Syntheses and biological evaluation of vinblastine congeners.

Article abstract of DOI:10.1039/b209990j

Sixty-two congeners of vinblastine (VLB), primarily with modifications of the piperidine ring in the carbomethoxycleavamine moiety of the binary alkaloid, were synthesized and evaluated for cytotoxicity against murine L1210 leukemia and RCC-2 rat colon cancer cells, and for their ability to inhibit polymerization of microtubular protein at < 10(-6) M, and for induction of spiralization of microtubular protein, and for microtubular disassembly at 10(-4) M concentrations. An ID50 range of >10(7) M concentrations was found for L1210 inhibition by these compounds, with the most active 1000x as potent as vinblastine.

Full text of DOI:10.1039/b209990j

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Syntheses and biological evaluation of vinblastine congeners†
Martin E. Kuehne,*a William G. Bornmann,a Istvan Markó,a Yong Qin,a Karen L. LeBoulluec,a
Deborah A. Frasier,a Feng Xu,a Tshilundu Mulamba,a Carol L. Ensinger,a Linda S. Borman,b
Anne E. Huot,c Christopher Exon,d Fred T. Bizzarro,d Julia B. Cheungd and Susan L. Banee
a Department of Chemistry, University of Vermont, Burlington, VT 05405, USA
b Department of Pharmacology and Vermont Regional Cancer Center, University of Vermont,
Burlington, VT 05405, USA
c Biomedical Technologies, University of Vermont, Burlington, VT 05405, USA
d Hoffmann La Roche, Inc., Nutley, NJ 07110, USA
e Department of Chemistry, SUNY, Binghamton, NY 13902, USA
Received 14th October 2002, Accepted 15th April 2003
First published as an Advance Article on the web 13th May 2003
Sixty-two congeners of vinblastine (VLB), primarily with modifications of the piperidine ring in the
carbomethoxycleavamine moiety of the binary alkaloid, were synthesized and evaluated for cytotoxicity against
murine L1210 leukemia and RCC-2 rat colon cancer cells, and for their ability to inhibit polymerization of
microtubular protein at <10Ϫ6 M, and for induction of spiralization of microtubular protein, and for microtubular
disassembly at 10Ϫ4 M concentrations. An ID50 range of >107 M concentrations was found for L1210 inhibition by
these compounds, with the most active 1000× as potent as vinblastine.
substoichiometric concentration (10Ϫ7 M), like VLB, while its
Introduction
C-20Ј epimer 5 is only as potent as the 20Ј-deethyl analogue 3
in that test. Like vinblastine, both compounds 4 and 5 gave
spiral aggregates with microtubular protein and disassembled
microtubules at 10Ϫ4 M concentration.
The development of practical, enantioselective total syntheses
of the clinically effective anticancer alkaloids vinblastine
(VLB, 1) and vincristine (VCR, 2, Scheme 1)1,2 provided the
first opportunity to systematically synthesize molecular
modifications for a study of structure–activity relationships.
Structural changes of the bridged piperidine ring of the carbo-
methoxycleavamine (top) segment of these binary alkaloids
Since the introduction of an equatorial 20Ј-ethyl substituent
resulted in a substantial increase in cytotoxic potency and
achieved a VLB-like substoichiometric level of inhibition of
tubulin polymerization, it was of interest to explore the struc-
ture–activity sensitivity to substitution at C-20Ј with other alkyl
substituents. Increased homologation of the C-20Ј ethyl group
gave a drop, rather than a further increase in L1210 and RCC-2
cytotoxicity for the 20Ј-equatorial n-propyl congener 6R, to
about one tenth of the potency of the 20Ј-deoxy VLB congener
4. Similar results were found for its C-20Ј epimer 7R relative to
20Ј-deoxyleurosidine (5). While the C-20Ј propyl congeners still
produced an inhibition of tubulin polymerization, no spiraliz-
ation was found at higher concentrations with microtubular
protein.
led to a range of cytotoxicities with ID50 > 10Ϫ6 M to ID50
=
10Ϫ13 M, thus establishing the importance of this segment of
the molecule for VLB-like biological activity. Key features of
our synthetic strategy are the facile, stereoselective assembly
of the structural moiety required for coupling to the vindoline
(bottom) segment and the high stereoselectivity of that process
for the specific C-14Ј–16Ј relative stereochemistry,3,4 which is
required for VLB-like biological activity (see Scheme 1). The
compounds of this paper were all prepared by this method-
ology. When designated with an R in the Tables, they were
synthesized at Hoffmann–La Roche.
Replacement of the equatorial C-20Ј substituents by a benzyl
group (8R) resulted in loss of all VLB-like activity, while an
axial C-20Ј benzyl group (9R) still provided modest L1210
cytotoxicity and inhibition of tubulin polymerization.
Biological results
Our initial synthesis of the simple vinblastine analogue lacking
the 20Ј-ethyl and hydroxyl functions (3),3,4 showed that this
compound, like VLB, exhibits cytotoxicity against L1210
leukemia cells (albeit about 300× less potently, Table 1), and
cytotoxicity against RCC rat colon cancer cells with 1/100 the
potency of VLB. At the biochemical level it inhibits tubulin
polymerization at a stoichiometric concentration (10Ϫ6 M) and
VLB-like disassembly of microtubules at higher concentration
(10Ϫ4 M). Microtubular protein single spirals were formed with
compound 3 at 10Ϫ4 M concentration, while VLB produces
spiral aggregates at that concentration.5,6
Decreasing the size of the C-20Ј substituent, on the other
hand, led to increased potency. Thus, both C-20Ј methyl con-
geners (10 and 11) are almost as active as vinblastine (1) in
cytotoxicity with L1210 cells and they are slightly more potent
in inhibition of tubulin polymerization, in disassembly of
microtubules and in formation of spiral aggregates with micro-
tubular protein. Indeed, single spirals were produced with low
concentrations (1–2 × 10Ϫ7 M) of each compound with micro-
tubular protein. Introduction of combined α and β-methyl sub-
stitution at C-20Ј (12), however, resulted in activities closer to
those of the 20Ј-deethyl-20Ј-deoxy congener 3, and loss of the
activity of microtubule disassembly. The introduction of two
C-20Ј ethyl substituents (13R) led to a further decrease in
activity.
The epimeric 20Ј-deoxyvinblastine (4) and 20Ј-deoxyl-
eurosidine (5) showed 1/10 and 1/100, respectively, of the cyto-
toxic potency of VLB against L1210 and RCC-2 cells,5–7 and
20Ј-deoxyvinblastine (4) inhibits tubulin polymerization at a
These results suggest that substitution at C-20Ј modulates
VLB-like activity at the biochemical and the cellular levels, not
primarily by affecting the conformational shape of the bridged
piperidine ring (deformation towards half-chair or boat)
but, rather by the need for a lipophylic substituent with limited
† Electronic supplementary information (ESI) available: Full experi-
mental procedures and UV, IR, 1H NMR, 13C NMR and MS data for
all compounds. See http://www.rsc.org/suppdata/ob/b2/b209990j/
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 2 1 2 0 – 2 1 3 6
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 3
2120

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Scheme 1 Typical reaction conditions for formation of C-20’ varied substitution of vinblastine congeners: (a) 60 (R4 = H), respective aldehyde
(X = TMS or tBDMS), THF, rt, 24 h; (b) C6H5CH2Br, THF, refl. 24 h; (c) MeOH, Et3N or DIBEA, refl. 6 h or 6 d; (d) 61 (R4 = C6H5CH2), respective
aldehyde, toluene, refl. 12 h; (e) 1) tBOCl, Et3N, CH2Cl2, 0 ЊC; 2) vindolineؒHCl, acetone, 0 ЊC, AgBF4, HBF4ؒEt2O, 25 min; 3) KBH4, HOAc, rt,
30 min; (f ) tBAF, THF, rt, 2 h; (g) Ts2O, Et3N, CH2Cl2, 0 ЊC to rt, 2.5 h; (h) toluene, refl. 6 h to 5 d; (i) 1) 10% Pd/C, H2, MeOH, 6 h, rt; 2) toluene refl.
