Novel C2-C3′ N-peptide linked macrocyclic taxoids. Part 1: Synthesis and biological activities of docetaxel analogues with a peptide side chain at C3′

Article abstract of DOI:10.1016/j.bmcl.2005.07.069

A series of novel docetaxel analogues possessing a peptide side chain at the C3′-N position was synthesized. These compounds were designed to mimic a region of the α-tubulin loop that is equivalent to the paclitaxel binding pocket in β-tubulin. Eight new

Full text of DOI:10.1016/j.bmcl.2005.07.069

Bioorganic & Medicinal Chemistry Letters 15 (2005) 4722–4726
Novel C2–C30 N-peptide linked macrocyclic taxoids. Part
1: Synthesis and biological activities of docetaxel analogues
with a peptide side chain at C30
`
Anne-Laure Larroque, Joelle Dubois, Sylviane Thoret, Genevieve Aubert,
*
¨
Daniel Guenard and Franc¸oise Gueritte
´
´
Institut de Chimie des Substanes Naturelles, CNRS, 1 avenue de la Terrasse, 91198 Gif sur Yvette Cedex, France
Received 23 May 2005; revised 20 July 2005; accepted 26 July 2005
Available online 13 September 2005
Abstract—A series of novel docetaxel analogues possessing a peptide side chain at the C30-N position was synthesized. These
compounds were designed to mimic a region of the a-tubulin loop that is equivalent to the paclitaxel binding pocket in b-tubulin.
Eight new peptidic taxoids were obtained and evaluated as inhibitors of microtubule disassembly, as well as for their cytotoxicity.
Ó 2005 Elsevier Ltd. All rights reserved.
TaxolÒ (paclitaxel)1 1a and its semi-synthetic analogue
TaxotereÒ (docetaxel)2 1b are two complex diterpenoids
useful as chemotherapeutic drugs in the treatment of
breast, ovarian, and nonsmall-cell lung cancers,3 and
are also active against prostate cancer.4 Their therapeu-
tic effect is due, at least in part, to their interaction with
microtubules. In vitro, paclitaxel and docetaxel promote
the assembly of tubulin into microtubules5 and inhibit
the disassembly of microtubules.6 Microtubules are
cytoskeletal elements essential in all eukaryotic cells,
with functions extending from cellular transport to cell
motility and mitosis. They are made of repeating ab-tu-
bulin heterodimers that bind head to tail into protofila-
ments. In 1998, the structure of tubulin was determined
by electron crystallography on zinc-induced sheets of
tubulin stabilized with paclitaxel 1a.7
The 3.7 A resolution allows localization of the paclitaxel
1a binding site on the b-subunit and also to visualize the
region of a-tubulin that is equivalent to the paclitaxel
binding pocket.8,9 Depending on their secondary struc-
ture, a- and b-tubulins could be superimposed with only
few differences. One of these is that the paclitaxel site on
b-tubulin is occupied in a-tubulin by a series of eight
amino acids, which form part of a loop connecting
strands B9 and B10 (Fig. 1).8,9
˚
We performed molecular modeling to study the super-
imposition of the a- and b-tubulin structures in this
region, and we observed conformational similarities
between the octapeptide Val362-Val-Pro-Gly-Gly-Asp-
Leu-Ala369 and the structure of paclitaxel when it is
bound to b-tubulin (Fig. 2). Thus, six amino acids,
Val363-Pro-Gly-Gly-Asp-Leu368, of the a-tubulin octa-
peptide showed a good insertion between positions 30
and 2 of paclitaxel 1a, while the other amino acids mim-
ic a part of the paclitaxel skeleton. Moreover, this super-
position could be related to that of the conformationally
restricted macrocyclic taxoids synthesized in our group
to mimic the ÔnonpolarÕ and ÔT-shapedÕ conformation
of paclitaxel and docetaxel.10–12
O
R1O
O
OH
R2
NH
O
O
Ph
OH
O
AcO
OBz
HO
1a R1= Ac, R2= Ph
1b R1= H, R2= OtBu
As shown earlier, bioactive taxoids bind to the b-tubulin
subunit in a specific binding conformation, and several
studies (X-rays, NMR, and molecular modeling)
have been performed to elucidate the nature of the
bioactive conformation as ÔnonpolarÕ,13,14 ÔpolarÕ15 or
Keywords: Microtubules; Docetaxel; Paclitaxel; Taxoids.
