water (10 mL), under argon was added AgBF4 (43 mg, 0.22 mmol).
The reaction mixture was stirred for 2 h at 50 ꢀC and cooled to
room temperature. The solvent was removed under reduced pres-
sure at 25–30 ꢀC, the mixture was diluted by a 10% sodium bicar-
bonate solution and extracted three times with dichloromethane.
The combined organic layer was dried over magnesium sulfate, fil-
tered and concentrated. The crude product was stirred in a 7:3 mix-
ture of heptane ⁄ ethyl acetate and filtered. The solvent was
removed in vacuo to afford pure benzofuranenorcleavamine com-
pound 17 as a pale yellow oil (86 mg, 60%): mmax(neat) ⁄ cm 3388,
2948, 1723, 1458, 1403, 1196 and 750; dH(500 MHz; CDCl3) 1.00
(3H, t, J 7.4, 21-H), 1.19 (3H, d, J 7.0, 10¢-H), 1.68 (1H, m, 2-H),
1.93 (2H, m, 20-H), 2.42 (1H, m, 19-H), 2.47 (1H, m, 1-H), 2.67 (1H,
172.4 (C-23); [a]D -4 (c 1.0 in CH2Cl2); m ⁄ z (ESI) 355.2028 (M+H+.
22
C21H27N2O3 requires 355.2022), 355.2 (100%).
General method for the preparation of the
quaternary ammonium salts (19-23 and 24–27)
A mixture of cleavamine derivatives 12-15 or norcleavamine
derivatives 16-18 and methyl bromoacetate or 'bromo-peptides'
(11) (1 eq) were stirred at room temperature in acetonitrile for 2 h.
The solvent was removed under reduced pressure and the crude
mixture was purified either by precipitation (1:2.5 AcOEt ⁄ Heptane)
Chem Biol Drug Des 2010; 75: 284–294
287

Ngo et al.
or by column chromatography on silica (97:3 dichloromethane ⁄ meth-
anol then a gradient of methanol).
Compound 21
The reaction was performed with 13 (32 mg, 0.06) and methyl
bromoacetate (9 mg, 0.06 mmol) to afford 21 as an amorphous
solid (27 mg, 65%): mmax(neat) ⁄ cm 2948, 1749, 1611, 1461, 1202,
1117; dH(500 MHz; CDCl3) 1.00 (3H, t, J 7.6, 21-H), 2.00 (3H, m,
20-H and 2-H), 2.26 (1H, m, 1-H), 2.70 (3H, s, 10¢-H), 3.05 (2H, m,
3¢-H), 3.16 (1H, m, 8-H), 3.20 (1H, m, 1-H), 3.61 (2H, m, 2¢-H), 3.65
(3H, s, 23-H), 3.75 (3H, s, 26-H), 3.79 (6H, s, 11¢-H and 12¢-H),
3.88 (1H, m, 7-H), 4.20 (2H, m, 5-H, 19-H), 4.36 (1H, m, 8-H), 4.61
(1H, d, J 16.2, 24-H), 4.63 (1H, m, 7-H), 4.72 (1H, m, 5-H), 4.88
(1H, m, 19-H), 4.89 (1H, d, J 16.2, 24-H), 5.55 (1H, s, 3-H), 5.73
(1H, s, 8¢-H), 7.05 – 7.23 (3H, m, 12-H,13-H and 14-H), 7.63 (1H, d,
J 7.9, 11-H), 8.32 (1H, s, NH), dC(75 MHz; CDCl3) 11.7 (C-21), 19.1
(C-8), 27.5 (C-3¢), 27.6 (C-20), 30.8 (C-2), 35.6 (C-10¢), 35.7 (C-1),
52.5 (C-11¢, C-12¢), 53.5 (C-23), 54.1 (C-18), 56.0 (C-2¢), 56.5 (C-26),
57.4 (C-7), 61.1 (C-5), 61.7 (C-24), 63.6 (C-19), 87.5 (C-8¢), 108.3
(C-4¢), 110.5 (C-14), 111.1 (C-9), 112.1 (C-6¢), 117.0 (C-11), 120.5
(C-12), 122.9 (C-3), 123.0 (C-13), 128.8 (C-10), 132.7 (C-17), 133.4
(C-4), 134.5 (C-15), 156.0 (C-9¢), 158.3 (C-5¢, C-7¢), 167.7 (C-25),
170.1 (C-22); [a]D22+52 (c 1.43 in CHCl3); m ⁄ z (ESI) 602.3219 (M+.
C35H44N3O6 requires 602.3230), 602.2 (100%); k(EtOH) (De)
223(+12), 257 (+7).
Compound 19
The reaction was performed with 12 (29 mg, 0.06 mmol) and
methyl bromoacetate (9 mg, 0.06 mmol). The crude mixture was dis-
solved in 1 mL of ethyl acetate and diluted by 2.5 mL of heptane.
