Synthesis, biological evaluations, and tubulin binding poses of C-2α sulfur linked taxol analogues

Article abstract of DOI:10.1016/j.bmcl.2007.03.026

In combination with chemical modifications, bioassays, and computational simulation techniques, C-2 benzoylthio, and benzylthio taxoids were synthesized, biologically evaluated, and their binding conformations rationalized, in order to probe the interacti

Full text of DOI:10.1016/j.bmcl.2007.03.026

Bioorganic & Medicinal Chemistry Letters 17 (2007) 3191–3194
Synthesis, biological evaluations, and tubulin binding poses
of C-2a sulfur linked taxol analogues
Lei Wang,a,e Ana A. Alcaraz,c Ruth Matesanz,d Chun-Gang Yang,a Isabel Barasoain,d
d
e
c
a,b,
*
´
J. Fernando Dıaz, Ye-Zhi Li, James P. Snyder and Wei-Shuo Fang
aInstitute of Materia Medica, Chinese Academy of Medical Sciences, 1 Xian Nong Tan Street, Beijing 100050, PR China
bKey Laboratory of Bioactive Substances and Resources Utilization of Chinese Herbal Medicine (Ministry of Education),
Peking Union Medical College, Beijing 100050, PR China
cDepartment of Chemistry, Emory University, Atlanta, GA 30322, USA
dCentro de Investigaciones Biologicas, Consejo Superior de Investigaciones Cientificas, Ramiro de Maeztu 9, 28040 Madrid, Spain
eDepartment of Chemistry, Jilin University, Changchun, Jilin 130012, PR China
Received 29 November 2006; revised 6 March 2007; accepted 8 March 2007
Available online 14 March 2007
Abstract—In combination with chemical modifications, bioassays, and computational simulation techniques, C-2 benzoylthio, and
benzylthio taxoids were synthesized, biologically evaluated, and their binding conformations rationalized, in order to probe the
interaction of taxane molecule with b-tubulin.
Ó 2007 Elsevier Ltd. All rights reserved.
Chemical and biological evaluation of the antitumor
agent paclitaxel (PTX, TaxolÒ, 1) has been a main-
stream focus for natural product research since the
mid 1990s. The subject remains an active field in the at-
tempt to overcome problems of resistance1a,b and toxic-
ity.1c Both PTX2 and its analogue docetaxel 2 (DTX)3a,b
are currently used clinically for the treatment of ovarian,
breast, and non-small lung cancers, especially at the
recurrent or metastatic stages.
eleutherobin, discodermolide, and laulimalide.4 Com-
pounds that prevent the formation of microtubules but
likewise perturb the tubulin-microtubule equilibrium
(e.g., colchicine congeners, vinca alkaloids,5a hemiaster-
lins5b, and noscapine analogues5c) have also contributed
to the rather large family of tubulin-based antineoplastic
agents in recent years.
With respect to PTX, extensive structure-activity rela-
tionship (SAR) studies over the past two decades have
led to several generalizations. Perhaps the most crucial
one concerns three key side chains on the southern hemi-
sphere of the molecule: the C-13 N-benzoyl phenylisos-
erne, the C-2 benzoate, and the C-4 acetate moieties.
Both in terms of atomic constitution and molecular con-
formation, all are critical to taxane tubulin binding and
cytotoxicity.6a,b At the conformational level, although
there are upwards of a dozen different forms of PTX
conformers in solution,7 three extremes have at times
been proposed as that bound to b-tubulin: the polar,
nonpolar, and T-Taxol conformations.8 Only the latter
has led to the design and synthesis of tethered taxanes
that show potency superior to PTX in a combination
of microtubule stabilization and cytotoxicity assays.9
R2O
R1
Ph
O
NH O
OH
OH
O
HO
BzO
H
O
AcO
2: R1 = Boc, R2 = H
1: R1 = Bz, R2 = Ac
Many other antitubulin agents possessing a similar
mechanism of action are also found, including the most
widely studied classes such as epothilones, sarcodictyins,
Keywords: Paclitaxel; Taxol analogues; Binding conformation; SAR
studies.