*
Corresponding author. Tel.: +33 1 69 82 45 80; fax: +33 1 69 07 72
47; e-mail: gueritte@icsn.cnrs-gif.fr
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.07.069

A.-L. Larroque et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4722–4726
4723
Figure 1. The paclitaxel binding site in b-tubulin (A) and the B9–B10 loop in a-tubulin (B).9
Figure 2. Overlapping of paclitaxel (green) and octapeptide A362–A369 (magenta) after manual superimposition of a- and b-tubulin secondary
structures, followed by an optimization of the position of the octapeptide (Sybyl software).
ÔT-shapedÕ9,16 forms. Our previous studies were based on
the synthesis of macrocyclic docetaxel analogues with
bridges linking the C30 nitrogen to the C2 oxygen using
variable chain sizes to describe the ÔactiveÕ conforma-
tion.10–12 The results obtained from this work seem to
confirm that the ÔT-shapedÕ form is preferred for a good
interaction with the b-tubulin binding pocket.12 With
the goal of synthesizing novel macrocyclic docetaxel
analogues, we designed new derivatives by insertion of
specific amino acids of the a-tubulin loop between the
30-NH and the 2-C positions of docetaxel 1b (Fig. 3).
These could constitute another approach to lock the
HO
O
OH
O
HO
Ph
O
O
AcO
HO
(AA)n
NH
O
O
O
Figure 3. Macrocyclic peptidic taxoids (AA, amino acids of the
a-tubulin region equivalent to the paclitaxel binding site).

4724
A.-L. Larroque et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4722–4726
docetaxel conformation and improve the taxoid b-tubu-
lin interaction in the same way that the octapeptide
interacts within a-tubulin.
Optimization of the conditions for the addition of valine
benzyl ester gave compound 3a in 54% yield. Hydrogen-
olysis of 3a led to the deprotected amino acid taxoid 5a
in 93% yield. The second amino acid (proline) was then
added by creation of a peptide bond with the free acid
function of valine using 1-ethyl-3-(30-dimethylamino-
propyl)carbodiimide hydrochloride22 (EDCI) as the
coupling reagent and 1-hydroxybenzotriazole23 (HOBt)
to limit undesirable side reactions.24 Moreover, we used
a low dielectric constant solvent, such as CH2Cl2, to
minimize possible racemization via formation of an oxa-
zolone intermediate.25 Thus, proline benzyl ester was
added to valine to give compound 3b (91%). To increase
the peptide chain length further, the benzyl ester was re-
moved by hydrogenolysis, and the other amino acids
were introduced using the same pathway (Scheme 2).
All these compounds were deprotected at C7, C10,
and C20 by HF/pyridine with good yields, affording
compounds 6a–6d, while the terminal amino acids were
all deprotected by hydrogenolysis, leading to com-
pounds 2a–2d in 57–98% yields. In the same way, we
also prepared analogues 8 and 11 bearing the tripeptides
Val-Phe-Met-OMe and Gly-Ser(OBn)-Tyr(OBn)-OBn,
respectively (Scheme 3). Compound 8 was obtained by
addition of the dipeptide Phe-Met-OMe26 to 5a. Prepa-
ration of compound 11 was initiated from the glycine
derivative 9 resulting from the attachment of glycine
to 4 by a urea function, as described earlier for the
formation of 3a (Scheme 2). Coupling of 9 with the
dipeptide Ser(OBn)-Tyr(OBn)-OBn led to 11 after
deprotection of the hydroxyl groups (Scheme 3). Final-
ly, the peptide part of taxoid 11 was deprotected by
hydrogenolysis to give 12.
In this paper, we describe, as a preliminary result, the
synthesis of novel taxoids 2a–2d bearing at the 30N po-
sition from one to four amino acids (Val-Pro-Gly-Gly)
which are part of the a-tubulin loop that is equivalent
to the b-tubulin paclitaxel binding site. The variable
length of the peptide allows us to correlate step by step
the biological activities to the number of added amino
acids and to verify if the addition of amino acids pos-
sessing a free or a protected amino group at the C30
position is not deleterious to the antitubulin activity.