The precipitated ammonium salt 19 was collected by filtration,
washed with heptane and dried under vacuum (23 mg, 70%): mmax(-
neat) ⁄ cm 3415, 2949, 1749, 1614, 1461, 1204, 746; dH(500 MHz;
CDCl3)-NH2 are missing- 1.02 (3H, t, J 7.3, 21-H), 1.99 (2H, m, 20-
H), 2.26 (2H, m, 1-H and 2-H), 3.15 (1H, m, 1-H), 3.16 (1H, m, 8-H),
3.43 (3H, s, 26-H), 3.70 (3H, s, 23-H), 3.79 (6H, s, 7¢-H and 8¢-H),
3.88 (1H, m, 7-H), 4.18 (1H, m, 5-H), 4.20 (1H, d, J 15.1, 19-H), 4.36
(1H, m, 8-H), 4.61 (1H, d, J 16.3, 24-H), 4.63 (1H, m, 7-H), 4.72 (1H,
m, 5-H), 4.81 (1H, d, J 15.1, 19-H), 4.91 (1H, d, J 16.3, 24-H), 5.54
(1H, s, 3-H), 6.02 (1H, s, 2¢-H), 6.09 (1H, s, 6¢-H), 7.06 – 7.21 (3H,
m, 12-H,13-H and 14-H), 7.63 (1H, d, J 8.8, 11-H), 8.32 (1H, s, NH);
dC(75 MHz; CDCl3) 11.9 (C-21), 19.1 (C-8), 27.5 (C-20), 30.9 (C-2),
35.4 (C-1), 53.0 (C-26), 53.6 (C-23), 53.7 (C-18), 55.9 (C-7¢), 56.3 (C-
8¢), 57.4 (C-7), 61.1 (C-5), 61.7 (C-24), 63.6 (C-19), 92.5 (C-2¢, C-6¢),
106.1 (C-4¢), 110.6 (C-14), 111.1 (C-9), 116.8 (C-11), 120.4 (C-12),
122.7 (C-3), 122.9 (C-13), 128.7 (C-10), 132.7 (C-17), 132.8 (C-4),
134.6 (C-15), 147.8 (C-1¢), 164.5 (C-25), 165.4 (C-3¢, C-5¢), 167.7 (C-
22); [a]D22+37 (c 1.3 in CHCl3); m ⁄ z (ESI) 562.2892 (M+. C32H40N3O6
requires 562.2917), 562.3 (100%); k(EtOH) (De)4 (+20), 256 (+12).
Compound 22
The reaction was performed with 14 (32 mg, 0.06 mmol) and
methyl bromoacetate (9 mg, 0.06 mmol) to afford 22 as an amor-
phous solid (25 mg, 70%): mmax(neat) ⁄ cm 2921, 2852, 1751,
1616,1459, 1210, 1097, 746; dH(500 MHz; CDCl3)1.00 (3H, t, J 7.6,
21-H), 1.22 (3H, d, J 7.1, 10¢-H), 2.00 (3H, m, 20-H, 2-H), 2.26 (1H,
m, 1-H), 3.16 (1H, m, 8-H), 3.20 (1H, m, 1-H), 3.40 (1H, m, 3¢-H),
3.65 (3H, s, 23-H), 3.75 (3H, s, 26-H), 3.79 (6H, s, 11¢-H, 12¢-H), 3.88
(1H, m, 7-H), 4.10 (1H, m, 2¢-H), 4.20 (2H, m, 5-H, 19-H), 4.36 (2H,
m, 8-H, 2¢-H), 4.61 (1H, d, J 15.2, 24-H), 4.63 (1H, m, 7-H), 4.72
(1H, m, 5-H), 4.88 (1H, m, 19-H), 4.89 (1H, d, J 15.2, 24-H), 5.55
(1H, s, 3-H), 6.03 (1H, s, 8¢-H), 7.05 – 7.23 (3H, m, 12-H,13-H and
14-H), 7.63 (1H, d, J 7.9, 11-H), 8.32 (1H, s, 16-H); dC(75 MHz;
CDCl3) 11.7 (C-21), 19.1 (C-8), 19.4 (C-10¢), 27.6 (C-20), 30.8 (C-2),
35.5 (C-3¢), 35.7 (C-1), 53.1 (C-18), 53.5 (C-23), 55.5 (C-11¢, C-12¢),
56.5 (C-26), 57.4 (C-7), 61.1 (C-5), 61.7 (C-24), 63.6 (C-19), 79.1 (C-
2¢), 89.4 (C-8¢), 106.4 (C-6¢), 110.5 (C-14), 111.1 (C-9), 111.4 (C-4¢),
117.0 (C-11), 120.5 (C-12), 122.9 (C-3), 123.0 (C-13), 128.8 (C-10),
132.7 (C-17), 133.4 (C-4), 134.5 (C-15), 156.0 (C-5¢, C-7¢), 159.3 (C-
9¢), 167.7 (C-25), 170.1 (C-22); m ⁄ z (ESI) 603.3095 (M + H+.
C35H44N2O7 requires 603.3070), 603.3 (100%); k(EtOH) (De) 223(+5),
254 (+1).