6 h; (j) CH2Cl2, HOAc, KMnO4, hexaoxocyclooctadecane, Ϫ72 ЊC, 1 h.
volume of radial motion. To test this hypothesis, we synthesized
C-15Ј–20Ј ring cis-fused deoxy-VLB congeners, where the C-20Ј
substituent is now tied back to the piperidine ring. Indeed, it
was found that VLB-like activity was substantially increased.
The C-20Ј S six-membered ring compound 14 (see Table 1 and
Scheme 7) is 500× more potent and the corresponding C-20Ј R
fused ring 15 is 1000× more potent than VLB in L1210 cyto-
toxicity, while the activity with RCC-2 cells is equivalent to that
of 20Ј-deoxyvinblastine (4). The corresponding five-membered
ring analogues 16 and 17 (see Table 1 and Scheme 6) gave
activity profiles similar to those of the C-20Ј ethyl compounds
4 and 5.
Even the C-15Ј–20Ј benzo analogue 18 (see Table 1 and
Scheme 8), with four coplanar carbon atoms of the piperidine
ring DЈ, still shows cytotoxicities equal to those of 20Ј-deoxy-
vinblastine (4). All of these ring-fused congeners also exhibit
full VLB-type activity at the biochemical level with tubulin and
with microtubules.
The extraordinary high L1210 cytotoxicity of the C-15Ј–20Ј
fused cyclohexane congeners 14 and 15 prompted us to syn-
thesize the corresponding tetrahydropyran analogues 19 and 20
(see Scheme 9). These compounds showed a 4–5 × 105 drop in
cytotoxic potency relative to the cyclohexane fused congeners
14,15, to the range of 20Ј-deoxy VLB activity.
In an alternative approach, the C-20Ј spiro five and six-
membered ring substituted congeners 21 and 22 (see Scheme 3)
were synthesized. While these compounds were not expected
to display the high activity of the C-15Ј–20Ј ring-fused com-
pounds (restricted rotation C-20Ј monoalkyl class), it could be
hoped that they would have greater VLB activity than the C-20Ј
diethyl compound 13R. Accordingly, these compounds showed
full VLB activity at the biochemical level (tubulin polymeriz-
ation inhibition at substoichiometric concentration with spiral
aggregation at higher concentration) and 20Ј-deethyl-20Ј-deoxy
VLB-like cytotoxicity with RCC-2 cells, but they were not cyto-
toxic to L1210 cells at concentrations considered for activity
(<10Ϫ6 M).
This same activity profile was also found with 20Ј-deethyl-
20Ј-deoxy VLB congeners in which the DЈ-piperidine ring had
been enlarged to a seven (23) or eight (24) membered ring
(see Table 2, Scheme 4). Molecular modeling suggests that in
common with the 20Ј-spiro substituted compounds 21 and 22,
compounds 23 and 24 have a conformation with a shielded
ring CЈ nine-membered ring cavity.
The lack of L1210 cytotoxicity, despite good inhibition of
tubulin polymerization, suggests a possible failure of the con-
geners 21–24 to penetrate the cell membrane. Accordingly, on
electroporation they were found to have very high cytotoxic
activity.8
Based on the hypothesis that shielding of NbЈ to protonation,
solvation, and/or binding might be responsible for inhibition of
transport of compounds 21–24 into the cell, and a consequent
lack of cytotoxicity, a more accessible basic nitrogen was intro-
duced by formation of the hydrazide 25 (see Scheme 4 and
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 2 1 2 0 – 2 1 3 6
2121

O
Table 1
Population growth IC50/M
Microtubule
L1210
leukemia
RCC-2 Colon
adenocarcinoma
Microtuble assembly
IC50/M
Disassembly into spiral
aggregates at 10Ϫ4 M
Spiralization of
MTP at 10Ϫ4 M
Compound
R1
R2
R3
R4
1a
3a
Et
H
Et
H
OH
H
H
Et
H
Propyl
H
CH2C6H5
H
Me
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
4 × 10Ϫ10
3 × 10Ϫ9
5 × 10Ϫ7
3 × 10Ϫ8
5 × 10Ϫ7
6 × 10Ϫ7
6 × 10Ϫ7
N.D.
3 × 10Ϫ7
ϩ
ϩ
ϩ
ϩ
1 × 10Ϫ7
3 × 10Ϫ9
2 × 10Ϫ8
5 × 10Ϫ8
8 × 10Ϫ8
>1 × 10Ϫ6
5 × 10Ϫ7
1 × 10Ϫ9
1 × 10Ϫ9
3 × 10Ϫ8
5 × 10Ϫ6
8 × 10Ϫ13
3 × 10Ϫ13
2 × 10Ϫ8
1 × 10Ϫ9
3 × 10Ϫ9
5 × 10Ϫ8
3 × 10Ϫ8
>10Ϫ6
2 × 10Ϫ6
ϩ, Single spirals
4a
2 × 10Ϫ7
ϩ
5a
1 × 10Ϫ6
ϩ (5 × 10Ϫ5 M)
ϩ (5 × 10Ϫ5M)
6Ra
7Ra
8Ra
9Ra
10a
11a
12a
13Ra
14
Propyl
H
CH2C6H5
H
Me
H
Me
Et
3 × 10Ϫ6
N.D.
Ϫ
, Amorphous aggregates
2 × 10Ϫ6
, Amorphous aggregates
No effect, at 1 × 10Ϫ5
3 × 10Ϫ6
Ϫ (5 × 10Ϫ5M)
N.D.
Ϫ
ϩ
ϩ
Ϫ
Ϫ
ϩ
ϩ
ϩ
Ϫ
ϩ
ϩ
ϩ
N.D.
ϩ
ϩ
ϩ
3 × 10Ϫ8
5 × 10Ϫ8
8 × 10Ϫ7
>1 × 10Ϫ6
2 × 10Ϫ8
2 × 10Ϫ8
N.D.
2 × 10Ϫ7 single spirals
1 × 10Ϫ7 single spirals
3 × 10Ϫ6
Me
Et
No effect at 2 × 10Ϫ5
1 × 10Ϫ7
R2 ϩ R4 = –(CH2)4–, R1 = R3 = H
R1 ϩ R3 = –(CH2)4–, R2 = R4 = H
R2 ϩ R4 = –(CH2)3–, R1 = R3 = H
R1 ϩ R3 = –(CH2)3–, R2 = R4 = H
15
1 × 10Ϫ7
16
3 × 10Ϫ6
17
5 × 10Ϫ7
5 × 10Ϫ8
N.D.
2 × 10Ϫ7
ϩ
ϩ
18
Benzo
Benzo
1 × 10Ϫ7
ϩ(5 × 10Ϫ5M)
ϩ(5 × 10Ϫ5M)
19
R1 ϩ R3 = (CH2)O(CH2)2, R2 = R4 = H
R2 ϩ R4 = (CH2)O(CH2)2, R1 = R3 = H
1 × 10Ϫ6
N.D.
N.D.
ϩ
ϩ
ϩ
N.D.
N.D.
ϩ
ϩ
ϩ
Ϫ
20
N.D.
