Synthesis and biological activity of macrocyclic taxane analogues

Article abstract of DOI:10.1016/j.bmcl.2004.02.086

A series of paclitaxel analogues possessing a macrocyclic structure between the 7 and 10 positions has been prepared. These compounds possess in vitro activity against a paclitaxel resistant cell line and have in vivo activity comparable to paclitaxel.

Full text of DOI:10.1016/j.bmcl.2004.02.086

Bioorganic & Medicinal Chemistry Letters 14 (2004) 2555–2558
Synthesis and biological activity of macrocyclic taxane analogues
James G. Tarrant,a,* Donald Cook,a Craig Fairchild,b John F. Kadow,a Byron H. Long,b
William C. Roseb and Dolatrai Vyasa
aDiscovery Chemistry, Bristol-Myers Squibb Pharmaceutical Research Institute, PO Box 5100, Wallingford, CT 06492-7660, USA
bOncology Drug Discovery, PO Box 4000, Princeton, NJ 08543-4000, USA
Received 8 April 2003; revised 24 February 2004; accepted 26 February 2004
Abstract—A series of paclitaxel analogues possessing a macrocyclic structure between the 7 and 10 positions has been prepared.
These compounds possess in vitro activity against a paclitaxel resistant cell line and have in vivo activity comparable to paclitaxel.
ꢀ 2004 Elsevier Ltd. All rights reserved.
First approved for clinical use in 1992, the taxane anti-
cancer drug TAXOLꢁ (paclitaxel 1, Fig. 1) has become
one of the most exciting advances in cancer chemo-
therapy ever discovered. Because TAXOLꢁ has dem-
onstrated activity against ovarian, breast, lung, Kaposi’s
sarcoma, bladder, prostate, esophageal, head and neck,
cervical, and endometrial cancers, considerable efforts
are still being expended on clinical trials to fully eluci-
date the usefulness of this drug.1 Recent efforts toward
the discovery of novel taxanes have introduced clinical
candidates endowed with in vivo efficacy superior to
paclitaxel in both paclitaxel resistant and sensitive
human tumor xenograft models.2
ment of novel taxane analogues with greater efficacy,
reduced toxicity, and an expanded spectrum of activity
may result in significant improvements in patient
response. TAXOTEREꢁ (docetaxel 2, Fig. 1), a semi-
synthetic taxane has also been approved for clinical use.
In the context of discovering analogues that overcome
paclitaxel resistance mediated through the P-glycopro-
tein efflux mechanism, modifications at the northern
hemisphere of the molecule, specifically, the 7 and 10
positions, have been implicated and pursued. SAR
studies performed by a number of different groups have
demonstrated that these positions are quite tolerant to
chemical manipulation; probably due to the fact that
these residues do not interact directly with tubulin.4 C7
paclitaxel ethers have shown in vitro potency against
both paclitaxel sensitive and resistant cell lines.5 Ana-
logues modified at the C10 position exhibit improved in
vitro potency against resistant cell lines.6 It has been
hypothesized that modifications of this region of
the molecule may affect paclitaxel’s susceptibility to
P-glycoprotein mediated efflux. With this information in
hand we contemplated the synthesis of novel analogues
7 and 13 derived by linking the C7 and C10 positions of
the taxane skeleton in a macrocyclic array.7 Such ana-
logues with the 7 and 10 positions simultaneously
blocked might be endowed with in vivo activity against
tumors expressing the MDR phenotype. P-glycoprotein
mediated efflux is thought to be responsible for devel-
opment of the MDR (multidrug resistant) phenotype.
Alteration of the binding motif between paclitaxel ana-
logues and P-glycoprotein could result in activity against
tumors that are unresponsive to treatment or that have
become resistant as a result of taxane treatment. It has
been proposed that the ability of C10 taxane analogues
Significant progress has also been made toward the
discovery of orally active taxanes.3 As the clinical
potential of TAXOLꢁ continues to grow, the develop-
O
R2O
R1
NH
O
O
OH
O
10
O
7
OH
HO
H
BzO
AcO
Figure 1. Paclitaxel 1 (R1 ¼ Ph, R2 ¼ Ac) docetaxel 2 (R1 ¼ O-tert-Bu,
R2 ¼ H).
Keywords: Taxanes; Paclitaxel; Macrocycles; P-glycoprotein.
* Corresponding author at present address: Neurogen Corporation, 35
NE Industrial Rd., Branford, CT 06405, USA. Tel.: +1-203-4888201-
x305; fax: +1-203-4837027; e-mail: jim_tarrant@nrgn.com
0960-894X/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.02.086