To confirm the specificity of the interaction between
tubulin and the peptide analogues, we also synthesized
three other compounds in which the amino acids at C30
(Val-Phe-Met-OMe, Gly-Ser(OBn)-Tyr(OBn)OBn, and
Gly-Ser-TyrOH) are different from those constituting
the octapeptide residue. To our knowledge, no taxoids
bearing a peptide side chain directly linked to the C30
position have been previously described. However,
linking of amino acids to taxoids at positions 20, 7,
and 10 has been reported with the goal of promoting
water solubility or of obtaining prodrugs of antitumor
taxoids.17–21
Our retrosynthetic scheme of C30 taxoid analogues
linked to amino acids 2a–2d is shown in Scheme 1. Com-
pounds 2a–2d could be obtained from compounds 3a–
3d, respectively, after deprotection of the C7, C10, and
C20 hydroxyl groups and hydrogenolysis of the benzyl
ester protecting the acid group of the amino acids, which
have been added step by step to compound 4. The latter
can be readily synthesized from docetaxel 1b through
double protection at C7 and C10, and removal of the
Boc group.10
The new compounds 2a–2d, 6a–6d, and 8, 9, and 12 as
well as docetaxel 1b, were evaluated for their inhibition
of cold-induced microtubule disassembly27 and for
their cytotoxicity against the KB cancer cell line28
(Table 1). In the series 2a–2d and 6a–6d, most of the
compounds exhibited very good interaction with
microtubules, showing IC50 values similar to that of
docetaxel. Except for analogue 2b, which is three to
four times less active than docetaxel, there were no
crucial differences in the antitubulin activity of
To mimic the desired a-tubulin loop, it was necessary to
have the N-terminal part of the peptide linked to the 30-
position and the C-terminal part to the 2-position. Thus,
the connection of 4 to the first amino acid (valine) was
realized by introduction of a urea function using car-
bonyldiimidazole (CDI) (Scheme 2).
O
O
TESO
HO
O
HO
O
OTES
O
OH
R
NH
O
R
NH
O
O
Ph
O
Ph
OTBDMS
OH
O
AcO
OBz
AcO
OBz
HO
2b R= Val-Pro-OH
2a R= Val-OH
3a R= Val-OBn
3b R= Val-Pro-OBn
3c R= Val-Pro-Gly-OBn
3d R= Val-Pro-Gly-GlyOBn
2c R= Val-Pro-Gly-OH
2d R= Val-Pro-Gly-GlyOH
TESO
O
OTES
O
NH2
O
1b
O
Ph
OTBDMS
AcO
OBz
HO
4
Scheme 1. Retrosynthetic pathway of docetaxel analogues containing amino acids at 30-NH.

A.-L. Larroque et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4722–4726
CO2Bn
4725
HN
O
TESO
O
TESO
O
OTES
OTES
O
O
NH
a
O
HO2C
b
N
H
NH
O
O
Ph
O
Ph
93%
O
OTBDMS
OTBDMS
AcO
OBz
AcO
OBz
HO
HO
5a
3a
d
c (91%)
54%
4
O
O
HO
O
TESO
OH
O
OTES
O
R
NH
O
N
H
NH
O
d
R
O
Ph
O
Ph
OH
O
OTBDMS
O
AcO
OBz
HO
AcO
OBz
HO
6a R= Val-OBn ((75%)
6b R= Val-Pro-OBn (76%)
6c R= Val-Pro-Gly-OBn (60%)
6d R= Val-Pro-Gly-GlyOBn(80%)
3b R= Pro-OBn
b (93%),c (79%)
3c R= Pro-Gly-OBn
b (97%),c (80%)
3d R= Pro-Gly-Gly-OBn
b
2a R= Val-OH (42%)
2b R= Val-Pro-OH (83%)
2c R= Val-Pro-Gly-OH (57%)
2d R= Val-Pro-Gly-GlyOH (98%)
Scheme 2. Reagents and conditions: (a) Val-OBn (10 equiv), CDI (20 equiv), NMM (33 equiv), CH2Cl2/CH3CN (1/1), 60 °C; (b) Pd/C 10%, H2,
MeOH/AcOH (9/1), rt; (c) AA-OBn (5 equiv), EDCI (5 equiv), HOBt (5 equiv), CH2Cl2, rt; (d) HF/pyridine 70% (45 equiv), CH3CN, 0 °C and rt.