2 × 10Ϫ6
21
–(CH2)4–
–(CH2)5–
H
H
H
H
H
H
H
H
H
H
5 × 10Ϫ7
5 × 10Ϫ7
1 × 10Ϫ8
6 × 10Ϫ7
>10Ϫ6
5 × 10Ϫ7
22
>10Ϫ6
5 × 10Ϫ7
27
CH3
OH
OH
OH
CH3
Et
1 × 10Ϫ9
4 × 10Ϫ7
1 × 10Ϫ6
1 × 10Ϫ6
28
No effect at 2 × 10Ϫ5
1 × 10Ϫ5
Ϫ
Ϫ
29
Ϫ, Short MT
a Denotes methanesulfonate salt. The corresponding compounds without designation R were also tested as free bases and found to have the same activities as these salts.

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Table 2
Population growth
microtubule IC50/M
Microtuble
L1210
leukemia
RCC-2 colon
adenocarcinoma
Microtuble
assembly IC50/M
Disassembly into spiral
aggregates at 10Ϫ4 M
Spiralization of
MTP at 10Ϫ4 M
Compound
23
DЈ7 ring
>10Ϫ6
5 × 10Ϫ7
N.D.
2 × 10Ϫ7
2 × 10Ϫ6
ϩ
N.D.
ϩ
ϩ
N.D.
ϩ
, amorphous
aggregates
23a
24
5 × 10Ϫ8
>10Ϫ6
DЈ8 ring
DЈ5 ring
5 × 10Ϫ7
>10Ϫ6
2 × 10Ϫ7
26R
>10Ϫ6
1.5 × 10Ϫ5
ϩ
a Denotes methanesulfonate salt.
Table 4) of the seven-membered ring DЈ congener 23. Indeed,
this compound, like VLB hydrazide (175), had almost the
L1210 cytotoxicity of VLB (1). Finally, administration of the
dimethanesulfonate salt of the base 23 also resulted in modest
L1210 cytotoxicity, comparable to that of the unsubstituted
piperidine ring DЈ compound 3.9
The corresponding lower homologue with a five-membered
ring DЈ (26R) in place of the piperidine ring, was found to lack
all VLB-like activity.
Oxidation of some of the preceding vinblastine congeners to
the corresponding vincristine series (Table 4) resulted only in
minor perturbation of the cytotoxicity and tubulin polymeriz-
ation inhibition values for 20Ј-deethyl-20Ј-deoxyvincristine
(37), 20Ј-deoxyvincristine (38) and its C-20Ј epimer (39). Of
interest, however, is the 100× increased inhibition of tubulin
polymerization seen with the C-20Ј propyl homologues (40R
and 41R). Indeed, these compounds were the most potent of all
examined in this test.
Considering that the methyl homologue 10 of 20Ј-deoxy-
vinblastine (4) has a relatively greater L1210 cytotoxicity than
the ethyl compound 4, and approaches that of vinblastine, it
seemed likely that introduction of an axial C-20Ј hydroxyl
substituent into this compound could provide another VLB
congener with superior activity. However, evaluation of 20Ј-
methyl-20Ј-deethyl vinblastine (27) showed that here the axial
C-20Ј hydroxyl substituent did not enhance cytotoxic potency.
The corresponding C-20Ј epimer, 20Ј-methyl-20Ј-deethylleuro-
sidine (28) showed no VLB-type tubulin activity, as expected
from the lack of such activity with leurosidine (29).
The syntheses and evaluation of a group of vinblastine con-
geners with decreased biological activities also provided a scale
for consideration of structure–activity changes based on
introduction of substituents on the aromatic ring of the carbo-
methoxycleavamine (top) moiety of the binary alkaloids
(Table 3). Thus syntheses of 11Ј-methoxy-20Ј-deethyl-20Ј-
deoxyvinblastine (30) gave a congener which is more potent in
inhibiting tubulin polymerization than the parent demethoxy
compound 3, but which lacks the tubulin spiralization effect at
high concentration and, which is also less cytotoxic. A decrease
or loss of activity was seen with 11Ј-methoxy-20Ј-deoxyleuro-
sidine (31R) and 11Ј-methoxy-20Ј-deoxyvinblastine (32R) was
even found to lack all VLB-like activity.
Since the tryptophan derived amide derivative of vinblastine
(42, Fig. 1) had been reported to have a better therapeutic index
than vinblastine (however, with decreased potency),10 we also
examined that compound and the corresponding 20Ј-deoxy and
20Ј-epi-20Ј-deoxy VLB tryptophan derivatives (43 and 44,
Fig. 1). All three compounds were found to be equivalent in
L1210 cytotoxicity and in inhibition of tubulin polymerization,
but the deoxy compounds 43 and 44 were less potent in RCC-2
cytotoxicity (Table 4). On the other hand, the carboxylic acid
45, derived from 20Ј-deoxyvinblastine (4), was as potent in
RCC-2 cytotoxicity as the methyl ester 4.
The isolation of peptides that home specifically to tumor
blood vessels has led to their attachment to doxorubicin for its
targeted drug delivery with diminished general cytotoxicity.11
We therefore attached such a peptide to vinblastine and to our
seven-membered ring DЈ congener deacetyl-23. While congener
23 was not transported into L1210 cells as the free base (see
above) the resulting bridged CRGNC peptide derivative 176
now gave some inhibition of L1210 cells as the free base. This
compound and a corresponding VLB derivative 177 (Fig. 1)
showed the anticipated attenuation of L1210 cell inhibition
(Table 4) that would suggest their in vivo investigation for
targeted delivery.
A unique feature of our synthetic route to vinblastine
congeners is the ability to generate these compounds as
atropisomers (isolable conformational isomers, Fig. 2),2,4,7
which can be thermally converted to products with the natural
conformation. Examination of the atropisomers of the potent
agents VLB (1), 20Ј-deoxy VLB (4) and its C-20Ј epimer 5,
of the corresponding methyl analogues 10 and 11, and of the
C-15Ј–16Ј cyclohexyl fused compound 14 showed that, as
expected from the drastically altered shape and the increased
binding ability of the piperidine nitrogen in the atropisomers,
In 10Ј-methoxy-20Ј-deoxyvinblastine (33R) moderate L1210
and RCC-2 cytotoxicity and inhibition of tubulin polymeriz-
ation are retained and with its C-20Ј epimer (34R) we also
observed disassembly of microtubules and tubulin spiraliz-
ation, as with the parent compound 5. Introduction of an 11Ј-
bromo substituent (35) led to loss of the tubulin spiralization
activity of 20Ј-deethyl-20Ј-deoxyvinblastine (3), while a 10Ј-
chloro derivative 36 showed an increase in L1210 cytotoxicity
and in potency of tubulin polymerization inhibition.
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 2 1 2 0 – 2 1 3 6
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Table 3
Population growth IC50/M
Microtubule
Disassembly into
spiral aggregates
at 10Ϫ4 M
L1210
leukemia
RCC-2 Colon
adenocarcinoma
Microtuble
assembly IC50/M
Spiralization of
MTP at 10Ϫ4 M
Compound
R1
R2
R3
R4
30
H
H
H
Et
OMe
OMe
H
H
1 × 10Ϫ6
1 × 10Ϫ6
>5 × 10Ϫ6
N.D.
2 × 10Ϫ7
2 × 10Ϫ6
Ϫ
Ϫ
Ϫ, short MT
, amorphous
aggregates
Ϫ
, amorphous
aggregates
ϩ
31Ra
32Ra
33Ra
Et
Et
H
H
OMe
H
H
OMe
>1 × 10Ϫ6
1 × 10Ϫ8
>10Ϫ6
no effect at 1.3 × 10Ϫ5
2 × 10Ϫ6
Ϫ
Ϫ
5 × 10Ϫ7
34Ra
35
H
H
H
Et
H
H
H
Br
H
OMe
H
Cl
5 × 10Ϫ8
1 × 10Ϫ7
2 × 10Ϫ8
5 × 10Ϫ8
1 × 10Ϫ6
N.D.