2556
J. G. Tarrant et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2555–2558
to overcome MDR in vitro is the result of reduced
binding affinity for P-glycoprotein.8 The proximity of
C7 to the P-glycoprotein binding site of paclitaxel has
been demonstrated by photoaffinity studies.9
Variation of the C13 side chain was accomplished uti-
lizing the standard sequence of reactions. Reductive
de-esterification of 7,10-bismethyl-thiomethoxypaclitaxel
(4) with tetrabutylammonium borohydride,14 provided
the analogous baccatin compound in good yield. The
13-hydroxy group was protected as its trimethylsilyl
derivative to give compound 10, which was treated with
ethylene glycol as outlined above. In this case, the acy-
clic by-product was not isolated. Desilylation of 11 was
effected by treatment with TBAF and the resulting
baccatin analogue was acylated with b-lactam 12 using a
variation of the Ojima–Holton methodology.15 Desilyl-
ation of the intermediate with TBAF afforded macro-
cyclic analogue 13 (Scheme 2).
Previously, researchers in our group had developed the
use of MTM ethers to introduce the MOM and other
ethers into the 7-position of paclitaxel.10 We believed
this technology would prove useful for the introduction
of macrocyclic elements into paclitaxel.
Our synthesis began with the preparation of 20-tert-
butyldimethylsilyl-paclitaxel, which was the converted
to 10-deacetylpaclitaxel (3) upon treatment with
hydrazine hydrate in ethanol.11 This compound was
then converted to the 7,10-bismethyl-thiomethoxy ana-
logue 4. Exposure of this compound to 10 equiv of a diol,
for example, ethylene glycol, in the presence of N-iodo-
The macrocyclic taxane analogues were evaluated for
inhibition of tubulin polymerization,16 and for in vitro
cytotoxicity against the paclitaxel sensitive human colon
carcinoma cell line HCT-116 and against the human
breast carcinoma cell line A2780. These analogues were
also evaluated against the paclitaxel resistant cell lines
HCT116/mdr17 and A2780/tub.18 Table 1 summarizes
this data.
ꢀ
succinimide, silver trifluoromethanesulfonate and 4 A
molecular sieves12 provided a 1:1 mixture of the 7,
10-macrocyclic analogue 5 along with the 7-substituted
10-deacetyl analogue 6, which were readily separable by
chromatography. The structures were confirmed by
spectroscopic methods. The superior reactivity of the 7-
position when compared to the 10-position parallels the
reactivity observed for acetylation and silylation.13
Desilylation was effected by treatment with TBAF at
)10 ꢂC to provide compound 7. Cyclization of com-
pound 6 could be accomplished by treatment with a
slight excess of triphosgene to afford the cyclic carbon-
ate 8 (Scheme 1). Table 1 lists the novel macrocyclic
taxanes prepared using this series of reactions.
The macrocyclic analogues were significantly more po-
tent against the paclitaxel resistant cell line, HCT116/
mdr. This cell line is known to express elevated levels of
P-glycoprotein and is about 100-fold resistant to paclit-
axel. This suggests that these analogues are less sus-
ceptible to efflux by P-glycoprotein. The precise
mechanism of this reduced susceptibility remains to be
elucidated. Macrocyclic analogues 8 and 13 were slightly
O
O
MTMO
HO
O
Ph
NH
O
O
Ph
NH
O
OMTM
O
OH
O
O
O
c
a,b
d
1
OTBDMS
OTBDMS
HO
HO
H
H
BzO
AcO
3
BzO
AcO
4
HO
O
O
O
O
O
O
O
OH
O
O
O
Ph
NH
O
Ph
NH
O
O
O
+
OTBDMS
OTBDMS
HO
HO
H
H
BzO
BzO
AcO
O
O
5
6
AcO
f, e
e
O
O
O
O
O
O
O
O
O
O
O
O
O
Ph
NH
O
Ph
NH
O
O
O
OH
OH
HO
HO
H
H
BzO
7
BzO
AcO
8
O
O
AcO
Scheme 1. Reagents and conditions: (a) TBSCl, imidazole, DMF (94%); (b) hydrazine hydrate, EtOH (95%); (c) dimethyl sulfide, benzoyl peroxide,
ꢀ
CH3CN (80%); (d) diol, NIS, AgOTf, 4 A sieves, THF (25–55%); (e) TBAF, THF, )10 ꢂC (76–95%); (f) triphosgene, pyridine, DCM, 0 ꢂC–rt (13%).

