Synthesis of novel macrocyclic docetaxel analogues. Influence of their macrocyclic ring size on tubulin activity

Article abstract of DOI:10.1021/jm030770w

This work describes the synthesis of a series of novel macrocyclic taxoids 3 and 3(H) designed to mimic the docetaxel solid-state ("nonpolar") conformation. These compounds, bearing 18-, 20-, 21-, and 22-membered rings connecting the C-2 OH and C-3′ NH mo

Full text of DOI:10.1021/jm030770w

J . Med. Chem. 2003, 46, 3623-3630
3623
Syn th esis of Novel Ma cr ocyclic Doceta xel An a logu es. In flu en ce of Th eir
Ma cr ocyclic Rin g Size on Tu bu lin Activity
Olivier Querolle,† J oe¨lle Dubois,*,† Sylviane Thoret,† Fanny Roussi,† Sara Montiel-Smith,‡
Franc¸oise Gue´ritte,† and Daniel Gue´nard†
Institut de Chimie des Substances Naturelles, CNRS, Avenue de la Terrasse, 91198 Gif sur Yvette Cedex, France,
and Facultad de Ciencias Qu´ımicas Beneme´rita Universidad Auto´noma de Puebla, Puebla, Me´xico
Received J anuary 17, 2003
This work describes the synthesis of a series of novel macrocyclic taxoids 3 and 3(H) designed
to mimic the docetaxel solid-state (“nonpolar”) conformation. These compounds, bearing 18-,
20-, 21-, and 22-membered rings connecting the C-2 OH and C-3′ NH moieties, were constructed
by ring-closing olefin metathesis of the taxoid-ω,ω′-dienes 4. Biological evaluation of these new
taxoids showed that activity is dependent on the ring size, and only the 22-membered ring
taxoid 3d exhibits significant tubulin binding. Synthesis of the open-chain analogues 7 and
7(H) and comparison of their biological activities with macrocyclic taxoids show that the carbon
tether between C-2 OH and C-3′ NH does not hamper tubulin binding. Computational studies
of the conformational behavior of the macrocyclic taxoids 3 indicate that the 18-, 20-, and 21-
membered-ring 3a -c adopt mainly conformations that are not recognized by tubulin. The most
active taxoid 3d appears to adopt a conformation that is between the “nonpolar” and T-shaped
forms.
In tr od u ction
Taxoids are a series of anticancer drugs1 that inhibit
cell growth by interacting with microtubules.2 Paclitaxel
(Taxol) 1a 3 and docetaxel (Taxotere) 1b4 (Figure 1), the
leading compounds of this series, are effective clinical
agents for the treatment of various cancers. Because of
their original activity and their efficiency in cancer
treatment, extensive studies have been made on struc-
ture-activity relationships.5 The recently determined
F igu r e 1.
structure of tubulin6 clearly shows the location of the
the centroid of the C-2 benzoyl phenyl ring is almost
equidistant from the centroids of both hydrophobic
groups at the C-3′ position (T-shaped structure). This
conformation has been recognized in the deconvolution
of the average spectra of paclitaxel in chloroform11 and
proposed to be the bound conformation on â-tubulin.12
To delineate the bound conformation of taxoids on
tubulin, the synthesis of analogues with built-in con-
formational restrictions has been designed.13 Several
compounds with bridges linking the C-2 and C-3′ phenyl
groups have been synthesized to mimic the “polar”
conformation.13a-c Only a few of them retain an interac-
tion with microtubules and to a lesser extent than
paclitaxel (19-36%).13c Analogues of the T-shaped con-
formation have been recently designed bearing either
a conformationally constrained side chain13e or a tether
between the C-4 acetyl group and the C-3′ phenyl
ring.13d These compounds show significant cytotoxicity
and tubulin binding. Conformational analysis of these
derivatives is compatible with the hypothesis of a
bioactive T-shaped conformation but does not rule out
the existence of another one. Finally, the first deriva-
tives designed to mimic the “nonpolar” conformation
have been described with a tether between the C-2 and
N-3′ groups.13f Though these new compounds were
reported to be cytotoxic, their binding to microtubules
was not reported.
taxoid binding site, but the 3.5 Å resolution does not
allow complete determination of the ligand conforma-
tion. The first information on the 3D structure of taxoids
was obtained by X-ray diffraction on the crystal of
docetaxel.7 Since the past 10 years, several conforma-
tional studies (X-rays, NMR, and molecular modeling)
have led to the proposal of three major “active” confor-
mations for antitumor taxoids. Whereas the taxane core
is conformationally rigid, the C-13 â-phenylisoserine
side chain possesses a high degree of freedom and the
three conformers refer to its relative orientation. Two
of these conformers show hydrophobic collapse between
the C-2 benzoyl group and the C-3′ substituents. The
“polar” form, observed by NMR in polar solvents8 and
in the crystal structure of paclitaxel,9 displays a hydro-
phobic interaction between the C-2 benzoyl moiety and
the phenyl group at position C-3′. Otherwise, this
interaction involves the C-3 NH substituent in the
“nonpolar” conformer observed in the docetaxel solid
state7 and by NMR in apolar solvents.10 The third
conformer is devoid of intramolecular interactions, and
* To whom correspondence should be addressed. Phone:
33 (0)1 69 82 30 58. Fax: 33 (0)1 69 07 72 47. E-mail: joelle.dubois@
icsn.cnrs-gif.fr.
† Institut de Chimie des Substances Naturelles.
‡ Facultad de Ciencias Qu´ımicas Beneme´rita Universidad Auto´noma
de Puebla.