2 × 10Ϫ6
1 × 10Ϫ6
5 × 10Ϫ7
ϩ
Ϫ
N.D.
Ϫ
N.D.
36a
a Denotes methanesulfonate salt. The corresponding compound without designation R was also tested as free base and found to have the same
activities as the salt.
Fig. 1
these compounds 46–51 did not inhibit tubulin polymeriz-
ation nor generate spiral aggregates from microtubular protein
(Table 5. The marginal cytotoxicities shown in the table may be
due to slight contamination by the corresponding very potent
active atropisomers)
Fig. 2
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 2 1 2 0 – 2 1 3 6
2124

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Table 4
Microtubule
Population growth IC50/M
Disassembly
into spiral
aggregates
at 10Ϫ4/M
L1210
RCC-2 Colon
Microtuble
tubulin IC50/M
Spiralization of
MTP at 10Ϫ4 M
Compound R1
R2
R3
R4 R5
n
leukemia adenocarcinoma
37a
H
Et
H
propyl
H
H
Et
H
OMe
OMe
OMe
OMe
Ac CHO
Ac CHO
Ac CHO
Ac CHO
1
1
1
1
4 × 10Ϫ7
N.D.
2 × 10Ϫ6
2 × 10Ϫ7
5 × 10Ϫ7
1 × 10Ϫ8
N.D.
ϩ
ϩ
N.D.
ϩ
ϩ
, amorphous
material
, amorphous
material
N.D.
(P)
N.D.
N.D.
N.D.
N.D.
N.D.
38a
7 × 10Ϫ10 7 × 10Ϫ9
39a
2 × 10Ϫ8
1 × 10Ϫ7
N.D.
40Ra
5 × 10Ϫ7
ϩ
41Ra
H
propyl OMe
Ac CHO
Ac Me
1
5 × 10Ϫ8
7 × 10Ϫ7
1 × 10Ϫ8
Ϫ
42a
43
Et
Et
H
Et
H
Et
H
Et
OH
H
Et
H
H
OH
H
OH
Tryp
Tryp
Tryp
OH
NHNH2
NHNH2
CRGNC
CRGNC
1
1
1
1
2
1
2
1
4 × 10Ϫ8
5 × 10Ϫ8
4 × 10Ϫ8
6 × 10Ϫ9
5 × 10Ϫ9
3 × 10Ϫ9
4 × 10Ϫ7
4 × 10Ϫ8
3 × 10Ϫ8
2 × 10Ϫ7
1 × 10Ϫ7
4 × 10Ϫ8
N.D.
N.D.
N.D.
N.D.
3 × 10Ϫ7
3 × 10Ϫ7
3 × 10Ϫ7
6 × 10Ϫ7
1 × 10Ϫ6
5 × 10Ϫ6
5 × 10Ϫ6
9 × 10Ϫ6
N.D.
(P)
H
H
H
H
H
H
H
Me
Me
Me
Me
Me
Me
Me
44a
45
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
25a
175
176
177
N.D.
a Denotes methanesulfonate salt. The corresponding compounds without designation R were also tested as free bases and found to have the same
activities as these salts.
Table 5
Microtubule
Population growth IC50(M)
Disassembly into
L1210
leukemia
RCC-2 Colon
adenocarcinoma
Microtuble
assembly IC50/M
spiral aggregates
Spiralization of
MTP at 10Ϫ4 M
Compound
R1
R2
R3
at 10Ϫ4 M
46
47
48
49
50
51
Et
Et
H
Me
H
OH
H
Et
H
Me
H
H
H
H
H
>10Ϫ6
>10Ϫ6
No effect, at 1 × 10Ϫ5
6 × 10Ϫ6
Ϫ
Ϫ
5 × 10Ϫ7
5 × 10Ϫ7
1.8 × 10Ϫ7
1.5 × 10Ϫ5
5 × 10Ϫ8
3 × 10Ϫ7
>10Ϫ6
,MT and spirals
ϩ, Single spirals
No effect, at 1 × 10Ϫ5
No effect, at 5 × 10Ϫ5
No effect, at 5 × 10Ϫ5
No effect, at 1 × 10Ϫ5
Ϫ
Ϫ
4 × 10Ϫ6
3 × 10Ϫ5
N.D.
Ϫ(5 × 10Ϫ5
Ϫ(5 × 10Ϫ5
Ϫ(5 × 10Ϫ5
)
)
)
Ϫ(5 × 10Ϫ5
Ϫ(5 × 10Ϫ5
)
)
H
–(CH2)4–
, Amorphous
aggregates (5 × 10Ϫ5M)
Possible contamination of 51 with 16.
This inactivity was also found with the C-14Ј,16Ј-epi-com-
pound vincovaline (52, Fig. 3, Table 6),12 its atropisomer 53
(Table 7), 20Ј-deoxyvincovaline (54), its atropisomer 55, the
corresponding C-20Ј epimer 56, the C14Ј,16Ј,20Ј-epi-isomers 57
and 58 of the α and β-cyclopentyl fused compounds 18 and 19
and the atropisomer 59 of the diastereomer of the cyclohexyl
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 2 1 2 0 – 2 1 3 6
2125

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Table 6
Microtubule
Population growth IC50/M
Microtuble
assembly
adenocarcinoma IC50/M
Disassembly into Spiralization
L1210
leukemia
RCC-2 Colon
spiral aggregates
of MTP at
Compound
R1
R2
R3
R4
at 10Ϫ4 M
10Ϫ4 M
52
54
57
Et
Et
OH
H
H
H
H
H
>1 × 10Ϫ4
1.5 × 10Ϫ5
8 × 10Ϫ8
N.D.
>10Ϫ4
Ϫ
Ϫ
Ϫ
Ϫ, Short MT
Ϫ
Ϫ, Short MT
5 × 10Ϫ6
No effect at 5 × 10Ϫ5
1 × 10Ϫ4
R2 ϩ R4 = –(CH2)3–, R1 = R3 = H
Possible contamination of 57 with 16
R1 ϩ R3 = –(CH2)3–, R2 = R4 = H
Possible contamination of 58 with 17
>10Ϫ6
>10Ϫ6
58
5 × 10Ϫ7
no effect at 1 × 10Ϫ5
Ϫ
Ϫ, Short MT
congeners, they were found to provide a new series of com-
pounds that can modulate the activity of active cytotoxic agents
and, that can overcome multidrug resistance. Those results are
described in a separate publication.13
Synthetic methodology
The carbomethoxy cleavamine (top) segment of the described
VLB congeners, with variations of C-20Ј substitution, were all
generated using procedures analogous to those developed for
the 20Ј-ethyl compounds.7 While condensations of aldehydes
containing variations of the eventual C-20Ј substituents were
initially carried out with the Nb-unsubstituted indoloazepine
60,14 with a following Nb-benzylation of the resulting epimeric
bridged azepines and rearrangement, later syntheses of the key
tetracyclic intermediates used condensations of the Nb-benzyl-
indoloazepine 61, cracked to an acrylate amine at 110 ЊC
(Scheme 1).15
The aldehyde precursors for 20Ј-deoxyvinblastine (4), 20Ј-
deoxyleurosidine (5) and their C-20Ј methyl analogues 10 and
11 were generated by alkylation of valerolactone, with eventual
selective reduction of an ester function after introduction into
tetracyclic vinylogous urethane intermediates 62 for generation
of the tetracyclic vindoline coupling precursors 63 (Scheme 2).
In syntheses of the C-20Ј propyl analogues 6R and 7R it was
found that the tetracyclic intermediates 64 could be synthesized
more effectively by making the intermediate aldehyde 65 by
reaction of a pyrrolidine enamine derivative of pentanal with
acrylonitrile, followed by borohydride reduction of the result-
ing aldehyde, O-silylation and reduction of the nitrile function
by diisobutylaluminum hydride (DIBAL-H).