*
SAR studies at the C-2 position have shown that both
the nature and stereochemistry of the 2-benzoyl group
Corresponding author. Tel.: + 86 10 63165237; fax: + 86 10
63017757; e-mail: wfang@imm.ac.cn
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.03.026

3192
L. Wang et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3191–3194
in PTX derivatives are determinants of activity.10,11
On the other hand, many paclitaxel analogues with
aroyl12 or alkyl13 ester groups at the C-2 position
have been reported with modest to highly potent
cytotoxicity.
H-13 chemical shifts in 10aa and 10ab appear at d4.9,
an upfield shift of 1.2 ppm by comparison with the
C-2 benzoates in 1 and 2. In addition, the quaternary
carbons at 198 ppm in the 13C NMR of 10aa and 10ab
were attributed to C-10 after careful assignment with
1D and 2D NMR techniques. These observations fall
outside expectations since taxoid H-13 and C-10 reso-
nances (C-13 esters or amides16) to date have been
observed at 5.5–6.2 ppm and 165–175 ppm, respectively.
This should be due to conformational distortion after
the introduction of C-2 thioester.
We also undertook the preparation of a series of C2 het-
eroatom-substituted taxoids to probe the PTX C-2
structural requirements. Based on the methodology re-
ported in our first communication,14a we were able to
develop a cytotoxicity SAR for the 2-amido ana-
logues,14b its C-10 modified analogues,14c and 2-deben-
zoyloxy-2a-phenylthio 10-acetyl docetaxel14d whose
inactivity can be attributed to its shorter chain length
at C-2 compared with benzoate.
C-2 sulfide baccatin 6b can also be obtained by
treatment of the mixture of 4a and 4b with sodium
benzylsulfide at room temperature in 83% yield. The
most significant changes in the 1H NMR for 6b are
found at H-2 and H-3. That the protons are shifted
upfield by 0.3 and 1.5 ppm, respectively, can be attrib-
uted to the loss of the C-2 carbonyl group. Following
reduction, coupling of side chains, and deprotection
reactions, the final products 10ba and10bb were afforded
(Scheme 1).
Although a photoaffinity labeling experiment indicated
that C-2 substituents are close to residues 217–231 of
b-tubulin15 and X-ray crystallographic data gave the
location of b-tubulin and its taxane ligand,18 the binding
conformation as well as the residues in b-tubulin and
functional groups in taxane involved in binding process
cannot be determined with precision.
Next, we determined the free energy changes of binding
for all four S-linked taxoids 10aa–10bb at 35 °C as ꢀ28
to ꢀ30 KJ/mol. This 28–35% reduction in DG for bind-
ing corresponds to 2000–10000 times loss of binding
affinity. The same compounds were subjected to MTT-
determined cytotoxicities and found to be much less ac-
tive in both sensitive and MDR tumor cell lines. In
agreement with the potency difference between PTX
(1) and DTX (2), the taxoids 10aa and 10ba with a
PTX side chain are 3–5 fold less active than 10ab and
10bb with the DTX side chain. But there appeared to
be no obvious difference in the activity between
2-PhCOS and 2-PhCH2S series (Table 1).
Here we report preparation of the first PTX analogues
bearing 2a-benzoylthio and benzylthio moieties de-
signed on the basis of isostere replacement principle,
and their initial SARs as well. Their conformations cor-
related with their binding affinity to b-tubulin are also
explored.
AcO
AcO
HO
O
O
O
OTES
O
OTES
O
OH
O
O
HO
R1
HO
BzO
R2
O
H
H
H
O
AcO
AcO
4b
AcO
4a: R1=OH, R2=MsO
5 : R1=OH, R2=N3
3
In order to explain the low activity of those taxanes, the
ligand-tubulin interaction was analyzed with molecular
dynamics based on T-conformation.
Following a known procedure,14a 10-DAB 3 was con-
verted into the key epoxide intermediate 4b. All reac-
tions proceeded in satisfactory yields from 88–97%.
Epoxide 4b exists as an inseparable mixture containing
2-mesylate 4a (ca. 2:1). Treatment of the 4a and 4b mix-
ture with NaN3 afforded the double SN2 product 2-azido
baccatin 5 in ca. 85% yield, implying that 4a was con-
verted to 4b during the course of the reaction.