10.1021/jm030770w CCC: $25.00 © 2003 American Chemical Society
Published on Web 07/23/2003

3624 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 17
Querolle et al.
Sch em e 1. Retrosynthesis of Compounds 3
F igu r e 2.
Then, after intermediate purification leading to the
N-acylated derivative (88% yield), esterification at C-2
has been achieved by addition of 5 equiv of 5-hexenoic
acid together with DCC and DMAP. Compounds 4a -d
have been thus synthesized in good yields (60-79%)
(Scheme 2). These diene precursors were subjected to
RCM using Grubbs’ catalyst in refluxing CH2Cl2 to
afford the macrocyclic taxoids 6a -d in 85-90% yield
(Table 1).
All compounds were obtained as an E/Z mixture with
a 0.5 to 4 ratio, determined by NMR, depending on the
macrocycle formed. Because of the superposition of
ethylenic 1H signals with other protons, the configura-
tion of the isolated compounds was determined either
by nuclear Overhauser effect spectrometry (NOESY) or
by comparison of the 13C chemical shifts of the allylic
carbons. The Z-isomer prevailed only for the 18-
membered-ring compound 6a , and the amount of the
E-isomer increased with the ring size. Both isomers
were found in similar amounts for 6b, whereas the
E-isomer was preferentially formed for 21- and 22-
membered macrocyclic taxoids 6c and 6d . Z and E
isomers were easily separated by silica gel chromatog-
raphy for 6a and 6b to give 6a -Z, 6a -E, 6b-Z, and 6b-
E. E and Z isomers were inseparable in 6c, and only
the E-isomer of 6d has been obtained in pure form.
Hydrogenation of macrocyclic taxoids 6a -d on Pd/C
afforded the corresponding macrocyclic taxoids 6(H)a -d
bearing a saturated linker in satisfactory yield (72-
84%) (Scheme 2).
In an attempt to model the “nonpolar” conformation,
we have designed macrocyclic taxoids with bridges
linking the C-2 benzoyl phenyl group and the N-3′ acyl
moiety. We have shown with 19- to 25-membered-ring
thio- or dithiomacrocyclic taxoids 2 that the macrocyclic
ring size had an influence on tubulin binding14 (Figure
2). The 21-membered-ring sulfide 2a and 20-membered-
ring disulfide 2b were the most active compounds
though much less than docetaxel on microtubule disas-
sembly and cytotoxicity. Suspecting the presence of
sulfur to be deleterious to microtubule binding, we
designed new C-2,C-3′ N-linked macrocyclic taxoids 3
with carbon tethers (Figure 2). With the 20- and 21-
membered-ring taxoids 2a and 2b being the most active
compounds in the sulfur-containing series, we decided
to vary the macrocyclic ring size below and above these
values in order to investigate its influence on biological
activities. Therefore, we present, in this paper, the
syntheses of compounds 3 bearing 18-, 20-, 21- and 22-
membered rings together with the examination of their
conformational behavior related to their biological ac-
tivities.
Ch em istr y
In the design of the synthesis of compounds 3, ring-
closing metathesis (RCM)15 methodology was chosen for
the crucial macrocyclization step. This strategy has
proven its efficacy for the synthesis of macrocyclic
taxoids. First employed by Ojima and his collabora-
tors for the synthesis of a large number of C-2,C-3′
linked macrocyclic taxoids,13b,c this methodology has
been also used in preparing analogues with C-4,C-3′13d
and C-2,C-3′N13f linkers. By use of the same strategy
as for compounds 2, macrocyclic taxoids 3 can be
obtained by RCM on the open-chain ω,ω′-diene precur-
sor 4, which can be synthesized from the C-2 OH and
C-3′ NH2 taxoid 5. This deacylated derivative 5 is easily
obtained from docetaxel as previously described14
(Scheme 1).
To compare the biological activities of the macrocyclic
taxoids with their open-chain precursors, compounds
4a -d were also subjected to hydrogenation on Pd/C to
afford the corresponding saturated taxoids 4(H)a -d in
70-80% yield (Scheme 3). All these compounds were
fully deprotected by HF/pyridine with moderate to good
yields, affording 19 compounds 3a -E and -Z, 3b-E and
-Z, 3c, 3d and 3d -E, 3(H)a -d , 7a -d (Scheme 2), and
7(H)a -d (Scheme 3).
Resu lts a n d Discu ssion
The introduction of the alkenyl chains on compound
5 was performed by acylation of C-2 OH and C-3′ NH2
with commercially available alkenoic acids in the pres-
ence of DCC and DMAP (Scheme 2). For the 21-
membered macrocyclic taxoid, acylation of the amine
has been first realized with 2 equiv of 6-heptenoic acid.
Biologica l Eva lu a tion . The 19 new compounds were
evaluated for their inhibition of cold-induced microtu-
bule disassembly16 and for their cytotoxicity against the
KB cell line17 (Table 2).
All compounds are less cytotoxic than docetaxel with
cytotoxicity in agreement with tubulin activity, in

Docetaxel Analogues
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 17 3625
Sch em e 2a
a
(i) For a , b, d : DCC, DMAP, CH2dCH(CH2)nCOOH (5 equiv), toluene, 60 °C, 3 h (5a , 62%; 5b, 60%; 5d , 77%). For c: DCC, DMAP,
CH2dCH(CH2)4COOH (2 equiv), toluene, room temp, 3 h, then DCC, DMAP, CH2dCH(CH2)3COOH (5 equiv), toluene, 60 °C, 3.5 h (5c,
79%). (ii) HF/pyridine, pyridine, CH3CN, 0 °C, 1 h. then room temp, 6.5-8 h (3a -Z, 57%; 3a -E, 51%; 3b-Z, 69%; 3b-E, 68%; 3c, 70%; 3d ,
50%; 3d -E, 61%; 3(H)a , 45%; 3(H)b, 55%; 3(H)c, 64%; 3(H)d , 56%; 7a , 46%; 7b, 71%; 7c, 75%; 7d , 75%). (iii) (Cy3P)2Ru(dCHPh)Cl2 (6%
mol), CH2Cl2, reflux, 4 h (6a , 89%; 6b, 90%; 6c, 90%; 6d , 74%); (iv) H2, Pd/C, EtOAc, 3.5 h (6(H)a , 84%; 6(H)b, 75%; 6(H)c, 76%; 6(H)d ,
72%).