In the C-20Јmethyl series, cyclization of the DЈ-seco-tosylate
intermediates 66–69 produced a mixture of “natural” (lower
energy) and “unnatural” (higher energy) atropisomers of the
final products, with the ratio depending on the diastereomeric
relationship of the seco-intermediates. From the corresponding
cyclization the C-20Ј ethyl compounds 20Ј-deoxyvinblastine
(4), its C-14Ј,16Ј,20Ј epimer 20Ј-deoxyvincovaline (54), 20Ј-
deoxyleurosidine (5) and its C-14Ј,16Ј,20Ј epimer had been
obtained as high–low energy atropisomers in respective ratios
of 3 : 97, 43 : 57, 28 : 72, and 60 : 40, while the ratios for the
C-20Ј methyl analogues 10,72,11,73, were 5 : 95, 46 : 54, 29 : 71
and 58 : 42, in good agreement, based on isolated product
Fig. 3
fused compound 14. While these diastereomers of active VLB
congeners lack the fundamental ability to affect tubulin
polymerization and therefore do not qualify as additional VLB
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 2 1 2 0 – 2 1 3 6
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Table 7
Microtubule
Population growth IC50/M
Disassembly into
spiral aggregates
at 10Ϫ4 M
L1210
leukemia
RCC-2 Colon
adenocarcinoma
Microtuble assembly
IC50/M
Spiralization of
MTP at 10Ϫ4 M
Compound R1
R2
R3
R4
53
55
56
59
Et
Et
H
OH
H
Et
H
H
H
H
H
H
>10Ϫ6
N.D.
No effect, at 1 × 10Ϫ4
1 × 10Ϫ4
Ϫ
Ϫ
Ϫ
Ϫ
Ϫ
5 × 10Ϫ7 >10Ϫ6
Ϫ, short MT
Ϫ
, Amorphous
aggregates
>10Ϫ6
>10Ϫ6
No effect, at 1 × 10Ϫ4
No effect, at 1.5 × 10Ϫ5
R2 ϩ R4 = –(CH2)4–,
R1 = R3 = H
5 × 10Ϫ7 N.D.
yields. Complete inversion of the high to low energy atrop-
isomer in refluxing toluene was seen in each case. In the C-20Ј
propyl series, the higher energy atropisomers were not
isolated but they were converted directly to the “natural” type
atropisomers by heating of the crude reaction products in
toluene.
[3.3.0]oct-6-ene-3-one (97,17 Scheme 5). Hydrogenation of the
double bond (100%) and Baeyer–Villiger oxidation (79%)
afforded the lactone 98. Its reduction with DIBAL-H (87%)
provided the lactol product 99. Attempts to condense this alde-
hyde equivalent with the indoloazepine 60 failed. Consequently,
the lactol ring was opened by formation of the dithiolan alco-
hol 100 (79%). Formation of its acetate (95%) and liberation of
the aldehyde function with mercuric acetate provided the alde-
hyde 96 (78%).
For synthesis of the C-20Ј dimethyl congener 12 of vinblast-
ine, the required 4,4-dimethyl-5-functionalized-valeraldehyde
74a (Scheme 3) was prepared by sodium hydroxide catalyzed
alkylation of isobutyraldehyde with acrylonitrile (49% of 75a),
followed by sodium borohydride reduction of of the aldehyde
function (95%) and protection of the resulting alcohol 76a as its
tert-butyldimethylsilyl ether (77a, 69%) prior to reduction of
the nitrile function (79%) with diisobutyl aluminum hydride
(DIBAL-H). Condensation of the aldehyde 74a with the indolo-
azepine 60,14 subsequent benzylation, rearrangement, coup-
ling to vindoline and reduction proceeded in the usual fashion
(i.e. Scheme 1).7 Cyclization of a C-21Ј tosylate 81a (R = Me)
required five days at reflux in toluene to furnish, after debenzyl-
ation, the product 12 in the “natural” atropisomeric form.16
The C-20Ј diethyl compound 13R and the spiro five and six-
membered ring congeners 21 and 22 were prepared analogously.
Here, a higher energy atropisomer (82), was isolated and could
be thermally converted to the lower energy atropisomer
(13R,21,22) in refluxing toluene.
Syntheses of the seven and eight-membered ring DЈ homo-
logues 23 and 24 of 20Ј-deethyl-20Ј-deoxyvinblastine (3) were
obtained, respectively, by condensation of 6-tert-butyldimethyl-
silyloxyhexanal and 7-tert-butyldimethylsilyloxy heptanal
(84,85, Scheme 4) with the indoloazepines 60 or 61, followed by
the rearrangement, vindoline coupling and cyclization steps
described for the lower homologue 3.4 Diastereomeric coupling
products 86,87, and 88,89, were obtained in 1 : 1 ratios in the
two homologous series. Again, atropisomeric products 90,92
and 91,93 were formed in the ring DЈ cyclization and inversion
of the higher energy atropisomers 90–93 to the lower energy
atropisomers 23,94, and 24,95 was found on heating. Conform-
ational inversion of these DЈ enlarged atropisomers to the lower
energy atropisomeric form still required refluxing toluene, thus
suggesting that the major barrier to this inversion is passage of
the C-3Ј methylene group through the cavity of the nine-mem-
bered ring CЈ, rather than a conformational change of ring DЈ.
For syntheses of the C-15Ј, 20Ј cyclopentyl cis-fused VLB
congeners 16 and 17 (Scheme 6), the cis-2-[(acetoxy)methyl]-
cyclopentaneacetaldehyde 96 was prepared from cis-bicyclo-
Condensation of the aldehyde 96 with the indoloazepine 60
gave the racemic acetates 101,102. They were methanolized to
the corresponding alcohols 103,104, which were chromato-
graphically separated and tosylated (105,106), and followed by
our usual sequence for elaboration of DЈ-seco-binary alkaloids.
Two sets of C-14Ј–15Ј diastereomeric tosylates 107,108 and
109,110 were obtained (Scheme 6). Their difference in rate of
cyclization allowed definition of their relative stereochemistry.
The C-14Ј–15Ј PREF,18 14Јα–H and the C-14Ј–15Ј PREF,14Јβ–
H fused products 109,110 were assigned to the slower cycliz-
ation (4 h at reflux in THF) and their corresponding C-14Ј–15Ј
PARF diastereomers 107,108 to the faster cyclization (at room
temperature and complete in <2 h in THF at reflux), based on
generation of 1,3-diaxial repulsions of ring DЈ substituents at
C-14Ј, C-20Ј and on NbЈ in generation of the quaternary salts
corresponding to the former pair 109,110. The absolute con-
figuration at C-16Ј,14Ј,15Ј,20Ј within each set of diastereomers
follows from the NMR chemical shift of the vindolinyl ethyl
group at δ 0.82 for the C-16ЈS, 14ЈR eventual debenzylation
products 16 and 17 as contrasted with their diasteromers (i.e. 16
vs. 111),4 as well as from the potent VLB-type biological activity
of products 16 and 17, that is absent in the C-16ЈR, 14ЈS com-
pounds. Atropisomers of these compounds (which are typically
found to have characteristic very broad NMR signals in all such
isolated VLB congeners,1,3 see below), could not be detected in
these syntheses.
The starting material for syntheses of the C-15Ј, 20Ј cis-fused
cyclohexyl VLB congeners 14 and 15, the atropisomer 51 of the
former and its diastereomer 59, was 2-indanol (112, Scheme 5).