Compound 20
The reaction was performed with 12 (29 mg, 0.06 mmol) and
BrCH2ProIleAsp(OBzl)2 (11) (38 mg, 0.06 mmol). The crude mixture
was dissolved in 2 mL of ethyl acetate and diluted by 5 mL of hep-
tane. The precipitated ammonium salt 20 was collected by filtra-
tion, washed with heptane and dried under vacuum (34 mg, 54%):
mmax(neat) ⁄ cm 2962, 1737, 1660, 1229; dH(500 MHz; CDCl3)-NH2 are
missing- 0.70 - 0.80 (6H, m, 36-H and 37-H), 1.02 (3H, t, J 7.3, 21-
H), 1.16 (1H, m, 35-H), 1.46 (1H, m, 35-H), 1.80 - 2.28 (9H, m, 1-H,
2-H, 20-H, 28-H, 29-H and 34-H), 2.80 (1H, m, 41-H), 3.02 (1H, m,
41-H), 3.15 (1H, m, 1-H), 3.16 (1H, m, 8-H), 3.50 (2H, m, 30-H), 3.70
(3H, s, 23-H), 3.79 (6H, s, 7¢-H and 8¢-H), 3.88 (1H, m, 7-H), 4.18
(1H, m, 5-H), 4.20 (1H, d, J 15.1, 19-H), 4.30 - 4.51 (5H, m, 8-H, 24-
H, 27-H and 33-H), 4.63 (1H, m, 7-H), 4.72 (1H, m, 5-H), 4.81 (1H, d,
J 15.1, 19-H), 4.84 (1H, m, 40-H), 5.00 (2H, d, J 9.1, 43-H), 5.04
(2H, d, J 5.3, 45-H), 5.54 (1H, s, 3-H), 6.02 (1H, s, 2¢-H), 6.09 (1H, s,
6¢-H), 6.75 (2H, m, 32-H and 39-H), 7.06 – 7.21 (13H, m, 12-H, 13-
H, 14-H and 10Har), 7.63 (1H, d, J 8.8, 11-H), 8.32 (1H, s, 16-H);
dC(75 MHz; CDCl3)-Main pics- 11.9 (C-21 and C-36), 15.2 (C-37),
19.1 (C-8), 24.8 (C-35), 27.5 (C-20), 29.0 (C-28), 30.9 (C-2), 35.4 (C-
1), 36.2 (C-41), 47.6 (C-30), 48.5 (C-40), 52.7 (C-23), 55.9 (C-7¢), 56.3
(C-8¢), 57.4 (C-7), 57.7 (C-33), 61.1 (C-5), 61.4 (C-27), 63.6 (C-19),
63.8 (C-24), 67.0 (C-43), 67.7 (C-45), 92.5 (C-2¢, C-6¢), 106.1 (C-4¢),
110.6 (C-14), 111.1 (C-9), 116.8 (C-11), 120.4 (C-12), 122.7 (C-3),
122.9 (C-13), 128.2 - 128.7 (10 Car + C-10), 132.7 (C-17), 132.8 (C-4),
134.6 (C-15), 135.0 (2 Cquat ar), 147.8 (C-1¢), 165.4 (C-3¢, C-5¢), 170.0
(C-42 and C-44), 170.5 (C-25, C-31 and C-38), 173.1 (C-22); [a]D22+40
(c 1.2 in CHCl3); m ⁄ z (ESI) 1053.5328 (M+. C60H73N6O11 requires
1053.5332), 1053.5 (100%).
Compound 23
The reaction was performed with methoxycleavamine 15 (30 mg,
0.08 mmol) and methyl bromoacetate (12 mg, 0.08 mmol) to afford
23, after purification by column chromatography on silica (7:3
dichloromethane ⁄ methanol then a gradient of methanol), as a
translucent oil (27 mg, 65%): mmax(neat) ⁄ cm 3236, 2952, 1745, 1214
and 747; dH(300 MHz; CDCl3) 1.03 (3H, t, J 7.0, H-21), 2.03 (2H, m,
H-20), 2.56 (1H, dt, J 16.3 and 3.9, H-1), 2.64 (1H, s, H-2), 2.86 (1H,
m, H-1), 2.95 (1H, m, H-8), 3.40 (3H, s, H-22), 3.60 (1H, m, H-7),
3.63 (3H, s, H-24), 3.79 (3H, s, H-27), 4.00 (1H, m, H-7), 4.22 (2H,
288
Chem Biol Drug Des 2010; 75: 284–294

Elaboration of Simplified Vinca Alkaloids and Phomopsin Hybrids
m, H-5 and H-19), 4.36 (1H, m, H-8), 4.57 (1H, m, H-25), 4.87 (2H,
669.1990 for 81Br (M+. C34H40N2O7Br requires 667.2019 for 79Br and
669.1998 for 81Br), 667.2 (100%).
m, H-5 and H-25), 4.97 (1H, m, H-19), 5.66 (1H, s, H-3), 7.10 – 7.23
(3H, m, H-12, H-13 and H-14), 7.37 (1H, d, J 8.0, H-11), 9.70 (1H, s,
H-16); dC(75 MHz; CDCl3) 11.6 (C-21), 18.1 (C-8), 27.3 (C-20), 29.3
(C-2), 30.5 (C-1), 52.9 (C-22), 53.2 (C-27), 53.6 (C-24), 56.0 (C-7),
59.9 (C-5), 62.0 (C-25), 63.5 (C-19), 80.7 (C-18), 103.7 (C-9), 111.8
(C-14), 117.7 (C-11), 121.2 (C-3), 121.6 (C-12), 124.3 (C-13), 129.0 (C-
10), 129.6 (C-17), 134.8 (C-15), 135.2 (C-4), 169.7 (C-26), 171.2 (C-
23); [a]D22+2.5 (c 1.1 in CHCl3) m ⁄ z (ESI) 441.2398 (M+. C25H33N2O5
requires 441.2389), 441.2 (100%).