Ta ble 1. Ring-Closure Metathesis Results on Compounds
4a -d
enable any IC50 measurement. In the reduced series,
only a weak microtubule disassembly inhibition was
compd
n
n′
ring size
yield, %
E/Z
observed with 3(H)c and 3(H)d . The difference between
3d and 3(H)c,d may be due to the presence of the double
bond in 3d that either induces a more favorable
conformation by additional constraint or promotes
hydrophobic interaction in the tubulin binding site. It
is noted that inhibition of microtubule disassembly
seems to be independent of the stereochemistry of the
double bond. Finally these results show that the pres-
ence of an alkenyl tether between C-2 and N-3′ does not
prevent tubulin binding as shown by the close values
obtained for 3d and its acyclic analogue 7d .
6a
6b
6c
6d
2
3
4
4
2
3
3
4
18
20
21
22
89
90
90
85
35/65
55/45
80/20
75/25
contrast to what has been observed with the sulfur-
containing macrocyclic taxoids 2.14 The only compounds
that are inactive on microtubule disassembly but still
cytotoxic are the E-isomer of compound 3b and its
reduced analogue 3(H)b. It is noted that the acyclic
taxoids are in the same range of activity except for 7b,
which is the most cytotoxic (only 10 times less than
docetaxel), and for 7(H)a , which is the less active
compound on KB cells. Compounds 7a -d and 7(H)a -d
are the first docetaxel derivatives substituted both at
C-2 and N-3′ by alkyl chains. They show similar
activities on microtubule disassembly compared to tax-
oids bearing a hydrophobic group either at C-2 or at
N-3′.18,19
The activities of macrocyclic compounds 3a -d and
3(H)a -d on microtubule disassembly are very interest-
ing. Their interaction with microtubules seems to be
dependent on the ring size because compounds bearing
less than 21 atoms in the macrocycle are devoid of any
activity. Only the 22-membered-ring compound 3d is
well recognized by tubulin in the unsaturated series
with an activity 3 and 6 times higher than, respectively,
the 20-membered-ring disulfide 2b and the 21-membered-
ring sulfide 2a .14 These results confirm that the pres-
ence of the sulfur atom is deleterious to microtubule
binding. Inhibition of microtubule disassembly has been
observed with the 21-membered-ring derivative 3c at
high concentration, but the activity was too low to
Con for m a tion a l Stu d ies. To investigate how these
macrocyclic taxoids were conformationally restricted, we
first studied their conformational behavior in solution.
As mentioned in the Introduction, taxoids may exist in
solution under different conformers. To clarify our
discussion, we have taken into account the highest
population conformations. Thus, the “nonpolar” form is
the major conformation in chloroform characterized by
a small H2′/H3′ coupling constant (J H2′/H3′ < 2 Hz),
whereas taxoids adopt preferentially the “polar” con-
formation in DMSO indicated by a larger coupling
constant (J H2′/H3′ ) 6-8 Hz).20
In contrast to paclitaxel and docetaxel, the H2′/H3′
coupling constant was very similar in both polar and
apolar solvents for sulfur-containing macrocyclic taxoids
2 bearing at the most a 23-membered ring (J H2′/H3′ < 2
Hz in CDCl3 and J H2′/H3′ ) 2.5 Hz in DMSO-d6). The
same coupling values were obtained for the 22-mem-
bered-ring taxoid 3d in both solvents. NMR conforma-
tional analysis (NOESY and rotating-frame Overhauser
enhancement spectroscopy (ROESY)) in polar and apo-
lar solvents showed an intense nuclear Overhauser

3626 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 17
Querolle et al.
Sch em e 3a
a
(i) H2, Pd/C, EtOAc, 4 h (4(H)a , 80%; 4(H)b, 78%; 4(H)c, 70%; 4(H)d , 74%). (ii) HF/pyridine, pyridine, CH3CN, 0 °C, 1 h, then room
temp, 6.5-8 h (7(H)a , 68%; 7(H)b, 71%; 7(H)c, 65%; 7(H)d , 64%).
Ta ble 2. Biological Activities of Macrocyclic Taxoids and of
Their Acyclic Analogues
microtubule
disassembly
cytotoxicity
against
ring
size
inhibitory activity
KB cell line
IC50 (µM)
b
compd
n
n′
IC50/IC50(paclitaxel) a
3a -Z
3a -E
3b-Z
3b-E
3c
2
2
3
3
4
4
4
2
3
4
4
2
3
4
4
2
3
4
4
2
2
3
3
3
4
4
2
3
3
4
2
3
3
4
2
3
3
4
18
18
20
20
21
22
22
18
20
21
22
inactive
inactive
inactive
inactive
>100c
7.3
>100
>100
>100
45
14
8
14
>100
55
13
23
1.6
0.01
0.65
0.5
10
1
3d -E
3d
7
3(H)a
3(H)b
3(H)c
3(H)d
7a
7b
7c
7d
7(H)a
7(H)b
7(H)c
7(H)d
inactive
inactive
30
45
5.1
4.6
2.4
1.1
2.9
F igu r e 3. Superimposition of conformer A (cyan) found for
3c and conformers B (yellow) and C (magenta) found for 3d .
