Synthesis and bioactivities of paclitaxel analogs with a cyclopropanated side-chain

Article abstract of DOI:10.1016/S0040-4039(03)00176-X

Four paclitaxel analogs with a cyclopropanated side-chain were synthesized by coupling of the spirocyclopropanated oxazoline-5-carboxylic acid 4 with 7-TES-baccatin III, followed by hydrolytic ring opening and rearrangement. The absolute configuration of

Full text of DOI:10.1016/S0040-4039(03)00176-X

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 44 (2003) 2049–2052
Synthesis and bioactivities of paclitaxel analogs with a
cyclopropanated side-chain
Changhui Liu,a Markus Tamm,b Marcus W. No¨tzel,b Armin de Meijere,b Jennifer K. Schillinga and
David G. I. Kingstona,*
aDepartment of Chemistry, M/C 0212, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA
bInstitut fu¨r Organische Chemie der Georg-August-Universita¨t Go¨ttingen, Tammannstrasse 2, D-37077 Go¨ttingen, Germany
Received 18 December 2002; revised 17 January 2003; accepted 17 January 2003
Abstract—Four paclitaxel analogs with a cyclopropanated side-chain were synthesized by coupling of the spirocyclopropanated
oxazoline-5-carboxylic acid 4 with 7-TES-baccatin III, followed by hydrolytic ring opening and rearrangement. The absolute
configuration of the 2%-position was determined by NMR analysis of the corresponding Mosher esters. The two new paclitaxel
analogs with the R configuration at C-2% were both active in the A2780 mammalian cell line cytotoxicity assay, but much less than
paclitaxel itself. © 2003 Elsevier Science Ltd. All rights reserved.
Paclitaxel (Taxol®) 1, originally isolated from the
Pacific yew (Taxus brevifolia),1 has become an impor-
tant anticancer drug, especially for the treatment of
breast and ovarian cancer.2 Many analogs of paclitaxel
have been prepared in studies directed at finding com-
pounds with improved activity, and several new analogs
are in clinical trials.3 Cyclopropyl groups have proved
to be highly effective in improving the activity of many
biologically active compounds,4,5 and several cyclo-
propane-bearing analogs of paclitaxel6 and epothilone7
have previously been synthesized and shown to have
improved or retained anticancer activity. The synthesis
of cyclopropanated paclitaxel analogs such as 3 was
thus of interest as a possible route to analogs with
enhanced bioactivity (Fig. 1). In particular, the 1,1-di-
substituted cyclopropyl group in the side-chain would
restrain the conformational mobility of the side-chain
and potentially bring it closer to the biologically active
binding conformation of paclitaxel. Thus a paclitaxel
analog with a methyl group at the 2%-position was
prepared by two groups,8,9 and was found to be slightly
more cytotoxic than paclitaxel; restricted conforma-
tional mobility was offered as one explanation for this
enhanced activity.8 On the other hand, a paclitaxel
analog with a deleted 3%-phenyl substituent was found
to be orders of magnitude less active than paclitaxel,10
but analogs with alkyl substituents in place of the
3%-phenyl ring and some additional modifications have
been shown to be more active than paclitaxel.11 Placing
the 1,1-disubstituted cyclopropyl group in the 3%-posi-
tion would also mimic gem-dimethyl substitution there,
the effect of which has not been tested yet.
The synthesis of 3 requires the attachment of a cyclo-
propanated side-chain to a protected baccatin III. The
preferred method of attaching paclitaxel side chains to
baccatin III is by ring-opening condensation with an
appropriately substituted b-lactam,12 but the b-lactam
required to apply this method to the synthesis of 3
cannot be prepared by the established [2+2] cycloaddi-
Figure 1. Structures of paclitaxel (1), 7-TES-baccatin III (2),
synthetic target (3), and ( )-2-phenylspiro(cyclopropane-1%,4-
oxazoline)-5-carboxylic acid (4).
* Corresponding author.