The diminutive binding affinities and cytotoxicities of 10
can be attributed to the alteration of local geometry
around the sulfur atoms by comparison with the classic
C-2 benzoyl ester. In the latter case, the C-2 phenyl ring
of PTX encapsulated by tubulin in the electron crystal-
lographic structure17 is bounded on three sides in a
rather narrow pocket. The opposite faces of the ring
are within van der Waals contact of His227 and
Leu215, while the para-position is in equally short con-
tact with the protein backbone at Asp224. Extension of
the C-2 substituent either perpendicular to the faces of
the ring or further from the baccatin core can be pre-
dicted to create one or more steric clashes. To test the
idea that the latter distance increment could cause a ste-
ric problem for series 10, thioester 10aa was constructed
by an O to S modification of T-Taxol, optimized with
the MMFF/GBSA force field18, and docked into b-tubu-
lin by superposing the taxane on the location of the ori-
ginal PTX ligand. To relax the protein-ligand complex,
20 °K molecular dynamics was performed for the thio-
Following the same strategy, the mixture of 4a and 4b
was treated with sodium thiobenzoate to furnish 6a in
56% yield. The H shifts for H-2 in 6a are almost iden-
1
tical to the 2-benzoate counterpart, while H-3 is shifted
slightly downfield by 0.2 ppm. The latter reflects a mod-
ified environment around H-3 arising from changes in
the C-2 thioester geometry (bond lengths and angles)
by comparison with the corresponding ester. Subsequent
reduction of 6a–7a (80%) and incorporation of the C-13
side chains with enantiopureb-lactams 8a–b afforded
products 9aa and 9ab, which were then deprotected at
C-20 to the desired 2-PhCOS analogues 10aa (PTX side
chain) and 10ab (DTX side chain) in 42–44% yields over
two steps (Scheme 1). It is interesting to note that the
˚
PTX ligand and nearby side chains in a 10 A sphere
around the binding site. Once unfavorable steric

L. Wang et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3191–3194
3193
O
O
R'
AcO
N
AcO
O
O
(8a) R'=Ph
OTES
O
OTES
O
Ph
(8b) R'=t-BuO
OTES
HO
b
a
O
4a + 4b
HO
HO
H
R
c
H
R
AcO
AcO
7a R=PhCOS
7b R=PhCH2S
6a R=PhCOS
6b R=PhCH2S
O
O
AcO
O
AcO
R'
NH O
R'
O
NH O
OH
OTES
O
OH
d
Ph
O
Ph
O
OTES
HO
HO
H
R
O
H
R
AcO
AcO
9aa R=PhCOS, R'=Ph 9ab R=PhCOS, R'=t-BuO
9ba R=PhCH2S, R'=Ph 9bb R=PhCH2S, R'=t-BuO
10aa R=PhCOS, R'=Ph 10ab R=PhCOS, R'=t-BuO
10ba R=PhCH2S, R'=Ph 10bb R=PhCH2S, R'=t-BuO
Scheme 1. Synthesis of C2 S-linked taxoids 10aa–bb. Reagents and conditions: (a) RNa, DMF, r.t; (b) NaBH4, MeOH–THF, ꢀ15 °C; (c) LHMDS,
THF, ꢀ50 ! ꢀ40 °C; (d) HF–Py, 0 °C ! r.t.
Table 1. Cytotoxicity and binding data for taxoids 10
Compound
R
R0
IC50 (lM)a,b
DG (KJ/mol)c
A2780
2.4 1.0
A2780/AD
10ab
10aa
PhCOS
PhCOS
PhCH2S
PhCH2S
PhCOO
Boc
Bz
6.96 0.67 (2.9)
27 7 (2.3)
ꢀ30.1 0.6
ꢀ28.4 1.1
ꢀ29.9 0.7
ꢀ28.6 0.1
ꢀ42.1 0.2
11.7 0.7
3.5 1.4
9.9 1.0
10bb
10ba
Boc
Bz
8.3 2.7 (2.3)
9.4 1.7 (0.94)
0.92 0.13 (575)
PTX(1)
Bz
0.0016 0.0002
a IC50 values in lM were determined after two days exposure to drugs using the MTT cell proliferation assay. Data are means SE of four
independent experiments.