Ta ble 3. Mole Fraction (MF) of the A, B, and C Conformations
for Compounds 3a -d
2.3
6.7
2.7
0.45
0.55
MF (%)
a
IC50 is the concentration that inhibits 50% of the rate of
microtubule disassembly. The ratio IC50/IC50(paclitaxel) gives the
compd
ring size (atoms)
A
B
C
3a
3b
3c
3d
18
20
21
22
93
0
0
27.5
37
7
12.5
0
b
activity with respect to paclitaxel. IC50(paclitaxel) ) 1 µM. IC50
87.5
72.5
0
measures the drug concentration required for the inhibition of 50%
cell proliferation after 72 h of incubation. IC50(docetaxel) ) 0.001
µM. c 3c showed a dose-response curve, but the 50% inhibition
of microtubule disassembly was only achieved at high concentra-
tion (>100 µM).
63
leading to similar results. Furthermore, though the
carbon chain of compounds 3a -d between N3′ and O2′
possesses a relatively high degree of freedom, it gener-
ates sufficient constraints to restrict the orientation of
the C2′ and C3′ substituents, which was the scope of
our study. For each compound, conformational searching
produced a great number of conformers and only the
conformers within 3.5 kcal/mol of the lowest energy
structure have been considered. These lower energy
conformers were clustered into three families (A, B, and
C) based on the orientation of the C-13 side chain
functional groups (mainly the C-2′ OH and C-3′ Ph)
(Figure 3).
The first two families A and B are very similar, the
main difference being the orientation of the phenyl
group. The C conformation has already been reported
in the literature8b but does not resemble any of the
proposed “polar”, “nonpolar”, or T-shaped forms, whereas
the B conformer is very similar to the “nonpolar”
conformation. The mole fraction (MF) of each conformer
was calculated according to the Boltzmann distribution
equation (Table 3).
effect (NOE) between 2′-H and 3′-H, characteristic of a
gauche interaction between these two protons, and other
NOE connectivity patterns were very similar to those
observed with docetaxel in apolar solvents. NMR spectra
of 3d obtained at low temperature were identical to
those obtained at room temperature. These NMR stud-
ies suggest that 3d adopts mainly the “nonpolar”
conformation, whatever the solvent polarity. Consider-
ing that 3d possesses the larger ring size of the series,
it is likely that the other macrocyclic derivatives 3a -c
are also sufficiently restricted to mimic a docetaxel-like
solid-state conformation, especially for the C2′ and C3′
substituents.
To better characterize the conformational behavior of
these macrocyclic taxoids, molecular dynamic modeling
studies were performed on compounds 3a -d . Though
it has been described for paclitaxel that such calcula-
tions could lead to different energy ranking of side chain
conformations according to the force field used,21 we
have always observed results in good agreement with
experimental data for docetaxel (ref 10 and unpublished
data). To minimize misinterpretation, two force fields
(MMFF94 and Tripos) have been used for this study,
This study shows that the major conformation of
compounds 3a -d changes from one derivative to an-

Docetaxel Analogues
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 17 3627
F igu r e 4. Superimposition the lowest energy conformer of compound 3d obtained by simulated annealing (yellow, torsion angles
T1 (O13-C1′-C2′-C3′) ) 63.9°; T2 (H2′-C2′-C3′-H3′) ) 78.2°) (a) with conformer B (blue, T1 ) 63.3°, T2 ) 85.2°); (b) with
docetaxel in “nonpolar form” (magenta, T1 ) 59.4°, T2 ) 89.2°) and T-shaped (cyan, T1 ) 55.7°, T2 ) 55.4°).
other depending on the ring size and consequently on
the ring rigidity. Since 3d is the only compound showing
significant tubulin activity, we can assume that the
bioactive conformation of these taxoids would be like
the B or C forms.
Exp er im en ta l Section
Gen er a l. 1H and 13C NMR spectra were recorded on a
Bruker AC 300 or AV 300 spectrometer. NOESY and ROESY
data were obtained from a Bruker AMX 400 spectrometer.
Chemical shifts are given as δ values and are referenced to
the residual solvent proton or carbon peak, i.e., chloroform
(C1HCl3 ) 7.27 ppm and 13CHCl3 ) 77.14 ppm). The spectra
were fully assigned using correlation spectroscopy (COSY),
heteronuclear multiple-quantum coherence (HMQC), and het-
eronuclear multiple-bond correlation (HMBC) on a Bruker AV
300. For compounds 3a -d , 3(H)a -d , 4a -d , 6a -d , 7a -d , and
7(H)a -d , all NMR spectra were very similar; the only
modifications were the added acyl chains. So, only the first
compound of each series will be fully described and the
characterization of the following ones can be found in the
Supporting Information. Mass spectra were obtained on an
AQA Navigator ThermoQuest. High-resolution mass spectra
were obtained from a Voyager-DE STR (PerSeptive Biosys-
tems) with gentisic acid as matrix and paclitaxel and docetaxel
as internal standards. Merck silica gel 60 (230-400 mesh) was
used for the flash chromatography purification of some com-
pounds. All chemicals were purchased from Fluka, Aldrich,
or Acros and were used without further purification unless
indicated otherwise. Solvents were purchased from SDS.