Compound 26
The reaction was performed with methoxy-norcleavamine (30 mg,
0.085 mmol) and BrCH2ProOMe (11, Microtubule assembly inhibitory
activity of the parent compound in the vinorelbine series: 3.2 mm
(IC50VLB: 1.6 mm): see reference 8) (21 mg, 0.085 mmol) to give,
after purification, compound 26 (38 mg, 75%) as a translucent oil:
mmax(neat) ⁄ cm 2359, 1652, 1254; dH(500 MHz; CDCl3) 1.08 (3H, m,
21-H), 1.91 (2H, m, 30-H), 1.96 (1H, m, 29-H), 2.10 (2H, m, 20-H),
2.16 (1H, m, 29-H), 2.37 (1H, dd, J 15.4 and 10.4, 1-H), 2.71 (1H, m,
2-H), 2.78 (1H, m, 1-H), 3.04 (1H, m, 31-H), 3.37 (3H, s, 22-H), 3.55
(1H, m, 19-H), 3.72 (3H, s, 24-H), 3.80 (3H, s, 33-H), 3.97 (1H, m,
31-H), 4.34 (1H, dd, J 4.7 and 13.2, 19-H), 4.44 (1H, m, 28-H), 4.49
(1H, d, J 14.2, 8-H), 4.68 (1H,d, J 16.5, 5-H), 4.94 (1H, d, J 16.5, 5-
H), 4.99 (1H, m, 25-H), 5.72 (1H, m, 25-H), 5.73 (1H, s, 3-H), 5.92
(1H, d, J 14.2, 8-H), 7.22 - 7.38 (3H, m, 12-H, 13-H and 14-H), 7.64
(1H, s, 11-H), 9.10 (1H, s, NH); dC(75 MHz; CDCl3) 11.6 (C-21), 24.7
(C-30), 27.1 (C-20), 27.9 (C-2), 28.8 (C-29), 31.8 (C-1), 47.0 (C-31),
51.1 (C-8), 52.4 (C-33), 53.1 (C-22), 53.4 (C-24), 55.3 (C-19), 59.3 (C-
25), 59.5 (C-28), 60.1 (C-5), 79.2 (C-18), 100.5 (C-9), 111.5 (C-14),
118.9 (C-11), 120.2 (C-3), 122.4 (C-12), 124.7 (C-13), 128.5 (C-10),
133.0 (C-4), 134.6 (C-15), 135.9 (C-17), 163.1 (C-26), 170.7 (C-23),
Compound 24
The reaction was performed with 16 (25 mg, 0.04 mmol) and
methyl bromoacetate (6 mg, 0.04 mmol). The crude mixture was dis-
solved in 1 mL of ethyl acetate and diluted by 2.5 mL of heptane.
The precipitated ammonium salt 24 was collected by filtration,
washed with heptane and dried under vacuum (6 mg, 20%): mmax(-
neat) ⁄ cm 3367, 2922, 1733, 1602, 1435 and 1193; dH(300 MHz;
CDCl3) -NH2 are missing- 1.09 (3H, t, J 7.5, H-21), 2.11 (3H, m, H-2,
H-20), 2.58 (1H, m, H-1), 2.92 (1H, m, H-1), 3.56 (1H, m, H-19), 3.73
(3H, s, H-26), 3.74 (3H, s, H-23), 3.83 (3H, s, H-7¢), 3.85 (3H, s, H-
8¢), 3.86 (1H, m, H-19), 4.69 (2H, d, J 2.7, H-24), 4.71 (1H, m, H-5),
4.80 (1H, m, H-8), 5.15 (1H, d, J 16.6, H-5), 5.66 (1H, m, J 14.8, H-
8), 5.69 (1H, s, H-3), 7.19 - 7.32 (3H, m, Hindol), 7.96 (1H, s, H-11),
9.16 (1H, s, NH-16); dC(75 MHz; CDCl3) 12.0 (C-21), 28.6 (C-20), 27.8
(C-2), 33.2 (C-1), 48.3 (C-18), 52.9 (C-26), 53.4 (C-23), 57.3 (C-8),
60.2 (C-5), 60.9 (C-7, C-8), 62.2 (C-24), 62.4 (C-19), 100.0 (C-2, C-6),
104.8 (C-4¢), 108.0 (C-9), 111.2 (C-14), 118.5 (C-11), 120.9 (C-3),
122.3 (C-12), 124.3 (C-13), 128.5 (C-10), 132.6 (C-4), 134.6 (C-15),
135.9 (C-17), 150.7 (C-1¢), 154.1 (C-3¢, C-5¢), 165.5 (C-25), 167.3 (C-
22); [a]D22+32 (c 0.42 in CHCl3) m ⁄ z (ESI) 704.0975 (79Br2); 706.0907
(79Br 81Br); 708.0930 (81Br2) (M+. C31H36Br2N3O6 requires 704.0971
(79Br2); 706.0950 (79Br 81Br); 708.0930 (81Br2)).
22
172.0 (C-32); [a]D -13 (c 0.65 in CHCl3); m ⁄ z (ESI) 524.2743
(M+.C29H38N3O6 requires 524.2761), 524.2 (100%).