0040-4039/03/$ - see front matter © 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0040-4039(03)00176-X

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C. Liu et al. / Tetrahedron Letters 44 (2003) 2049–2052
tion of an ester enolate to an N-silylimine. Recently, a
new simple access to the spirocyclopropanated oxazo-
line-5-carboxylic acid 4 was disclosed.13 Compound 4 is
a potential precursor to 3, since oxazolines have been
shown to be suitable precursors of the paclitaxel side-
chain.14 It can also be noted that the synthetic route
used to prepare 4 probably could not be used to
prepare its gem-dimethyl analog because of the steric
problems associated with doing a Michael reaction on a
3,3-dimethylacrylate.
four of them had the same mass and elemental compo-
sition. The chemical shift differences between the 7-H
and 10-H protons in the 1H NMR spectra of com-
pounds 9 and 10 (or 3 and 11) were quite large. By
comparison with literature values,20 it could be con-
cluded that 10 and 11 had the 7-(R) (7-epi ) configura-
tion rather than the normal 7-(S) configuration.
In order to determine the absolute configuration at the
2%-position, compound 10 was converted to two esters
with (R)- or (S)-methoxyphenylacetic acid (MPA),
using the EDC/DMAP coupling conditions. Since the
7-hydroxy group in this compound is highly hindered,21
reaction occurred only at the 2%-OH position to yield
the Mosher esters 12 and 13. This observation also
supported the assigned configuration being (R) at C-7
for 10 and 11, since 7-epi-paclitaxel is much less reac-
tive at this position than paclitaxel itself. The resulting
2%-MPA esters 12 and 13 were subjected to NMR
analysis.22 Fortunately, the chemical shift differences
DlRS were significant (Scheme 2). From these data it
was concluded that the taxoid 10 must have the (S)-
configuration at C-2%, which is the configuration of
2%-epi-paclitaxel. Thus, the diastereomers 3 and 11 must
be 2%-(R), and 9 must be 2%-(S).
The coupling of 7-TES-baccatin III 2 with ( )-2-phenyl-
spiro(cyclopropane-1%,4-oxazoline)-5-carboxylic acid (4)
using DCC/4-PP occurred in good yield (85%),15 and
the two diastereomers 5 and 6 were obtained in a ratio
of 2:3 and were easily separated by thin-layer chro-
matography. Unfortunately, 5 and 6 proved resistant to
hydrolysis under the conditions previously used [0.1N
HCl/dioxane (1:1)].14 Only starting material and vari-
ous decomposition products could be detected under
the literature conditions, while prolonged reaction times
led to cleavage of the side-chain. Surprisingly, however,
when the triethylsilyl groups in 5 and 6 were first
removed by treatment with HF–pyridine, and the
product subsequently hydrolyzed with 0.1N HCl/diox-
ane (1:1) at 50°C, the O-benzoyl derivatives 7 and 8
with free amino groups were obtained (Scheme 1).16
These compounds were less prone to rearrange by
benzoyl migration from the 2%-oxy group to the 3%-
amino group than other taxol analogs.17 Thus, under
neutral aqueous conditions and basic non-aqueous con-
ditions, migration of the benzoyl group did not take
place, but treatment with 0.1N NaHCO3/dioxane (1:1)
led to rearrangement accompanied by epimerization at
C-7,18 and the four cyclopropane-containing paclitaxel
analogs (9, 10, 3, and 11)19 were obtained. High resolu-
tion FAB mass spectra (HRFABMS) showed that all
The cytotoxic activity of these novel taxoids was evalu-
ated in vitro using the A2780 ovarian cancer cell line.
This assay showed that compounds 9 and 10, with the
unnatural configuration at 2%, are essentially inactive,
while the isomers 3 and 11 with the natural configura-
tion at 2% are active, but much less so than paclitaxel
itself (Table 1).
This finding could be due to a conformational restric-
tion such that the cyclopropyl analogs do not bind well
Scheme 1. Keys: (a) DCC, 4-PP, toluene, rt, 24 h, 85%; (b) HF–pyridine, THF, 0°C to rt, 24 h, 90%; (c) 0.1N HCl, 1,4-dioxane
(1:1), 50°C, 1 h, 85%; (d) 0.1N NaHCO3, 1,4-dioxane (1:1), rt, 6 h, 80%.