Toluene and THF were dried and distilled before use. Standard
workup means extraction with a suitable solvent (EtOAc
unless otherwise specified), washing the extract with H2O or
brine, drying over Na2SO4, and evaporation under reduced
pressure. Docetaxel 1b was a gift from Alain Commerc¸on
(Aventis-Pharma), and compound 5 has been prepared accord-
ing to the previously described procedure.14 Microtubular
proteins were purified from mammalian brain as previously
described.22 Cytotoxicity and microtubule disassembly inhibi-
tion were carried out according to established literature
protocols.16,17
Syn th esis of 2-Deben zoyl-2-a lk en oyl-2′-O-ter t-bu tyld i-
m eth ylsilyl-3′-d e-ter t-bu toxyca r bon yl-3′-a lk en oyl-7,10-d i-
tr ieth ylsilyld oceta xel (4a -d ). Gen er a l P r oced u r e for
com p ou n d s 4a ,b,d . A typical procedure is described for the
synthesis of 2-debenzoyl-2-pent-4-enoyl-2′-O-tert-butyldimeth-
ylsilyl-3′-de-tert-butoxycarbonyl-3′-pentenoyl-7,10-ditriethylsi-
lyldocetaxel (4a ). Pent-4-enoic acid (81 µL, 0.8 mmol, 5 equiv)
was added dropwise to a toluene (1 mL) solution of 5 (150 mg,
0.16 mmol) and DCC (163 mg, 0.8 mmol, 5 equiv) and 4-DMAP
(97 mg, 0.8 mmol, 5 equiv). The reaction mixture was stirred
at 60 °C for 3 h and then filtered through a Celite/silica gel
(1/1) column in EtOAc/heptane (1/1) and concentrated in vacuo.
After standard workup, the residue was purified by flash
chromatography (CH2Cl2/acetone, 98/2) to afford pure 4a (110
mg, 62%) as a white amorphous solid. 1H NMR (300 MHz,
CDCl3): δ -0.30 (s, 3H), -0.11 (s, 3H), 0.52-0.73 (m, 12H),
To have more information on the solution conforma-
tion of 3d , this compound was then subjected to con-
straint simulated annealing using NMR data. After
minimization, the lowest energy conformer was very
similar to the B conformation (Figure 4a) and situated
between the “nonpolar” and T-shaped docetaxel forms
(Figure 4b).
Su m m a r y a n d Con clu sion s
Nineteen new taxoids have been synthesized includ-
ing 11 18-membered- to 22-membered-ring macrocyclic
compounds. As expected, replacement of the sulfur atom
on the tether by a double bond greatly increased
microtubule interaction. Though the reduced macro-
cyclic taxoids 3(H)c and 3(H)d (21- and 22-membered
ring, respectively) exhibit weak activity on microtubule
disassembly, the unsaturated 22-membered-ring deriva-
tive 3d is the only compound showing a real inhibition.
The small difference between the IC50 of 3d and its
acyclic analogue 7d proves that the carbon tether
between C-2 and N-3′ does not hamper tubulin binding.
Examination of the conformation of the unsaturated
derivatives reveals that the macrocyclic taxoids 3a -c
adopt mainly a conformation that is not recognized by
tubulin. Molecular modeling studies have shown that
compound 3d can adopt two conformations B and C, the
former being very similar to the “nonpolar” conforma-
tion. In solution, this macrocyclic taxoid seems to adopt
a conformation situated between the “nonpolar” and
T-shaped forms. It is noted that these two conformations
are not so different, the main discrepancy being the
position of the C-3′ phenyl group with respect to the
median plane of the taxane core. All these results
suggest that the bioactive conformation of taxoids
interacting with microtubules is either the “nonpolar”
or the T-shaped form or another conformation between
these two forms. Other analogues bearing a more
restricted conformation must be designed to definitively
discriminate between these two forms.

3628 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 17
Querolle et al.
0.78 (s, 9H), 0.93-1.0 (m, 18H), 1.16 (s, 3H), 1.20 (s, 3H), 1.52
(s, 3H), 1.81 (s, 3H), 1.88 (m, 1H), 2.05-2.23 (m, 2H), 2.31-
2.69 (m, 12H), 3.7 (d, 3J H,H ) 7.0 Hz), 4.20 and 4.46 (qAB, 2J H,H
solution of taxoid 4a (52.7 mg, 0.047 mmol) in CH2Cl2 (4 mL)
was added dropwise a solution of bis(tricyclohexylphosphine)-
benzylideneruthenium(IV) dichloride (2.3 mg, 0.0028 mmol)
in CH2Cl2 (1.3 mL). The solution was refluxed for 4 h and
concentrated in vacuo. After standard workup, the residue was
purified by silica gel chromatography (EtOAc/heptane, 50/50)
to afford pure 6a -Z (30 mg, 58%) and 6a -E (16.3 mg, 31%) as
white amorphous solids.