Compound 27
The reaction was performed with methoxy-norcleavamine (30 mg,
0.085 mmol) and BrCH2ProIleAsp(OBzl)2 (11) (55 mg, 0.085 mmol) to
give, after purification, compound 27 (54 mg, 64%) as a translucent
oil: mmax(neat) ⁄ cm 2962, 1736 and 1650; dH(500 MHz; CDCl3)-40-NH
is missing- 0.82 (3H, t, 37-H), 0.88 (3H, t, 38- H), 1.07 (3H, m, 21-
H), 1.17 (1H, m, 36-H), 1.41 (1H, m, 36-H), 1.83 - 2.12 (7H, m, 20-H,
29-H, 30-H and 35-H), 2.32 (1H, m, 1-H), 2.71 (1H, m, 2-H), 2.78
(1H, m, 1-H), 2.83 (1H, dd, J 4.6 and 17.1, 42-H), 3.03 (1H, dd, J
4.6 and 17.1, 42-H), 3.22 (2H, m, 31-H), 3.37 (3H, s, 22-H), 3.46 (1H,
m, 19-H), 3.71 (3H, s, 24-H), 4.25 – 4.42 (3H, m, 19-H, 28-H and 34-
H), 4.53 (1H, d, J 13.9, 8-H), 4.65 (1H, d, J 16.0, 5-H), 4.84 (1H, d, J
8.0, 41-H), 4.97 (1H, d, J 16.0, 5-H), 5.00 (2H, s, 44-H), 5.06 (2H,s,
45-H), 5.57 (2H, s, 25-H), 5.72 (1H, s, 3-H), 5.86 (1H, d, J 13.9, 8-H),
6.94 (1H, d, J 8.0, 33-NH), 7.07–7.36 (13H, m, Har), 7.67 (1H, s, 11-
H), 9.14 (1H, s, 16-NH); dC(75 MHz; CDCl3) 11.7 (C-37), 11.9 (C-21),
15.6 (C-38), 24.8 (C-30), 27.0 (C-20), 28.2 (C-2), 28.8 (C-29), 29.7 (C-
36), 32.2 (C-1), 36.5 (C-42), 38.1 (C-35), 47.3 (C-31), 48.8 (C-41), 51.1
(C-8), 53.4 (C-22), 53.6 (C-24), 55.3 (C-19), 58.2 (C-34), 59.3 (C-25),
60.3 (C-5), 61.3 (C-28), 67.1 (C-45), 67.8 (C-44), 79.3 (C-18), 100.5
Compound 25
Reaction was performed with benzofuran-norcleavamine 17 (36 mg,
0.06 mmol) and methyl bromoacetate (9 mg, 0.06 mmol). The crude
mixture was dissolved in 1 mL of ethyl acetate and diluted by
2.5 mL of heptane. The precipitated ammonium salt was collected
by filtration, washed with heptane and dried under vacuum (26 mg,
65%): mmax(neat) ⁄ cm 2360, 1742, 1233 and 1059; dH(500 MHz;
CDCl3)1.08 (3H, t, J 7.7, 21-H), 1.22 (3H, d, J 6.3, 10-H), 2.09 (2H,
m, 20-H), 2.26 (1H, m, 2-H), 2.59 (1H, m, 1-H), 3.04 (1H, m, 1-H),
3.49 (1H, m, 3¢-H), 3.54 (1H, m, 19-H), 3.71 (3H, s, 26-H), 3.72 (3H,
s, 23-H), 3.82 (3H, s, 11¢-H), 3.83 (3H, s, 12¢-H), 4.09 (1H, m, 2¢-H),
4.28 (1H, m, 2¢-H), 4.34 (1H, m, 19-H), 4.64 (2H, d, J 1.8, 24 -H),
4.71 (1H, m, 5-H), 4.80 (1H, m, 8-H), 4.89 (1H,m, 5-H), 5.56 (1H, m,
8-H), 5.73 (1H, s, 3-H), 7.12 - 7.32 (3H, m, Har), 7.86 (1H, s, 11-H),
9.00 (1H, s, 16-NH); dC(75 MHz; CDCl3) 12.0 (C-21), 18.9 (C-10¢),
27.6 (C-20), 28.4 (C-2), 30.2 (C-1), 35.4 (C-3¢), 52.9 (C-26), 53.4 (C-
23), 55.0 (C-18), 55.3 (C-8), 59.8 (C-5), 60.4 (C-19), 60.9 (C-11¢, C-
12¢), 62.2 (C-24), 79.2 (C-18), 79.7 (C-2¢), 100.0 (C-8¢), 106.4 (C-6¢),
107.2 (C-9), 111.2 (C-14), 118.5 (C-11), 120.9 (C-3), 121.4 (C-4¢),
122.3 (C-12), 124.3 (C-13), 128.5 (C-10), 132.6 (C-4), 134.6 (C-15),
135.9 (C-17), 154.1 (C-5¢, C-7¢), 159.3 (C-9¢), 165.5 (C-25), 167.3 (C-
22); [a]D22+4 (c 0.9 in CHCl3); m ⁄ z (ESI) 667.1996 for 79Br and
(C-9), 120.2 (C-3), 125.0 -128.8 (18 Car), 134.8 (C-4), 135.9 (C-17),
22
163.2 (C-26), 170.7 (2 CO2), 170.9 (C-23), 171.9 (C-32, C-39); [a]D
-
37 (c 1.05 in CH2Cl2); m ⁄ z (ESI) 918.4661 (M+. C52H64N5O10 requires
918.4653), 918.7 (100%).