C. Liu et al. / Tetrahedron Letters 44 (2003) 2049–2052
2051
Scheme 2. Key: (a) (R)-Methoxyphenylacetic acid or (S)-methoxyphenylacetic acid, EDC, DMAP, CH2Cl2, rt, 24 h, 85%.
*DlRS=lR (7) −lS (8).
Table 1. Cytotoxicities (IC50 values) of compounds 9, 10,
3, and 11, in the A2780 mammalian cell assaya
the German group was supported by the Deutsche
Forschungsgemeinschaft (SFB 416, Project A3) and the
Fonds der Chemischen Industrie. We are grateful to
Karsten Rauch for help with the synthesis of com-
pound 4.
Compound
Cytotoxicity (IC50, mg/mL)
1
0.02
9
10
3
18
\20
7
References
11
8
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a Data are the mean of at least three determinations, and are rounded
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to the receptor, but it is perhaps more likely that the
cyclopropyl group is too small to have good van der
Waals or aromatic interactions at the binding site.23 In
the closest published analogy to this work, a 9-dihy-
drodocetaxel analog with a methyl group in place of the
3%-phenyl group was an average of 30-fold less cytotoxic
than the corresponding 3%-phenyl derivative, while the
3%-isobutyl derivative was significantly more active than
the 3%-phenyl derivative.24 The synthesis of substituted
cyclopropyl derivatives of 3 might thus yield some
compounds with interesting bioactivities.
The observation that 9 and 10 are even less cytotoxic
than 3 and 11 is consistent with the above-mentioned
configurational assignments, as taxoids with a 2%-(S)-
configuration are usually less active than those with a
2%-(R)-configuration.17,25
Acknowledgements
Work at Virginia Polytechnic Institute and State Uni-
versity was supported by a research grant from the
National Institutes of Health (CA-69571), and this
support is gratefully acknowledged. Mass spectra were
obtained by Mr. William Bebout at Virginia Polytech-
nic Institute and State University Institute. The work of
8. Kant, J.; Schwartz, W. S.; Fairchild, C.; Gao, Q.; Huang,
S.; Long, B. H.; Kadow, J. F.; Langley, D. R.; Farina,
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J. Med. Chem. 1991, 34, 1176–1184.
11. Ojima, I.; Slater, J. C.; Michaud, E.; Kuduk, S. C.;
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M.; Pera, P.; Bernacki, R. J. J. Med. Chem. 1996, 39,
3889–3896.
12. Ojima, I.; Habus, I.; Zhao, M.; Zucco, M.; Park, Y. H.;
Sun, C. M.; Brigaud, T. Tetrahedron 1992, 48, 6985–7012.
13. No¨tzel, M. W.; Tamm, M.; Labahn, T.; Noltemeyer, M.;
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4484.
7.33–7.52 (5H, m), 7.61 (1H, m), 7.67 (2H, m), 8.05 (2H,
m); 13C NMR l (ppm) 9.7, 13.7, 14.7, 15.9, 21.1, 21.9,
22.8, 26.9, 35.8, 36.3, 36.8, 43.3, 46.0, 58.8, 72.0, 72.5,
75.1, 75.9, 76.6, 77.3, 79.6, 81.2, 84.6, 127.2, 128.93,
128.94, 129.3, 130.3, 132.5, 132.8, 133.4, 134.0, 143.0,
167.2, 170.3, 170.4, 171.5, 172.3, 204.0; HRFABMS,
MH+ calcd 804.3231, found 804.3197. 10: [h]2D0=−56° (c