3
3
) 8.0 Hz, 2H), 4.35 (dd, J H,H ) 6.5 Hz and J ′H,H ) 10.5 Hz),
3
4.49 (br s, 1H), 4.93 (d, J H,H ) 9.0 Hz, 1H, C-5H), 4.99-5.08
3
(m, 4H), 5.12 (s, 1H), 5.44 (d, J H,H ) 7.0 Hz, 1H), 5.47 (br d,
3
3J H,H ) 9.0 Hz, 1H), 5.75-5.92 (m, 2H), 6.15 (t, J H,H ) 9.0
3
Hz, 1 H), 6.44 (d, J H,H ) 9.0 Hz, 1H), 7.17-7.41 (m, 5H). 13C
6a -Z. 1H NMR (300 MHz, CDCl3): δ -0.33 (s, 3H), -0.11
(s, 3H), 0.54-0.78 (m, 12H), 0.71 (s, 9H), 0.93-1.07 (m, 18H),
1.13 (s, 3H), 1.25 (s, 3H), 1.64 (s, 3H), 1.89 (s, 3H), 1.89-2.14
(m, 5H), 2.26-2.59 (m, 4H), 2.50 (s, 3H), 2.59-2.74 (m, 3H),
NMR (75 MHz, CDCl3): δ -5.7, -5.3, 5.4, 6.1, 6.9, 7.0, 10.5,
13.8, 18.3, 20.7, 23.3, 25.6, 26.6, 28.5, 29.4, 34.1, 35.4, 35.6,
37.4, 43.4, 46.6, 55.3, 58.5, 72.0, 72.7, 74.8, 75.2, 75.5, 76.9,
78.7, 81.1, 84.3, 115.8, 116.0, 126.6, 127.9, 128.6, 134.3, 136.7,
137.0, 138.0, 138.6, 170.1, 171.7, 171.9, 174.0, 205.4. MS
(ESI+), m/z: 1132 [M + Na+].
3
3
3.76 (d, J H,H ) 7.8 Hz, 1H), 4.16 and 4.39 (qAB, J H,H ) 8.0
3
3
Hz, 2H), 4.44 (dd, J H,H ) 10.0 Hz and J H,H ) 6.3 Hz, 1H),
4.52 (br s, 1H), 4.86 (d, 3J H,H ) 8.0 Hz, 1H), 5.11 (s, 1H), 5.24-
5.42 (m, 2H), 5.44-5.53 (m, 2H), 6.21-6.36 (m, 2H), 7.16-
7.40 (m, 5H). 13C NMR (75 MHz, CDCl3): δ -6.0, -5.3, 5.5,
6.1, 7.1, 10.0, 13.7, 21.6, 22.6, 22.7, 23.2, 25.5, 26.7, 34.7, 35.5,
35.8, 37.1, 43.3, 47.0, 55.3, 58.1, 70.9, 72.5, 74.9, 75.4, 77.6,
80.7, 84.6, 126.5, 127.6, 128.5, 129.1, 130.1, 133.7, 137.5, 138.4,
170.9, 171.4, 173.9, 205.1. MS (ESI+), m/z: 1104 [M + Na+].
For 4b and 4d , see the Supporting Information.
Syn th esis of 2-Deben zoyl-2-h ex-5-en oyl-2′-O-ter t-bu -
tyldim eth ylsilyl-3′-de-ter t-bu toxycar bon yl-3′-h ept-6-en oyl-
7,10-d it r iet h ylsilyld ocet a xel (4c). Hept-6-enoic acid (111
µL, 0.82 mmol, 2 equiv) was added to a solution of 5 (390 mg,
0.41 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
(158.2 mg, 0.82 mmol, 2 equiv), and DMAP (152.3 mg, 1.25
mmol, 3 equiv) in CH2Cl2 (3.9 mL). The reaction mixture was
stirred at room temperature for 3 h, and the reaction mixture
was concentrated in vacuo. After standard workup, the residue
was purified by flash chromatography (CH2Cl2/MeOH, 95/5)
to afford pure 2-debenzoyl-2′-O-tert-butyldimethylsilyl-3′-de-
tert-butoxycarbonyl-3′-hept-6-enoyl-7,10-ditriethylsilyldoce-
taxel (335.5 mg, 77%) as a white amorphous solid. 1H NMR
(300 MHz, CDCl3): δ -0.28 (s, 3H), -0.07 (s, 3H), 0.48-0.70
(m, 12H), 0.77 (s, 9H), 0.93-1.01 (m, 18H), 1.07 (s, 3H), 1.20
(s, 3H), 1.41 (m, 2H), 1.53 (s, 3H), 1.60 (m, 2H), 1.78 (s, 3H),
1.93 (m, 1H), 1.99-2.16 (m, 4H), 2.26 (m, 2H), 2.36 (s, 3H),
2.49 (m, 1H), 3.49 (d, 3J H,H ) 6.0 Hz, 1H), 3.69 (d, 3J H,H ) 11.3
In the same manner, 6b-d were synthesized (see the
Supporting Information).
Gen er a l P r oced u r e for th e Red u ction of th e Dou ble
Bon d (s). A typical procedure is described for the synthesis of
macrocyclic taxoid 6(H)a . A solution of 6a -Z (32.5 mg, 0.03
mmol) in EtOAc (3.1 mL) was added to 10% palladium on
carbon (32.5 mg) under a hydrogen atmosphere, and the
reaction mixture was stirred for 3.5 h at room temperature.