Computational procedures
All calculations were performed on a PC workstation. All modeling
studies were performed using Sybyl 7.3 software. The MMFF94 force
Chem Biol Drug Des 2010; 75: 284–294
289

Ngo et al.
field was used for minimization and partial charge calculations, a
dielectric constant of 1.0 being employed. Compounds 20 and 27 were
subjected to an unrestrained molecular dynamics simulation at 1600 K
for 20 000 fs. Conformations were sampled every 100 fs during the
simulation resulting in 200 randomized structures. Each of these con-
formers was minimized and compared with others with a RMS of 0.3 ꢀ.
The obtained structures were ranked according to energy. They were
analyzed using Sybyl 7.3 software and superimposition of conformers
was based on the backbone atoms of vinblastine. Docking experiments
on compound 22 were realized using the DOCK software of Sybyl 7.3
with Tripos force field for minimization.
bromoacetate. In fact, we have shown previously that vinblastine–
phomopsin hybrids carrying a tripeptide-protected side chain 1 and 2
were very potent on tubulin (almost as potent as vinblastine) (11).
The benzyl-protecting groups probably interact with the amino acids
of the active site as hybrids with a deprotected side chain were less
potent on tubulin. In addition, we have evaluated the microtubule
assembly inhibitory activity of compounds 4 and 5 (11) (Figure 2)
elaborated in one step and very good yields from anhydrovinblastine
and vinorelbine (scheme 1). Both compounds are very active.
The replacement of vindoline by a simpler pattern has been scar-
cely studied, and only a few examples of such compounds have
been published so far (see also 18–21). The selectivity of the direct
coupling of small groups to catharantine was always very low. For
example, Potier, Langlois and coworkers have synthesized indoline-
type compounds (21) 3 with a modest 30% yield and no selectivity
on C18.
Inhibition of tubulin assembly
Measurement of the inhibition of tubulin assembly was carried out
on Shimidazu UV 2401 PC spectrophotometer equipped with a tem-
perature-controlled cell. The drug, dissolved in ethanol at different
concentrations was added to a solution of free tubulin (prepared
according to the procedure of Shelansky and coll (15,16).) at 0 ꢀC.
Then the solution was placed in a temperature-controlled cell at
37 ꢀC (microtubule assembly), and the increase in the optical den-
sity was monitored in a UV spectrophotometer at 350 nm for
1 min. The maximum rate of assembly was recorded and compared
to a sample without drug. The IC50 of the compound is theoretically
calculated from the effect of several concentrations and compared
to the IC50 of vinblastine obtained within the same day with the
same tubulin preparation.
To improve the enantioselectivity of the direct coupling of simple
aromatics with catharantine and to obtain selectively the correct S
stereochemistry at the quaternary C18 chiral center, we have inves-
tigated the direct coupling of more electron-rich aromatics (22) to
catharantine (Scheme 2). Three aromatics were chosen for that pur-
pose: commercial 3,5-dimethoxyaniline, 5,7-dimethoxyindoline (8)
and 5,7-dimethoxy-2,3-dihydrobenzofuran 10 as indoline and
dihydrobenzofuran are bioisosteres (see for example 23). Compound
8 was easily synthesized from commercially available compound 6
(Scheme 3). Compound 10 was obtained by a three-step sequence
from commercial 3,5-dimethoxyphenol 9: iodation in position 2
(along with 10% of 2,6-diiodation), allylation of the alcohol and
5-exo trig radical cyclization (along with the dehalogenation in
position 6 of the minor compound) (24).
Cytotoxicity assays
The human tumor cell line KB (mouth epidermoid carcinoma) was
originally obtained from the ATCC. The cytotoxicity assays were per-
formed according to a published procedure (17).
As 3,5-dimethoxyaniline is water soluble, its condensation on cath-
arantine was performed in an aqueous medium in the presence of
ferric chloride (25) to give compound 12 as one diastereoisomer
and with an excellent yield (85%). The reaction had to be run at
35 ꢀC for completion. Nevertheless, when the same procedure was
applied to 8 and 10, expected products were not formed, and the
Polonovsky–Potier conditions were then applied. In the case of 5,7-
dimethoxyindoline (8), the expected compound 13 was obtained as
one diastereoisomer. However, the experimental conditions had to
Results and Discussion
To determine the impact of the side chain on biological activity, we
have simplified the vindoline moiety and elaborated quaternary
ammonium salts in the anhydrovinblastine and the vinorelbine
series by condensation either with the tripeptide-protected side
chain of phomopsin A (ProIleAsp(Obzl)2) or with commercial methyl
Figure 2: Tubulin assembly curves for compounds 4 and 5.
290
Chem Biol Drug Des 2010; 75: 284–294

Elaboration of Simplified Vinca Alkaloids and Phomopsin Hybrids
Scheme 1: (A) BrCH2CO2Me, CH3CN, RT.
Scheme 2: (a) (i) DMA, FeCl3, glycine buffer, HCl 0.1 N, (ii) NaBH4, NH4OH; (b) (i) m-CPBA, (ii) (CF3CO)2, (iii) MeOH, (iv) NaBH4, MeOH; (c)
BrCH2COR1, CH3CN; (D) (i) CF3CO2H then NBS (ii) AgBF4 then H2O ⁄ THF 50 ꢀC.
be modified to allow a total conversion of catharantine N-oxide.