Its hydrogenation over 5% Rh/C provided a diastereomeric
mixture of alcohols (59%), which was oxidized to the ketone
113 (96%). A Baeyer–Villiger oxidation to the lactone 114
(95%) and its reduction with DIBAL-H provided a lactol prod-
uct 115 (80%). Again, direct condensation of this product with
the indoloazepine 60 could not be achieved. On derivatization
with 1,2-ethanedithiol, the dithiolan alcohol 116 was formed
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 2 1 2 0 – 2 1 3 6
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Scheme 2 Reagents, conditions and yields: (R = Me), (a) LDA, MeI, HMPA, Ϫ78 ЊC to Ϫ40 ЊC, 3 h (66–77%); (b) 1) MeOH, conc. H2SO4, 45 min;
2) PCC, NaOAc, CH2Cl2, 30 min (40%); (c) 1) 60, MeOH, rt, 20 h; 2) C6H5CH2Br, THF, refl. 2 d; 3) Et3N, THF, refl. 10 h (85–90%); (d) 1) LAH, THF,
0 ЊC (55%); 2) Ts2O, Et3N, CH2Cl2, 0 ЊC to rt (62–71%); (i) 1) tBOCl, Et3N, CH2Cl2, 0 ЊC; 2) vindolineؒHCl, acetone, 0 ЊC, AgBF4, HBF4ؒEt2O,
25 min; 3) KBH4, HOAc, rt, 30 min (89%/72% R = Me); (j) 1) a: toluene, 110 ЊC, 17 h; b: toluene, 110 ЊC, 5 d; 2) 10% Pd/C, H2, MeOH a: 81% 10, 4%
49; b: 61% 11, 25% 50; (k) 1) a: toluene, 110 ЊC, 20 h; b: toluene, 110 ЊC, 7 d; 2) 10% Pd/C, H2, MeOH a: 37% 72, 51% 70; b: 41% 71, 47% 73);
(l) toluene, refl. (100%) (R = n-propyl), (e) 1) dioxane, refl. 16 h; 2) H2O, refl. 8 h, 3) aq. HCl 15 min (33%); (f ) 1) NaBH4, MeOH, 5 ЊC; 2) aq. HCl
(96%); 3) tBDMSCl, CH2Cl2, 4 ЊC, DIPEA, 4) DMAP, rt, 66 h (83%); (g) DIBAL-H, toluene, Ϫ70 ЊC, 3 h (81%); (h) 1) 60, MeOH, rt, 19 h;
2) C6H5CH2Br, THF, rt, 19 h; 3) MeOH, DIPEA. refl. 3 h (82%); 4) KF (H2O), CH3CN, BnEt3NCl, refl. 48 h, (82%); 5) TsCl, DMAP, PYD, rt, 26 h
(77%); (i) 1) tBOCl, Et3N, CH2Cl2, 0 ЊC; 2) vindolineؒHCl, acetone, rt, AgBF4, HBF4ؒEt2O, 80 min; 3) NaBH4, HOAc, rt, 30 min (38% 66, 42% 67);
(j + l) 1) xylene, 140 ЊC, 22 h; 2) 10% Pd/C, H2, MeOH, 11 or 6.5 h; 3) tol. refl. 4 h (37% 6R, 41% 7R).
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 2 1 2 0 – 2 1 3 6
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Scheme 3 Reagents, conditions and yields: (a) for 75, ref. 24, R = Me, isobutyraldehyde, hydroquinone, acrylonitrile, dioxane, aq. NaOH, 65 ЊC,
150 min (49% yield); (b) for 76, NaBH4, MeOH, 1 h, 0 ЊC (R = Me, 95%; R = (CH2)4, 85%; R = (CH2)5, 98%); (c) for 77, TBDMSCl, Et3N or DIEA,
CH2Cl2, DMAP, 0 ЊC to 20 ЊC, 16 h (R = Me, 69%; R = (CH2)4, 92%; R = (CH2)5, 75%); (d) 1) DBAL-H, CH2Cl2, Ϫ78 ЊC, 6 h; 2) 1 M HCl, 0 ЊC (74,
R = Me, 79%; R = (CH2)4, 72%; R = (CH2)5, 70%); (e) 1) 60, THF or MeOH or CH2Cl2, rt, 24 h; 2) C6H5CH2Br, THF, refl. 24 h; 3) MeOH, Et3N, refl.
6 d (78, R = Me, 70%; R = Et, 88%; R = (CH2)4, 98%; R = (CH2)5, 99%); (f ) TsOH or tBAF, THF, rt, 2 h (79, R = Me, 98%; R = Et, 86% (made with
48% aq. HF); R = (CH2)4, 91%; R = (CH2)5, 92%); (g) Ts2O or TsCl, Et3N, DMAP, CH2Cl2, 0 ЊC to rt, 2.5 h (80, R = Me, 72%; R = Et, 96%;
R = (CH2)4, 94%; R = (CH2)5, 95%; (h) 1) tBOCl, Et3N, CH2Cl2, 0 ЊC 15 min; 2) vindolineؒHCl, acetone, 0 ЊC, AgBF4, HBF4ؒEt2O, 30 min; 3) KBH4,
HOAc, rt, 20 min (81, R = Me, two diastereomers, 75%; R = Et, 47%, R = (CH2)5, 34%, its 14ЈR,16ЈR diastereomer 53%; (i) 1) toluene, refl. R = Me,
5 days, R = (CH2)4, 4 h for 14ЈS,16ЈS series; refl 13 h for R = Me, 14ЈR,16ЈR series; 2) 10% Pd/C, H2, MeOH; 3) toluene, reflux 6 h (12, 29%; its
14ЈR,16ЈR epimer, 29%; 13R, 45%; 22, 82%; its 14ЈR, 16ЈR diastereomer, 85%).
(64%). Acetylation of the hydroxyl group (98%) and liberation
of the aldehyde function with HBF4in aqueous THF pro-
vided the aldehyde 117 (95%). After generation of two racemic
cyclohexane C-15–20 cis-fused D-seco-ψ-vincadifformine
congeners 118,119 (Scheme 7), chromatographic separation of
diastereomers and formation of C-21 tosylates, coupling to
vindoline and reduction with potassium borohydride, two sets
of diastereomeric intermediates 124,125 and 126,127 were
obtained. Their respective cyclizations required 22 h vs. 3 h in
refluxing toluene. After debenzylation and chromatography,
pure samples of the VLB congeners 14 and 15, the atropisomer
51 of 14 and its C-16Ј,14Ј,15Ј,20Ј-epi-diastereomer (59) were
obtained.
sations with the lactols 99 and 115. (Lactol ring stabilization by
ring fusion in lactols 99 and 115, in contrast to the unsubsti-
tuted δ-lactol,3 is cancelled by introduction of two trigonal
centers in the benzolactol 130). After N-benzylation and
rearrangement (50% overall, the alcohol function in the
-seco-ψ-vincadifformine congener 131 was protected with a
tert-butyldimethylsilyl group (132, 93%) before coupling to
vindoline. The eventual diastereomeric products 133 (41%) and
134 (47%) were desilylated with p-toluenesulfonic acid in
aqueous THF (88%) and the resulting alcohols 135,136 cyclized
by reaction with p-toluenesulfonic anhydride to give, after
debenzylation (52% overall), the diastereomeric benzo VLB
congeners 18 and 137.