The reaction mixture was then filtered through Celite and
concentrated in vacuo. The residue was purified by silica gel
chromatography (EtOAc/heptane, 40/60) to afford pure 6(H)a
(27.2 mg, 84%) as a white amorphous solid. 1H NMR (300 MHz,
CDCl3): δ -0.33 (s, 3H), -0.14 (s, 3H), 0.54-0.81 (m, 12H),
0.72 (s, 9H), 0.93-1.08 (m, 18H), 1.15 (s, 3H), 1.27 (s, 3H),
1.22-1.43 (m, 4H), 1.47-1.69 (m, 2H), 1.60 (s, 3H), 1.76 (m,
1H), 1.89 (m, 1H), 1.92 (s, 3H), 2.00 (m, 1H), 2.16 (m, 1H),
2.22-2.37 (m, 3H), 2.43-2.58 (m, 2H), 2.47 (s, 3H), 3.77 (d,
3J H,H ) 7.5 Hz, 1H), 4.13 (qAB, 3J H,H ) 8.0 Hz, 2H), 4.39-4.50
3
3
Hz, 1H), 3.95 (dd, J H,H ) 11.3 Hz and J H,H ) 6.0 Hz, 1H),
3
3
4.34 (dd, J H,H ) 10.4 Hz and J H,H ) 6.5 Hz, 1H), 4.49 (br s,
3
1H), 4.66 and 4.73 (qAB, J H,H ) 9.0 Hz, 2H), 4.91-5.07 (m,
3
4H), 5.56 (br d, J H,H ) 9.6 Hz, 1H), 5.66-5.92 (m, 1H), 6.18
3
3
(t, J H,H ) 8.8 Hz, 1 H), 6.40 (d, J H,H ) 9.6 Hz, 1H), 7.06-
7.37 (m, 5H). 13C NMR (75 MHz, CDCl3): δ -5.8, -5.2, 5.4,
6.1, 7.0, 7.1, 10.8, 13.7, 21.0, 23.4, 25.4, 25.6, 26.6, 28.4, 33.4,
35.7, 36.7, 37.6, 43.2, 47.1, 54.7, 58.3, 72.4, 72.9, 74.1, 75.1,
75.1, 78.1, 78.7, 82.6, 84.0, 115.0, 126.5, 128.0, 128.7, 133.6,
138.1, 138.4, 138.5, 170.1, 171.6, 173.0, 206.3. MS (ESI+),
m/z: 1078 [M + Na+].
3
3
(m, 2H), 4.51 (d, J H,H ) 2.5 Hz, 1H), 4.88 (d, J H,H ) 8.0 Hz,
3
1H), 5.12 (s, 1H), 5.32-5.40 (m, 2H), 6.27 (t, J H,H ) 8.5 Hz,
1H), 6.36 (d, 3J H,H ) 8.0 Hz, 1H), 7.18-7.40 (m, 5H). 13C NMR
(75 MHz, CDCl3): δ -6.0, -5.5, 5.5, 6.0, 7.0, 7.1, 10.9, 13.7,
18.2, 21.6, 23.5, 24.1, 25.3, 25.5, 26.8, 27.0, 28.1, 34.1, 36.1,
36.3, 37.2, 43.5, 47.0, 53,5, 58.1, 71.4, 72.5, 74.8, 75.0, 76.7,
80.3, 80.8, 84.5, 126.8, 127.7, 128.5, 133.7, 137.6, 138.3, 171.2,
172.7, 175.0, 205.1. MS (ESI+), m/z: 1106 [M + Na+].
Hex-5-enoic acid (140 µL, 1.17 mmol, 5 equiv) was added to
a toluene (2.25 mL) solution of 2-debenzoyl-2′-O-tert-butyl-
dimethylsilyl-3′-de-tert-butoxycarbonyl-3′-hept-6-enoyl-7,10-
ditriethylsilyldocetaxel (247 mg, 0.23 mmol), DCC (241 mg,
1,17 mmol, 5 equiv), and DMAP (142 mg, 1.16 mmol, 5 equiv).
The reaction mixture was stirred at 60 °C for 3.5 h and then
filtered through a Celite/silica gel (1/1) column in EtOAc/
heptane (1/1) and concentrated in vacuo. After standard
workup, the residue was purified by flash chromatography
(CH2Cl2/acetone, 98/2) to afford pure 4c (209 mg, 79%) as a
Compounds 6(H)b-d and 4(H)a -d were prepared accord-
ing to this method. For experimental details and spectral data,
see the Supporting Information.
Gen er a l P r oced u r e for Rem ova l of th e Silyl P r otect-
in g Gr ou p s. A typical procedure is described for the synthesis
of macrocyclic taxoid 3a -Z. To a cooled solution of 6a -Z (38.5
mg, 0.035 mmol) in pyridine/acetonitrile (0.08/1, 1 mL) was
added dropwise HF/pyridine (70%, 170 µL, 1.4 mmol, 40 equiv)
at 0 °C. The solution was stirred for 1 h at 0 °C and then
warmed to room temperature and stirred for 6.5 h more at
room temperature. The reaction was quenched by addition of
saturated aqueous sodium hydrogenocarbonate (25 mL), and
the aqueous layer was extracted three times with EtOAc (50
mL). After standard workup, the crude residue was purified
by silica gel chromatography (CH2Cl2/MeOH, 93/7) to afford
pure 3a -Z (14.7 mg, 57%) as a white amorphous solid. 1H NMR
(300 MHz, CDCl3): δ 1.05 (s, 3H), 1.29 (s, 3H), 1.71 (s, 3H),
1.86 (m, 1H), 1.94 (s, 3H), 2.11 (m, 2H), 2.17-2.88 (m, 8H),
1
white amorphous solid. H NMR (300 MHz, CDCl3): δ -0.29
(s, 3H), -0.11 (s, 3H), 0.50-0.71 (m, 12H), 0.79 (s, 9H), 0.88-
1.06 (m, 18H), 1.16 (s, 3H), 1.19 (s, 3H), 1.43 (m, 2H), 1.62 (s,
3H), 1.67 (m, 2H), 1.76 (m, 2H), 1.80 (s, 3H), 1.88 (m, 1H),
1.96-2.21 (m, 6H), 2.24-2.35 (m, 3H), 2.40 (m, 1H), 2.41 (s,
3
3H), 2.52 (m, 1H), 3.72 (d, J H,H ) 7.0 Hz, 1H), 4.19 and 4.45
3
3
3
(qAB, J H,H ) 8.0 Hz, 2H), 4.34 (dd, J H,H ) 10.5 Hz and J H,H
) 6.5 Hz, 1H), 4.49 (bs, 1H), 4.92-5.09 (m, 5H), 5.12 (s, 1H),
3
3
5.42 (d, J H,H ) 7.0 Hz, 5.45 (br d, J H,H ) 9.0 Hz, 1H), 5.70-
3
3
5.87 (m, 2H), 6.14 (t, J H,H ) 8.5 Hz, 1 H), 6.37 (d, J H,H ) 9.0
Hz, 1H), 7.16-7.41 (m, 5H). 13C NMR (75 MHz, CDCl3): δ
-5.7, -5.3, 5.4, 6.1, 7.0, 7.1, 10.5, 13.8, 18.3, 20.7, 23.3, 23.8,
25.2, 25.6, 26.6, 28.4, 33.0, 33.5, 34.8, 35.3, 36.5, 37.4, 43.4,
46.6, 55.3, 58.5, 72.1, 72.7, 74.6, 75.2, 75.5, 76.9, 78.7, 81.1,
84.3, 114.9, 115.6, 126.6, 127.9, 128.7, 134.3, 137.8, 138.0,
138.5, 138.7, 170.1, 171.7, 172.6, 174.7, 205.4. MS (ESI+),
m/z: 1174 [M + Na+].