b The numbers in parentheses are the calculated relative resistance obtained by dividing the IC50 value of the resistant line by the IC50 value of the
parental line.
c Free energy changes derived from equilibrium binding constants of the ligands to microtubules at 35 °C (ꢀDG0app = RTlnK(binding)).
contacts were removed, the temperature was slowly
increased to 300 °K and the system allowed to reach a
stable state. At this point, the entire taxane molecule
˚
had risen out of the binding site by 1–2 A, and the
terminal C-2 phenyl ring had slipped from the tight
His/Leu/Asp sub-pocket to be repositioned in the first
water shell around the protein.
A similar set of calculations for thioether 10ba causes a
comparable extrusion of the molecule from the binding
pocket. In this case, however, the saturated C-2 side
chain deviates from planarity and causes a significant
rearrangement of the surrounding residues. Of particu-
lar note, the terminal phenyl ring is now perpendicular
to His227 and located in a pocket opened by the move-
ment of the Leu217 and Leu215 residues. Figure 1 de-
picts the final conformation of the C2–S–CH2–Ph
moiety and the displacement of the baccatin core by
comparison with PTX.
Figure 1. A superposition of PTX (blue) and thioether 10ba (light gold)
in the b-tubulin taxane binding site. For PTX, the C-2 phenyl is sandwi-
ched between His227 and Leu215 (red arrow). Compound 10ba’s C-2
In conclusion, C2 S-linked paclitaxel analogues are
found to be much less active in both tubulin binding
ability and cytotoxicity, although for different reasons
as revealed by molecular simulations. It is noteworthy
SCH2Ph group is predicted to adopt a staggered conformation that
relocates the terminal phenyl upward (red arrow) while causing His227
to shift to the left (See text and supplement for computational details).

3194
L. Wang et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3191–3194
that T-Taxol conformation has been proven9,19,20 to be
a good predictor for the tubulin binding/bioactivity of
taxanes, and here it gives another example for its
powerfulness.
7. Snyder, J. P.; Nevins, N.; Cicero, D. O.; Jansen, J. J. Am.
Chem. Soc. 2000, 122, 724.
8. (a) Vande Valde, D. G.; Georg, G. I.; Grunewald, G. L.;
Gunn, C. W.; Mitscher, L. A. J. Am. Chem. Soc. 1993,
115, 11650; (b) Dubois, J.; Guenard, D.; Gueritte-Voege-
lein, F.; Guerida, N.; Potier, P.; Gillet, B.; Beloeil, J.-C.
Tetrahedron 1993, 49, 6533; (c) Paloma, L. G.; Guy, R. K.;
Wrasidlo, W.; Nicolaou, K. C. Chem. Biol. 1994, 1, 107;
(d) Wang, M.; Cornett, B.; Nettles, J.; Liotta, D. C.;
Snyder, J. P. J. Org. Chem. 2000, 65, 1059; (e) Ojima, I.;
Chadravarty, S.; Inoue, T. Proc. Natl. Acad. Sci. U.S.A.
1999, 96, 4256.
9. 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. U.S.A. 2004, 101, 10006.
10. Chen, S.-H.; Wei, J.-M.; Farina, V. Tetrahedron Lett.
1993, 34, 3205.
Acknowledgments
The authors thank FANEDD (Grant No. 199949),
NSFC (Grant No. 20572135), and Fok Ying Tong Educa-
tion Foundation (Grant No. 91037) (WSF), Comunidad
Autonoma de Madrid (BIPPED-CM), Ministerio de
´
Educacion y Ciencia of Spain (Grant BFU-2004-0038)
(JFD) for financial supports. JPS and AAA are grateful
to Professor Dennis Liotta (Emory University)
for encouragement and support, JFD thanks Prof. J.M.
Andreu for useful discussions and support and Matadero
Madrid Norte S.A. and Carnicas Madrid-Canarias S.L.
for providing the brains for tubulin purification.
11. Chordia, M. D.; Kingston, D. G. I. J. Org. Chem. 1996,
61, 799.
12. (a) Chaudhary, A. G.; Gharpure, M. M.; Rimoldi, J. M.;
Chordia, M. D.; Gunatilaka, L. A. A.; Kingston, D. G. I.