0.41); 1H NMR l (ppm) 1.14 (3H, s), 1.17 (3H, s),
1.07–1.31 (4H, m), 1.65 (3H, s), 1.65–1.75 (1H, m), 2.00
(3H, d, J=1.3 Hz), 2.19 (3H, s), 2.20–2.39 (3H, m), 2.39
(3H, s), 3.66–3.73 (1H, m), 3.72 (1H, s), 3.94 (1H, d,
J=7.4 Hz), 4.33 (1H, d, J=8.6 Hz), 4.38 (1H, d, J=8.6
Hz), 4.70 (1H, d, J=10.3 Hz), 4.92 (1H, dd, J=5.7, 3.3
Hz), 5.73 (1H, d, J=7.4 Hz), 6.11 (1H, td, J=8.9, 1.5
Hz), 6.71 (1H, s), 6.83 (1H, s), 7.32–7.52 (5H, m), 7.62
(1H, m), 7.68 (2H, m), 8.06 (2H, m); 13C NMR l (ppm)
14.0, 14.6, 15.9, 16.4, 21.1, 21.3, 22.7, 26.2, 35.6, 36.6,
37.0, 40.6, 42.8, 57.8, 71.5, 75.4, 75.9, 77.8, 78.4, 79.5,
82.2, 82.9, 127.3, 128.96, 129.00, 129.4, 130.2, 132.6,
133.1, 133.4, 134.0, 140.3, 167.2, 169.7, 170.9, 172.1,
172.3, 207.5; HRFABMS, MNa+ calcd 826.3050, found
15. Preparation of compounds 5 and 6: To a solution of 4 (70
mg, 0.323 mmol) in 5 ml toluene was added DCC (68 mg,
0.323 mmol). After 15 min stirring, 4-PP (2 mg, cat.) was
added and keep stirring for 5 min before 2 (70 mg, 0.100
mmol) was added. The reaction mixture was allowed to
stir at room temperature for 24 h, followed by washing
through a short silica gel column using EtOAc as solvent.
The organic phase was concentrated in vacuum. The
residue was applied to PTLC (30% EtOAc/hexane) to
give 5 (33 mg, 36%) and 6 (44 mg, 49%).
16. Preparation of compounds 7 and 8. To a solution of 5 (32
mg, 0.0355 mmol) in 2.5 ml THF was added pyridine (0.5
ml) and HF–pyridine (0.5 ml) at 0°C. The reaction mix-
ture was then allowed to warm up to room temperature
and stirred overnight. The reaction mixture was diluted
with EtOAc (30 ml), and then washed with sodium
bicarbonate, water, and brine. The organic phase was
concentrated in vacuum. The residue was applied to
PTLC (50% EtOAc/hexane) to give the desired deprotec-
tion product (25.5 mg, 90%). To a solution of the depro-
tection product (25 mg, 0.032 mmol) in 5 ml 1,4-dioxane
was added HCl (0.1N, 5 ml) and stirring was continued
at 50°C for 1 h. The reaction mixture was diluted with
EtOAc (30 ml), and then washed with sodium bicarbon-
ate, water, and brine. The organic phase was dried with
sodium sulfate, concentrated in vacuum, and applied to
PTLC (60% EtOAc/hexane) to give 7 (21.3 mg, 85%).
Compound 8 was prepared in similar yield using the same
procedure.
17. Cabri, W. Tetrahedron Lett. 1996, 37, 4785–4786.
18. Preparation of compounds 9, 10, 3, and 11: To a solution
of 7 (16 mg, 0.02 mmol) in 1,4-dioxane (5 ml) was added
NaHCO3 solution (0.1N, 5 ml) at room temperature. The
reaction was allowed to stir for 6 h and TLC showed no
starting material was left. The reaction mixture was
diluted with EtOAc (20 ml), and then washed with water,
brine, and dried with sodium sulfate. The organic phase
was concentrated in vacuum, and the residue was applied
to PTLC (50% EtOAc/hexane) to give 9 (7.7 mg, 48%)
and 10 (5.1 mg, 32%). Compounds 3 and 11 were pre-
pared in similar yield by this procedure.