Synthesis of the benzo analogue 18 of VLB was derived from
Baeyer–Villiger oxidation of 2-indanone (128, Scheme 8). The
resulting lactone 129 (90%), on DIBAL-H reduction, provided
a lactol product 130, which could be condensed directly with
the indoloazepine 60, in contrast to failure of such conden-
For syntheses of the tetrahydropyran fused congeners 19 and
20, tetrahydropyran-4-one was subjected to a Horner–Emmons
condensation, followed by ester hydrolysis. The resulting
mixture of conjugated and unconjugated acids 138 and 139
(17 : 83) was condensed with formaldehyde in acetic acid and
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 2 1 2 0 – 2 1 3 6
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Scheme 4 Reagents, conditions and yields are for 7-membered ring products 23 and 94; products 24 and 95 are made analogously: (a) tBDMSiCl,
Et3N, DMAP, 54%; (b) Swern oxidation, 94%; (c) 61, neat, 130 ЊC; (d) 1) 60, MeOH; 2) PhCH2Br; 3) Et3N, MeOH, refl. (e) HF, aq. CH3CN, c + e
97%; (f ) Ts2O, Et3N, DMAP, 98%; (g) 1) tBuOCl, Et3N; 2) vindoline, AgBF4; 3) KBH4, AcOH, 80%, 1 : 1 86 and 87; (h) 1) sealed tube, CH3CN,
110 ЊC; 2) Pd/C, H2, 67% 90, 12% 23; (i) toluene, 110 ЊC, >98%; (h,i) 1) toluene, reflux; 2) Pd/C, H2; 3) toluene reflux, 84%; (j) hydrazine, MeOH,
reflux, 94%.
1.5% H2SO4 to give a 48% yield of the α,β–unsaturated lactone
140. Its hydrogenation over Pt led to a 7 : 1 ratio of cis–trans
fused lactones 141a,b in 88% yield. A more favorable 14 : 1
ratio, but only an 18% yield, was obtained from hydrogenation
of the β,γ–unsaturated lactone 142, that could be generated by
isomerization of the double bond with KOH and recyclization
of the acid (57%). While its hydrogenation with Pt in ethyl
acetate now produced an anticipated more favorable 14 : 1 ratio
of cis–trans lactones 141a,b, the major products of the
reaction were derived from hydrogenolysis to form cis- and
trans-3-methyltetrahydropyran-4-yl-acetic acids in a 9 :1 ratio.
Complete hydrogenolysis was obtained, as expected, by
hydrogenation with 10% Pd/C in ethanol (see Table 8).
A reduction of the 7 : 1 lactone mixture 141a,b with DIBAL-
H provided the corresponding lactols 143a,b. In order to
liberate a free aldehyde function, desired for optimum conden-
sation with an N-alkylindoloazepine, the lactols 143a,b were
opened with ethanedithiol, and the resulting thioacetal alcohols
144a,b were then converted to their acetate derivatives 145a,b.
Thioacetal cleavage with mercuric oxide then provided the
aldehydes 146a,b (7 : 1 cis–trans).
Condensation of the aldehydes 146a,b with the indolo-
azepine 14719 bearing a chiral ferrocenyl Nb-substituent, dia-
stereoselectively generated the tetracyclic amines 148,149
(54%), which were separated chromatographically from their
minor diastereomers 150,151 (9%) The major diastereomers
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 2 1 2 0 – 2 1 3 6
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Scheme 5 Reagents, conditions and yields: (a) 1) 10% Pd/C, H2, EtOAc, 25 psi, 90 min (100%); 2) MCPBA, NaHCO3, CH2Cl2, 20 ЊC, 24 h;
3) Na2S2O3, 20 min (79% 98); (b) DIBAL-H, CH2Cl2, Ϫ78 ЊC to 0 ЊC, 1 h (87% 99, 80% 115); (c) (CH2)2(SH)2, 12 M HCl, 0 ЊC to 20 ЊC (79% 100, 64%
116); (d) 1) AcCl, Pyd. CH2Cl2, 0 ЊC to 20 ЊC, 0.5 h; (95%, 98%); 2) HgO, THF, HBF4, 5 min (78% 96, 94% 117); (e) 1) 5% Rh/C, H2, MeOH, 20 psi,
20 ЊC (59%); 2) PCC, CH2Cl2, 2 h, 20 ЊC (96%); (f ) 1) MCPBA, CH2Cl2, NaHCO3, 20 ЊC, 17 h; 2) Na2S2O3,15 min (95% 114).
Scheme 6 a) 1) CH2Cl2, 20 ЊC, 18 h; 2) CHCl3, C6H5CH2Br, refl. 4 h; 3) MeOH, Et3N, refl. 18 h (84% 101, 102, 1 : 1); (b) K2CO3, MeOH, H2O, refl.
0.5 h, chromatographic separation (52% 103, 46% 104); (c) Ts2O, CH2Cl2, Et3N, 0 ЊC to 20 ЊC, 18 h (91% 105, 74% 106); (d) 1) tBOCl, Et3N,
2) vindoline, AgBF4, HBF4ؒEt2O; 3) KBH4, HOAc (48% 107 + 108, including their quaternary salt cyclization products); 67% 109 + 110); (e) 1) THF,
refl. 2 h; 2) 10% Pd/C, H2, MeOH, 2 h (39% 17, 37% 14Ј,15Ј,16Ј,20Ј epi-17, 35% 16, 36% 111).
were then subjected to replacement of the ferrocenyl Nb-substi-
tuent by a benzyl group (152,153, 36%, 2 steps), and to acetate
methanolysis. Chromatographic separation of the diastereo-
meric alcohols 154,155 and their tosylation (156,157, 82%, 2
steps) was followed by our usual coupling sequence with vindo-
line. The indoloazonines 158 and 159 were then cyclized at 110
ЊC. Hydrogenolysis of the quaternary Nb-benzyl substituent
provided the tetrahydropyran derivatives 19 and 20 (22% and
24% respectively, 5 steps). Here the final cyclization required
more strenuous conditions than those used with the corre-
sponding cyclohexyl substituted indoloazonines 124–127.
Consequently, debenzylation of the resulting pentacyclic quater-
nary salts gave only 5% of the higher energy atropisomer 160a
in addition to the lower energy conformer 160b of product 20.
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Table 8 Hydrogenation of unsaturated lactones 140 and 142. Product a: 3-methyltetrahydropyran-4-yl-acetic acid. Product b: ethyl 3-hydroxy-
methyltetrahydropyran-4-yl-acetate. All reactions at 1 atm H2
Product yield (the ratio of cis to trans)
Lactone
Catalyst
Solvent
141a/141b
a
b
140
10% Pd/C
10% Pd/C
10% Pd/C
PtO2
PtO2
PtO2
10% Pd/C
PtO2
Pd/C or PtO2
EtOH
AcOEt
AcOH
AcOEt
THF
t-BuOH
EtOH
AcOEt
CCl4
12%, 9:1
3.8%, 4:1
62.3%, 3.6:1
140
84.6%, 4.5:1
74.0%, 3.5:1
88.3%, 7.2:1
87.1%, 6.9:1
68.3%, 7.2:1
140
140
140
140
142
77.9%, 3.6:1
48.3%, 8.5:1
142
18.2%, 14.3:1
No reaction
140 or 142
Scheme 7 (a) 1) CH2Cl2, 20 ЊC, 16 h (95%); 2) CH2Cl2, C6H5CH2Br, refl. 3 h; 3) MeOH, Et3N, refl. 4 h (90% 118, 119, 1 : 1); (b) NaOMe, MeOH, rt,
3 h, chromatographic separation (120, 41% 121, 47%); (c) Ts2O, CH2Cl2, Et3N, DMAP, 0 ЊC, 48 h (86% 122, 82% 123); (d) 1) tBOCl, Et3N, CH2Cl2,
0 ЊC; 2) vindolineؒHCl, acetone, 0 ЊC, AgBF4, HBF4ؒEt2O, 30 min; 3) KBH4, HOAc, rt, 35 min; (e) 1) toluene, reflux 3 h; 2) 10%Pd/C, H2, MeOH,
3) toluene, reflux, 8 h, (d + e 20% 15, 20% 14Ј,15Ј,16Ј, 20Ј-epi-15); (f ) 1) toluene, refl. 22 h; 2) 10%Pd/C, H2, MeOH (d + f 27% 14, 23% 14Ј,15Ј,16Ј,
20Ј-epi-14); (g) toluene, reflux, 8 h.