J. Am. Chem. Soc. 1994, 116, 4097; (b) Nicolaou, K. C.;
Renaud, J.; Nantermet, P. G.; Couladouros, E. A.; Guy,
R. K.; Wrasidlo, W. J. Am. Chem. Soc. 1995, 117, 2409.
13. Ojima, I.; Kuduk, S. D.; Pera, P.; Veith, J. M.; Bernacki,
R. J. J. Med. Chem. 1997, 40, 279.
14. (a) Fang, W.-S.; Fang, Q.-C.; Liang, X.-T. Tetrahedron
Lett. 2001, 42, 1331; (b) Fang, W.-S.; Liu, Y.; Liu, H. Y.;
Xu, S.-F.; Wang, L.; Fang, Q.-C. Bioorg. Med. Chem.
Lett. 2002, 12, 1543; (c) Fang, W.-S.; Liu, H.-Y.; Fang, Q.-
C. Chin. Chem. Lett. 2005, 16, 38; (d) Fang, W.-S.; Liu, Y.;
Fang, Q.-C. Chin. Chem. Lett. 2002, 13, 708.
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmcl.2007.03.026.
References and notes
15. Rao, S.; Orr, G. A.; Chaudhary, A. G.; Kingston, D. G. I.;
Horwitz, S. B. J. Biol. Chem. 1995, 270, 20235.
16. Chen, S.-H.; Farina, V.; Vyas, D. M.; Doyle, T. W.; Long,
B. H.; Fairchild, C. J. Org. Chem. 1996, 61, 2065.
17. (a) Nogales, E.; Wolf, S. G.; Downing, K. H. Nature 1998,
391, 199; (b) Lowe, J.; Li, H.; Downing, K. H.; Nogales,
E. J. Mol. Biol. 2001, 313, 1045; (c) Snyder, J. P.; Nettles,
J. H.; Cornett, B.; Downing, K. H.; Nogales, E. Proc.
Natl. Acad. Sci. U.S.A. 2001, 98, 5312.
1. (a) Orr, G. A.; Verdier-Pinard, P.; McDaid, H.; Horwitz,
S. B. Oncogene 2003, 22, 7280; (b) Gottesman, M. M.
Annu. Rev. Med. 2002, 53, 615; (c) Weiss, R.; Donehower,
R. C.; Wiernik, P. H. J. Clin. Oncol. 1990, 8, 1263.
2. Mekhail, T.; Markman, M. Expert. Opin. Pharmacother.
2002, 3, 755.
3. (a) Ringel, I.; Horwitz, S. B. J. Natl. Cancer Inst. 1991, 83,
288; (b) Figgitt, D. P.; Wiseman, L. R. Drugs 2000, 59,
621.
18. See Ref. S9 in supplement materials.
4. Altmann, K. H. Curr. Opin. Chem. Biol. 2001, 5, 424.
5. (a) Lobert, S.; Boyd, C. A.; Correia, J. Biophys. J. 1997,
72, 416; (b) Arif, J. M.; AI-Hazzani, A. A.; Kunhi, M.; AI-
Khodairy, F. J. Biomed. Biotechnol. 2004, 2, 93; (c) Panda,
D.; Rathinasamy, K.; Santra, M. K.; Wilson, L. Proc.
Natl. Acad. Sci. U.S.A. 2005, 102, 9878.
19. Tang, S.; Yang, C.; Brodie, P.; Bane, S.; Ravindra, R.;
Sharma, S.; Jiang, Y.; Snyder, J. P.; Kingston, D. G. I.
Org. Lett. 2006, 8, 3983.
20. Ganesh, T.; Yang, C.; Norris, A.; Glass, T.; Bane, S.;
Ravindra, R.; Banerjee, A.; Metaferia, B.; Thomas, S. L.;
Giannakakou, P.; Alcaraz, A. A.; Lakdawala, A. S.;
Snyder, J. P.; Kingston, D. G. I. J. Med. Chem. 2007, 50,
713.
6. (a) Kingston, D. G. I. Chem. Commun. 2001, 10, 867; (b)
Gueritte, F. Curr. Pharm. Des. 2001, 7, 1229.