826.3050. 3: [h]2D0=−68° (c 0.30); H NMR l (ppm) 1.15
1
(3H, s), 1.27 (3H, s), 1.07–1.35 (4H, m), 1.69 (3H, s), 1.79
(3H, d, J=1.3 Hz), 1.88 (1H, m), 2.23 (3H, s), 2.25–2.38
(2H, m), 2.46 (3H, s), 2.57 (1H, m), 3.82 (1H, d, J=7.1
Hz), 3.91 (1H, s), 4.18 (1H, d, J=8.5 Hz) 4.33 (1H, d,
J=8.5 Hz), 4.44 (1H, dd, J=10.8, 6.7 Hz), 4.99 (1H, dd,
J=7.5, 2.1 Hz), 5.68 (1H, d, J=7.1 Hz), 6.24 (1H, td,
J=9.2, 1.5 Hz), 6.25 (1H, s 6.83), 1H (s), 7.44–7.60 (5H,
m), 7.64 (1H, m), 7.74 (2H, m), 8.10 (2H, m); 13C NMR
l (ppm) 9.6, 13.6, 14.3, 14.8, 20.9, 22.01, 22.04, 26.7,
35.5, 35.9, 37.2, 43.2, 45.6, 58.5, 71.0, 72.1, 75.1, 75.6,
76.4, 77.9, 79.5, 80.7, 84.5, 127.2,128.7, 128.9, 129.2,
129.3, 130.1, 132.59, 132.60, 132.7, 132.8, 133.8, 142.8,
167.0, 170.6, 170.8, 171.2, 172.3, 203.8, 207.4;
HRFABMS, MH+ calcd 804.3231, found 804.3239. 11:
1
[h]2D0=−70° (c 0.24); H NMR l (ppm) 1.16 (3H, s), 1.22
(3H, s), 1.08–1.37 (4H, m) 1.66 (3H, s), 1.71 (3H, d,
J=1.4 Hz), 2.18 (3H, s), 2.23–2.41 (3H, m), 2.57 (3H, s),
3.69 (1H, m), 3.89 (1H, s), 3.92 (1H, d, J=7.5 Hz), 4.35
(1H, d, J=8.5 Hz), 4.41 (1H, d, J=8.5 Hz), 4.78 (1H, d,
J=11.7 Hz), 4.96 (1H, dd, J=5.7, 3.4 Hz), 5.76 (1H, d,
J=7.5 Hz), 6.21 (1H, td, J=9.0, 1.4 Hz), 6.74 (1H, s)
6.80 (1H, s) 7.44–7.60 (5H, m) 7.65 (1H, m), 7.73 (2H,
m), 8.12 (2H, m); 13C NMR l (ppm) 13.8, 14.6, 14.8,
16.2, 20.9, 21.3, 22.1, 25.9, 35.3, 36.2, 37.5, 40.4, 42.7,
57.6, 70.9, 75.4, 75.7, 77.5, 78.2, 78.6, 79.5, 81.6, 82.8,
127.2, 128.7, 128.9, 129.3, 130.1, 132.5, 132.7, 133.8,
140.3, 167.1, 169.3, 171.2, 172.3, 172.6, 207.4;
HRFABMS, MH+ calcd 804.3231, found 804.3226.
20. Chmurny, G. N.; Hilton, B. D.; Brobst, S.; Look, S. A.;
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19. Compound 9: [h]D20=−60° (c 0.23); 1H NMR l (ppm)
1.14 (3H, s), 1.24 (3H, s), 1.05–1.40 (4H, m), 1.68 (3H, s),
1.88 (1H, m), 2.12 (3H, d, J=1.1 Hz), 2.15–2.35 (2H, m),
2.24 (3H, s), 2.28 (3H, s), 2.51–2.61 (1H, m), 3.68 (1H, s),
3.84 (1H, d, J=7.1 Hz), 4.16 (1H, d, J=8.3 Hz), 4.29
(1H, d, J=8.3 Hz), 4.45 (1H, dd, J=10.7, 6.6 Hz), 4.96
(1H, dd, J=7.7, 1.8 Hz), 5.66 (1H, d, J=7.1 Hz), 6.15
(1H, td, J=8.9, 1.3 Hz), 6.34 (1H, s), 6.66 (1H, s),
metry 2001, 12, 2915–2925.
23. We thank a referee for this suggestion.
24. Li, L.; Thomas, S. A.; Klein, L. L.; Yeung, C. M.;
Maring, C. J.; Grampovnik, D. J.; Lartey, P. A.; Plattner,
J. J. J. Med. Chem. 1994, 37, 2655–2663.
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