O
O
HO
O
TESO
HO
O
OH
OTES
O
R
NH
O
R
NH
O
b
O
Ph
O
Ph
OH
OTBDMS
O
AcO
OBz
AcO
OBz
HO
a
8 R= Val-Phe-Met-OMe
7 R= Val-Phe-Met-OMe
5a R= Val-OH
(33%)
(56%)
11 R= Gly-Ser(OBn)-Tyr(OBn)-OBn
c
9 R= Gly-OH
10 R= Gly-Ser(OBn)-Tyr(OBn)-OBn
(71%)
d
51%
12 R= Gly-Ser-Tyr-OH
(28%)
Scheme 3. Reagents and conditions: (a) Phe-Met-OMe (5 equiv), ECDI (5 equiv), HOBt (5 equiv), CH2Cl2, rt; (b) HF/pyridine 70% (45 equiv),
CH3CN, 0 °C and rt; (c) Ser(OBn)-Tyr(OBn)-OBn (5 equiv), ECDI (5 equiv), HOBt (5 equiv), CH2Cl2, rt; (d) Pd/C 10%, H2, MeOH, rt.
Table 1. Microtubule disassembly inhibition and cytotoxicity of
docetaxel 1b and compounds 2a–2d, 6a–6d, and 8, 11, and 12
protected and unprotected analogues, showing that
hydrophobicity of the side chain is not necessary for
Compounds
Microtubules disassembly Cytotoxicity against
IC50 (lM)a
the interaction with tubulin. We also noted a slight
systematic increase in antitubulin activity, following
the step-by-step addition of amino acids. With regard
to cytotoxicity, compounds 2a–2d bearing a deprotec-
ted amino acid side chain are less cytotoxic than their
corresponding protected analogues 6a–6d. This is
probably due to the presence of the free acidic func-
tion in compounds 2a–2d that hampers cell penetra-
tion. Among these compounds, 6a has a cytotoxicity
comparable to that of docetaxel, and it is noteworthy
that the cytotoxic effects of the compounds decrease
with the length of the amino acid side chain. Com-
pounds 8 and 12 also interact with microtubules but
to a lesser extent than the previous series, and, curi-
ously, compound 11, which was found inactive on
microtubules, showed significant cytotoxicity toward
KB cells.
KB IC50 (lM)b
Docetaxel 1b 0.5
0.001
0.67
0.004
4.50
0.05
>10
2a
6a
2b
6b
2c
6c
2d
6d
8
0.8
0.8
1.7
0.5
0.6
0.4
0.10
>10
0.5
0.3
4.00
1.10
0.09
1.05
2.6
Inactive
1.0
11
12
a IC50 is the concentration that inhibits 50% of the rate of microtubule
disassembly.
b IC50 measures the drug concentration required for the inhibition of
50% cell proliferation after 72 h incubation.

4726
A.-L. Larroque et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4722–4726
10. Querolle, O.; Dubois, J.; Thoret, S.; Dupont, C.; Gueritte,
´
In summary, we have synthesized 11 new taxoids with a
peptide side chain at C30, and we observed that the sub-
stitution of the Boc group of docetaxel by these amino
acids does not induce any loss of activity on microtubule
disassembly but leads, except for compound 6a, to a de-
crease in cytotoxicity, especially when the groups are
hydrophilic. These preliminary results have encouraged
us to continue our work on the synthesis of macrocyclic
peptide analogues of docetaxel mimicking the amino
acid region of a-tubulin that is similar to that of the taxol
binding site. This study will be presented as the second
part of this research program in a future publication.
´
F.; Guenard, D. Eur. J. Org. Chem. 2003, 542.
11. Querolle, O.; Dubois, J.; Thoret, S.; Roussi, F.; Gueritte,
´
´
F.; Guenard, D. J. Med. Chem. 2004, 47, 5937.
12. Querolle, O.; Dubois, J.; Thoret, S.; Roussy, F.; Montiel-
´ ´
Smith, S.; Gueritte, F.; Guenard, D. J. Med. Chem. 2003,
46, 3623.
´
´
13. Dubois, J.; Guenard, D.; Gueritte-Voeglein, F.; Guedira,
N.; Potier, P.; Gillet, B.; Beloeil, J.-C. Tetrahedron 1993,
49, 6533.