No conformational high energy isomer could be isolated from
cyclization of the diastereomeric tosylate 158, where only the
lower energy atropisomer 19 was obtained in analogy to the
cyclohexane fused congener 15.
The methyl analogues 27 and 28 of vinblastine (1) and leuro-
sidine (29), their C-14Ј,16Ј epimeric diastereomers (i.e. 161) and
corresponding atropisomers of these compounds (i.e. 162a,b,)
were obtained by the procedures used for syntheses of the
parent C-20Ј ethyl compounds.1,2 Enantioselectivity at C-20Ј
was achieved by use of the chiral aldehydes 167,168 on con-
densation with the N-benzyl indoloazepine 61 (Scheme 10).
The use of trimethylsilyl protected alcohols was found to be
preferable for synthesis of the vinblastine homologue 27 and
the homologue 161 of 20Ј-epi-vincovaline because of a
cleaner final cleavage of the silyl ether function in the final
reaction step. However, the tert-butyldimethylsilyl derivatives
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Scheme 8 (a) 1) MCPBA, NaHCO3, CH2Cl2, 20 ЊC; 2) Na2S2O3 (90%); (b) DIBAL-H, CH2Cl2, Ϫ78 ЊC, 2 h, 0 ЊC, 15 min (91%); (c) 1) CH2Cl2,
B(OH)3, rt, 3 d; 2) C6H5CH2Br, CHCl3, 8 h refl. 3) Et3N, MeOH, 10 h refl. (50%); (d) tBDMSCl, Et3N, CH2Cl2, rt, 5 min (93%); (e) 1) tBOCl, Et3N,
CH2Cl2, 0 ЊC, 5 min; 2) vindolineؒHCl, acetone, 0 ЊC, AgBF4, HBF4ؒEt2O, 30 min; 3) KBH4, HOAc, rt, 35 min (41% 133, 47% 134); (f ) TsOH, THF,
H2O, 20 ЊC, 45 min (88% 135, 89% 136); (g) 1) Ts2O, Et3N, CH2Cl2, 20 ЊC, 35 min; 2) 10% Pd/C, H2, MeOH, 20 ЊC, 30 min (52% 18).
could be used in the C-20Ј epimeric series for synthesis of the
homologue 28 of leurosidine (29). Coupling of the tetracyclic
intermediates 169a,b and 170a,b with vindoline, and reduc-
tion, provided the binary tosylates 171a,b and 172a,b.1,2 On
treatment with tetrabutylammonium fluoride these furnished
the epoxides 173a,b. Their cyclization and debenzylation gave
a preponderance of the higher energy atropisomeric products
162a,b over the lower energy conformers 27 and 161. Heating
in toluene at reflux resulted in complete transformation to the
latter products.
Alternatively, the C-21Ј tosylates 171a,b and 172a,b cyclized
on heating in methanol to give the quaternary salts 163a,b and
164a,b. Their debenzylation provided the silyl ethers 165a,b
and 166a,b. A final cleavage of the trimethylsilyl ether 165a
provided the 20Ј-methyl homologue 27 of vinblastine in its
lower energy conformation. The TBDMS ether 166a required
more forcing conditions for cleavage, thus resulting in partial
decomposition.
Noteworthy was the formation of a substantial amount of
C-16Ј–14Ј PREF products in the vindoline coupling reac-
tion of the C-11 methoxylated D-seco-ψ-vincadifformines. This
decreased C-16Ј–14Ј PARF selectivity is in agreement with
the reaction mechanism postulated for these coupling
reactions,4,7 which is based on a carbocation intermediate. On
stabilization by a C-11 methoxy substituent this cation has
an increased lifetime that allows instability of its initial
conformation.
Compounds in which the vindoline moiety is oxidized to the
vincristine series, 37–41, or are converted to tryptophan deriv-
atives, 42–44, and the acid 45, were obtained by the method-
ologies known for such transformations.21–23
Acknowledgements
This work was supported by the National Cancer Institute of
the National Institutes of Health with grant R01CA12010.
Vindoline was generously supplied by Dr. A. J. Hannart of
Omnichem.
The ring AЈ methoxy and halogen subtituted compounds
30–35 were obtained from the corresponding indoloazepines.20
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Scheme 9 Reagents and conditions: (a) 1) Ph3P=CHCO2Me, toluene, 20% PhCO2H, refl. 8 h, 96%; 2) 50% KOH, EtOH, refl. 20 h; 3) dil. HCl, 94%
(138 : 139 = 17 : 83); (b) (CH2O), AcOH, 1.5% conc. H2SO4, refl. 10 h, 48%; (c) 1) 5% KOH–EtOH, refl. 25 h; 2) dil. HCl, 53%; (d, e) see Table 8;
(f ) DIBAL-H, CH2Cl2, Ϫ78 to 0 ЊC, 77%; (g) 1) 1,2-ethanedithiol, conc. HCl, 60%; 2) AcCl, pyridine, CH2Cl2, 99%; (h) 48% HBF4, THF–H2O, 84%.
(i) R” = ferrocenylethyl, C6H6, 36 h refl. 63%; R” = Bn, toluene, 110 ЊC 70%; (j) 1) AcOH, 70 ЊC; 2) PhCH2Br, Et3N, K2CO3, 36%; (k) 1) NaOMe,
MeOH refl. chromatographic separation of diastereomers 154, 155; 2) Ts2O, DMAP, 84% 156, 82% 157; (l) 1) tBuOCl, Et3N; 2) vindoline, HBF4,
AgBF4; 3) KBH4; (m) 1) 110 ЊC, CH3CN, sealed tube; 2) Pd/C, H2, 22% 19 from 154, 24% 20 from 155; (n) toluene, 110 ЊC.
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Scheme 10 (a) CuI, CH2=CHCH2MgCl, THF, Ϫ60 ЊC to Ϫ20 ЊC (66%); (b) Et3N, DMAP, TsCl, CH2Cl2, 20 h, rt (88%); (c) 1) TMSCl, DIEA, THF,
30 min (86%); TBDMSTf (95%); 2) O3, CH2Cl2, Ϫ78 ЊC, 10 min; 3) (C6H5)3P, Ϫ78 ЊC to rt, 18 h (81%, 74%); (d) toluene, refl. 15 h (57%, 65%);
(e) 1) tBOCl, Et3N, CH2Cl2, 0 ЊC; 2) vindoline, acetone, 0 ЊC, 15 min, AgBF4, HBF4.Et2O, 15 min; 3) KBH4, HOAc, rt, 30 min (58%); (f ) TBAF, THF,
rt, 15 min (85%, 73% 173a, 91%, 77% 173b); (g) 1) MeOH, 1.5 eq. HOAc, refl. 12 h; 2) 10% Pd/C, H2, MeOH, 3 h (52% 162a, 30% 27); 46% 162b, 17%
161; (h) 1) 171a, MeOH, refl. 36 h, 2) 10% Pd/C, H2, MeOH, 2.5 h (67% 165a); 1) 172a/172b, MeOH, 120 ЊC, 19/29 h; 2) Pd/C, H2, MeOH, 72% 166a,
63% 166b; 3) TBAF, THF, 45 min (76% 27); (i) toluene, refl. 6 h (100%); (j) TBAF, THF, 1.5 h refl. Analogous conditions for transformations from
aldehyde 174 to product 28.
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sample gave spectroscopic data that matched those provided by
Dr Potier.7
13 L. S. Borman, W. G. Bornmann and M. E. Kuehne,
Cancer Chemother. Pharmacol., 1993, 31, 343.
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The sample may have been contaminated by VLB. A pure synthetic
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 2 1 2 0 – 2 1 3 6
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