Usually, catharantine N-oxide, trifluoroacetic anhydride and vindo-
line are reacted at )78 ꢀC for 1 h. In our case, we had to stir cath-
arantine N-oxide and trifluoroacetic anhydride at )78 ꢀC for 40 min
before the addition of compound 8, and the reaction mixture was
stirred at )78 ꢀC for 30 min and at room temperature for 30 addi-
tional min. In addition, as compound 13 decomposed during purifi-
cation on column chromatography, it was purified by an acid–base
extraction and isolated with 56% yield after conversion to the free
base. The great improvement in the enantioselectivity of the cou-
pling compared to that of compounds 3a and 3b (Figure 3) con-
Scheme 3: (a) CH3I, NaH, DMF, rt, 100%; (b) NaBH3CN,
CH3COOH, 10 ꢀC, 90%; (c) NIS, CH2Cl2, 0 ꢀC, 70%; (d) Allyl bro-
mide,K2CO3, CH3CN, reflux, 83%; (e) n-Bu3SnH, AIBN, toluene,
reflux, 77%.
firmed the importance of the reactivity of the aromatic. In the case
of dimethoxybenzofuran 10, the same conditions were used to give
the expected compound 14 with a good 60% yield. This compound
was obtained as a mixture of diastereomers as there was no con-
Chem Biol Drug Des 2010; 75: 284–294
291

Ngo et al.
were stable enough to provide vinorelbine intermediates 16–18
with modest to good yields for two steps (Scheme 2). It should be
noted that for compounds 12 and 14 two equivalents of NBS were
necessary as aniline and benzofuran were brominated first. The
ammonium ions 24–27 were obtained as one diastereomer.
Because of the gramine bridge, they could be in equilibrium with
an open enaminium intermediate leading to either a R or a S con-
figuration at N6. Thus, the configuration at the N6 quaternary cen-
ter had to be determined as it is a determining factor for the
orientation of the peptide chain. NOESY experiments on compound
26 showed one nuclear Overhauser effect between H1 and H8,
which is only consistent with a S configuration (Figure 4).
Figure 3: Former simplified vinca alkaloids.
trol of C3¢ asymetric center. Nevertheless, the NMR chemical shifts
of both diastereomers were the same as the distant position of C3¢
may abstract it from the influence of the chirality of the rest of the
molecule. These unique NMR spectra indicated also that the config-
uration at C-18 was totally controlled during the reaction.
Molecular dynamics studies (using SYBYL 7.3 software and
MMFF94 force field) were performed on compounds 20 and 27
possessing a three amino acids peptide chain to compare their con-
formation with those of compounds 1 and 2, and to determine
whether the simplification of the vindoline moiety has an impact on
the orientation of the added peptide chain (Figure 5). For the meth-
oxy hybrid 27, all the conformers produced by conformational
searching adopt a folded structure: the peptide side chain is falling
The 18S methoxy-cleavamine derivative 15 (20) was also synthe-
sized by a Polonovsky–Potier coupling of catharantine N-oxide with
anhydrous methanol followed by a reduction in the imminium inter-
mediate. As expected, a mixture of 18S and 18R methoxycleav-
amine was obtained with a 75 ⁄ 25 ratio. The desired 18S compound
15 could be separated and obtained with 43% yield after a careful
purification (Scheme 2).
All these simplified vinca-type alkaloids 12–15 were easily trans-
formed into the corresponding quaternary ammonium salts 19–23
by condensation with one equivalent of methyl bromoacetate or
BrCH2ProIleAsp(OBzl)2 (6) at room temperature in acetonitrile
(Scheme 2) (26). The S absolute configuration at C18 for compounds
19, 21 and 22 was confirmed by comparison of the signs of their
Cotton effect curves (compounds 19: 224 (+20) and 256 (+12), 21:
223 (+12) and 257 (+7) and 22: 223 (+5) and 254 (+1)) with thus of
vinblastine (27) (228 (+21) and 254 (+9)).
Subsequently, we have elaborated the quaternary ammonium salts
24–27 in the vinorelbine series. Only compounds 12, 14 and 15
Figure 4: Observed nuclear Overhauser effect for 26.
A
B
C
Figure 5: (A) Superimposition of the lowest energy conformer of compound 27 (magenta) and vinblastine (cyan) in its active conformation;
(B) Superimposition of the lowest energy conformer of compound 20 (yellow) and compound 1 (green) before docking with vinblastine (cyan)
and phomopsin (violet) in their active conformation; (C) Superimposition of the lowest energy conformer of compound 20 (yellow) and com-
pound 1 (green) after docking with vinblastine (cyan) and phomopsin (violet) in tubulin binding site.
292
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Elaboration of Simplified Vinca Alkaloids and Phomopsin Hybrids
down in place of vindoline for the methoxy group is too small to
2. Blaskꢃ G., Borman L.S., Cordell G.A., Kuehne M.E., Markꢃ I.,
McCormack J.J., Neuss N., Pearce H.L. (1990) Antitumor bis-
indole alkaloids from Catharantus roseus (L.). In: Brossi A.
editor. The Alkaloids 37. San Diego, CA: Academic Press Inc.;
1–240.
3. Noble R.L., Beer C.T., Cutts J.H. (1958) Role of chance observa-
tion in chemotherapy: Vinca Rosea. Ann N Y Acad Sci;76:882–
894.
4. Svoboda G.H., Neuss N., Gorman M.J. (1959) Alkaloids of Vinca
Rosea Linn. V. Preparation and characterization of alkaloids. J
Am Pharm Assoc;48:659–666.