14. Williams, H. J.; Scott, A. I.; Dieden, R. A.; Swindell, C. S.;
Chirlian, L. E.; Francl, M. M.; Heerding, J. M.; Krauss,
N. E. Tetrahedron 1993, 49, 6545.
15. Vander Velde, D. G.; Georg, G. I.; Grunewald, G. L.;
Gunn, C. W.; Mitscher, L. A. A. J. Am. Chem. Soc. 1993,
115, 11650.
Acknowledgments
16. Ganesh, T.; Guza, R. C.; Bane, S.; Ravindra, R.; Shanker,
N.; Lakdawala, A. S.; Snyder, J. P.; Kingston, D. G. I.
Proc. Natl. Acad. Sci. USA 2004, 101, 10006.
17. Deutsch, H. M.; Glinski, J. A.; Hernandez, M.; Haugwitz,
R. D.; Narayanan, V. L.; Suffness, M.; Zalkow, L. H.
J. Med. Chem. 1989, 32, 788.
The ICSN gratefully acknowledges the NMR depart-
ment for performing NMR spectra and the mass spec-
trometry department for high-resolution mass spectra.
The authors are also grateful for financial support from
CNRS.
´
´
18. Gueritte-Voegelein, F.; Guenard, D.; Lavelle, F.; Le Goff,
M.-T.; Mangatal, L.; Potier, P. J. Med. Chem. 1991, 34,
992.
References and notes
19. Mathew, A. E.; Mejillano, M. R.; Nath, J. P.; Himes, R.
H.; Stella, V. J. J. Med. Chem. 1992, 35, 145.
20. de Groot, F. M. H.; van Berkom, L. W. A.; Scheeren, H.
W. J. Med. Chem. 2000, 43, 3093.
1. Wani, M. C.; Taylor, H. L.; Wall, M. E.; Coggon, P.;
McPhail, A. T. J. Am. Chem. Soc. 1971, 93, 2325.
´
´
2. Guenard, D.; Gueritte-Voegelein, F.; Potier, P. Acc.
Chem. Res. 1993, 26, 160.
21. Yamaguchi, T.; Harada, N.; Ozaki, K.; Arakawa, H.;
Oda, K.; Nakanishi, N.; Tsujihara, K.; Hashiyama, T.
Bioorg. Med. Chem. Lett. 1999, 9, 1639.
22. Sheedan, J. C.; Cruickshank, P. A.; Boshart, G. L. J. Org.
Chem. 1961, 26, 2525.
23. Ko¨nig, W.; Geiger, R. Chem. Ber. 1970, 103, 2024.
24. Balcom, B. P.; Perterson, N. O. J. Org. Chem. 1989, 54,
1922.
´
´
3. Guenard, D.; Gueritte-Voegelein, F.; Lavelle, F. Curr.
Pharm. Des. 1995, 1, 95.
4. de Vit, R. Eur. J. Cancer 2005, 41, 502.
5. Schiff, P. B.; Fant, J.; Horwitz, S. B. Nature 1979, 277,
665.
´
6. Senilh, V.; Blechert, S.; Colin, M.; Guenard, D.; Picot, F.;
Potier, P. J. Nat. Prod. 1984, 47, 131.
7. Nogales, E.; Wolf, S. G.; Downing, K. H. Nature 1998,
391(6663), 199.
25. Mader, O.; Albericio, F. Chim. Oggi 2003, 6.
26. Kessler, H.; Ho¨lzemann, G. Liebigs Ann. Chem. 1981,
2028.
´
´
8. Lifeng, H.; George, A. O.; Horwitz, S. B. DDT 2001,
6(22), 1153.
27. Lataste, H.; Senihl, M.; Wright, M.; Guenard, D.; Potier,
P. Proc. Natl. Acad. Sci. USA 1984, 81, 4090.
ˆ
9. Snyder, J. P.; Nettles, J. H.; Cornett, B.; Dowing, K. H.;
Nogales, E. Proc. Natl. Acad. Sci. USA 2001, 98, 5312.
28. Da Silva, A. D.; Machado, A. S.; Tempete, C.; Robert-
Gero, M. Eur. J. Med. Chem. 1994, 29, 149.