3
2.51 (s, 3H), 3.82 (d, J H,H ) 7.7 Hz, 1H), 4.19 and 4.46 (qAB,
3
3
3J H,H ) 8.1 Hz, 2H), 4.29 (dd, J H,H ) 11.3 Hz and J H,H ) 6.5
3
Hz, 1H), 4.71 (br s, 1H), 4.92 (d, J H,H ) 9.5 Hz, 1H), 5.14 (s,
3
1H), 5.31 (m, 1H), 5.40 (d, J H,H ) 7.7 Hz, 1H), 5.48 (m, 1H),
3
3
5.58 (br d, J H,H ) 9.4 Hz, 1H), 6.23 (d, J H,H ) 9.4 Hz, 1H),
6.44 (t, J H,H ) 9.3 Hz, 1H), 7.27-7.44 (m, 5H). 13C NMR (75
3
Gen er a l P r oced u r e for th e Syn th esis of Ma cr ocyclic
Ta xoid s 6 by Rin g-Closu r e Meta th esis. A typical procedure
is described for the synthesis of macrocyclic taxoid 6a . To a
MHz, CDCl3): δ 10.6, 14.0, 22.0, 22.5, 22.6, 22.8, 26.7, 34.6,
35.5, 36.1, 36.6, 43.1, 46.4, 55.8, 57.4, 71.6, 72.0, 72.8, 74.9,
75.2, 76.5, 80.5, 80.6, 84.7, 126.6, 128.0, 128.8, 129.4, 129.8,

Docetaxel Analogues
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 17 3629
J agtap, P. J .; Samala, L. The Chemistry of Taxol and Related
Taxoids. In Progress in the Chemistry of Organic Natural
Products; Herz, W., Kirby, G. W., Moore, R. E., Steglich, W.,
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135.5, 138.2, 138.4, 171.3, 171.7, 172.9, 174.0, 211.6. HRMS
(MALDI-TOF), m/z: calcd for
found, 762.3087 (∆ ) 2.0 ppm).
In the same manner macrocylic taxoids 3b-d and 3(H)a -d
were synthesized as the acyclic taxoids 7a -d and 7(H)a -d .
For experimental details and spectral data, see the Supporting
Information.
C
39H49NO13‚Na+, 762.3102;
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Com p u ta tion a l P r oced u r es. All calculations were per-
formed on a SiliconGraphics Indigo2 Extreme workstation. All
modeling was done using Sybyl 6.8 software. The MMFF94
and Tripos force fields were used for minimization and partial
charge calculations, a dielectric constant of 1.0 being em-
ployed.22 In all cases, the A, B, C, and D rings of the taxoids
were considered as an aggregate. Compounds 3a -d -E 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 conformers was minimized and
compared with others with a rms of 0.3 Å. The obtained
structures were then ranked according to energy, and the
conformers within 3.5 kcal/mol of the lowest energy structure
were retained for further analysis. The structures were
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conformers was based on the backbone atoms of the taxane
core.
Compound 3d -E was subjected to 10 cycles of constraint
simulated annealing. The taxane skeleton was considered as
an aggregate, and the constraints were derived from NOE data
between H2′ and H3′ and between these protons and the
taxane core. The annealing protocol consisted of an initial
equilibration step during which the temperature was gradually
increased to 1000 K, followed by a production step of 1000 fs
and by gradual cooling to 100 K over a 1000 fs period. After
minimization using the previously described protocol, the 10
obtained conformer families were ranked by energy.
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Ack n ow led gm en t. The authors thank Dr. Alain
Commerc¸on (Aventis-Pharma) for the gift of docetaxel.
Christiane Gaspard is acknowledged for cytotoxicity
evaluations, J ean-Franc¸ois Gallard and Marie-The´re`se
Martin are acknowledged for performing NMR experi-
ments, and Vincent Gue´rineau is acknowledged for
high-resolution mass spectra. The authors acknowledge
the fellowship awarded by SEP-ANUIES (Me´xico) to
S.M. to carry out a part of her Ph.D. study at ICSN-
CNRS, France, and the authors acknowledge Dr. J .
Sandoval-Ram´ırez for his constant interest in this work.
Su p p or t in g In for m a t ion Ava ila b le: Experimental
details and characterization data for new compounds 4b, 4d ,
6b-d , 6(H)b-d , 4(H)a -d , 3b-d , 3(H)a -d , 7a -d , and
7(H)a -d . This material is available free of charge via the
Internet at http://pubs.acs.org.
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