5. Chen S.-H., Hong J. (2006) Novel tubulin-interacting agents: a
tale of Taxus brevifolia and Catharantus roseus-based drug dis-
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6. Guꢄritte F., Fahy J. (2005) The vinca alkaloids. In: Gragg G.M.,
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7. Fahy J. (2001) Modifications in the ``upper'' velbenamine part of
the vinca alkaloids have major implications for tubuline interact-
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8. Gigant B., Wang C., Ravelli B.G.R., Roussi F., Steinmetz
M.O., Curmi P.A., Sobel A., Knossow M. (2005) Structural
basis for the regulation of tubuline by vinblastine. Nat-
ure;435:519–522.
push it up (Figure 5A). For compound 20, all the generated con-
formers are much less folded: the nitrogen of aniline is surperim-
posed with the indolic nitrogen of vindoline and aniline is bulky
enough to push up the peptide chain (Figure 5B). This peptide chain
is more or less oriented as that of hybrids 1 and 2, and docking
experiments showed that it resides at the interface of tubulin b
and between that of hybrids 1 and 2 and that of phomopsin (Fig-
ure 5C).
Inhibition of tubulin assembly and cytotoxicity on KB cell line was
then evaluated for these new compounds 19–27. Both were very
disappointing as all these compounds were inactive, whereas their
equivalents in the anhydrovinblastine-phomopsin (1, 4) and vinorel-
bine-phomopsin (2, 5) series were very potent.
If the inactivity in the methoxy series (compounds 23 and 26–27)
can be explained by their conformation and the fact that their pep-
tide chains are not oriented as their anhydrovinblastine or vinorel-
bine equivalents, the inactivity in the aniline series clearly indicates
that the vinorelbine moiety or a more complex aromatic than dime-
thoxyaniline is necessary to inhibit tubulin assembly and that the
added peptide chains have a restrained impact on this activity.
Conclusion
9. Cormier A., Marchand M., Ravelli R.B.G., Knossow M., Gigant B.
(2008) Structural insight into the inhibition of tubulin by vinca
domain peptide ligands. EMBO Rep;9:1101–1106.
10. Natarajan A., Guo Y., Harbinski F., Fan Y.-H., Chen H., Luus L.,
Diercks J., Aktas H., Chorev M., Halperin J.A. (2004) Novel aryl-
sulfonamide-oxindole hybrid as an anticancer agent that inhibits
translation initiation. J Med Chem;47:4979–4982.
11. Ngo Q.A., Roussi F., Cormier A., Thoret S., Knossow M., Guꢄnard
D., Guꢄritte F. (2009) Synthesis and biological evaluation of
vinca alkaloids and phomopsin hybrids. J Med Chem;52:134–
142.
12. Potier P., Langlois N., Langlois Y., Guꢄritte F. (1975) Partial
synthesis of vinblastine-type alkaloids. J Chem Soc Chem Com-
mun:670–671.
In this report, we have described the concise elaboration of new
vinca-type and vinorelbine-type compounds by substitution of the
vindoline moiety either by a methoxy group or by simpler aromatics
to synthezise modified hybrids of vinca alkaloids and phomopsin A.
These compounds were transformed into nine simplified vinca-pho-
mospin A hybrids. None of these compounds was active on tubulin
polymerization inhibition. Molecular modeling studies showed that
the peptide chain of the aromatic hybrids is oriented in the same
direction as those of hybrids 1 and 2, which means that the addi-
tion of such a chain is not enough to induce a biological activity.
Work is currently underway in our laboratory to elaborate hybrids in
which added chain superimposes correctly on those of phomopsin
and in which aromatic southern part has been dressed out.
13. Langlois N., Guꢄritte F., Langlois Y., Potier P. (1976) Applica-
tion of a modification of the Polonovski reaction to the syn-
thesis of vinblastine-type alkaloids.
7017–7024.
J Am Chem Soc;98:
Acknowledgments
14. Mangeney P., Andriamialisoa R.Z., Lallemand J.-Y., Langlois N.,
Langlois Y., Potier P. (1979) 5¢-Nor-anhydrovinblastine. Tetrahe-
dron;35:2175–2179.
15. Berry R.W., Shelanski M.L. (1972) Interactions of tubulin with
vinblastine and guanosine triphosphate. J Mol Biol;71:71–80.
16. Shelanski M.L., Gaskin F., Cantor C.R. (1973) Microtubule assem-
bly in the absence of added nucleotides. Proc Natl Acad Sci U
S A;70:765–768.
Jacques Fahy and Philippe Maillas (Pierre Fabre) are gratefully
acknowledged for a generous gift of catharantine. The authors
acknowledge the Vietnamese government and Pr. Tran Van Sung,
director of the Institute of Chemistry of the NAST, Hanoi as well as
the CNRS for the fellowship awarded to Quoc Anh Ngo.
17. TempÞte C., Werner G.H., Favre F., Rojas A., Langlois N. (1995)
In vitro cytostatic activity of 9-demethoxyporothramycin B. Eur J
Med Chem;30:647–650.
18. Sundberg R.J., Gadamasetti K.G., Hunt P.J. (1992) Mechanistic
aspects of the formation of anhydrovinblastine by Potier–Polo-
novski oxidative coupling of catharantine and vindoline. Spectro-
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