J. G. Tarrant et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2555–2558
2557
Table 1. In vitro activity of macrocyclic taxane analogues20
O
R2
O
R1
NH
O
O
O
O
OH
HO
H
BzO
O
AcO
Compound
R1
R2
Tubulina
HCT116
IC50 (nM)b
HCT/mdr
R/S ratioc
A2780
IC50 (nM)d
A2780/tub
R/S ratioe
1
Ph
Ph
10-Ac, 7-H
1.0
2.2f
3.0
126f
10
2.4f
2.6
31f
15
32
6
7
CH2OCH2CH2OCH2
(CO)OCH2CH2OCH2
CH2OCH2CH2OCH2
1.49
0.73
0.67
8
Ph
t-BuO
Ph
1.9
0.8
31
12
2.1
13
14
18.5
18.0
CH2OCH2C(CH3)2CH2OCH2 2.87
17.2
11
23
a Concentration of test compound to give a change of 0.01 OD/h expressed as a ratio to the concentration of paclitaxel.
b IC50 values determined by MTS assay after 72 h drug exposure.
c HCT ratio Ana/PT ¼ HCT116 IC50 value of analogue/HCT116 IC50 value of paclitaxel in the same experiment (or A2780 cell line).
d HCT/mdr R/S ratio ¼ HCT116/mdr IC50 value/HCT116 IC50 value.
e A2780/tub R/S ratio ¼ A2780/tub IC50 value/A2780 IC50 value.
f Mean values of five tests.
O
O
MTMO
O
O
O
O
O
O
O
OMTM
O
O
O
NH
O
O
O
d, e,f
a,b
TMSO
c
4
TMSO
O
TESO
Ph
HO
OH
H
BzO
HO
HO
H
11
13
H
10
AcO
N
BzO
AcO
BzO
AcO
O
O
O
O
O
12
Scheme 2. Reagents and conditions: (a) tetrabutylammonium borohydride, DCM, 0 ꢂC–rt (50%); (b) TMSOTf, DIPEA, DMF, 0 ꢂC–rt (68%); (c)
ꢀ
ethylene glycol, NIS, AgOTf, 4 A sieves, THF (53%); (d) TBAF, THF, )10 ꢂC (55%); (e) LHMDS, 12, THF, )55 ꢂC (98%); (f) TBAF, THF, )10 ꢂC
(95%).
Table 2. In vivo activity of macrocyclic taxane analogues 7, 13, and 14
evaluated concomitantly with 1
more potent than paclitaxel in inhibiting tubulin poly-
merization, while analogues 7 and 14 were slightly less
potent than paclitaxel, indicating that the macrocycle is
not interfering with tubulin binding or altering the
mechanism of action. The lack of improved efficacy
against the paclitaxel resistant cell line A2780/tub is
consistent with this hypothesis.
Compound
T/C % (dose in mg/kg/injection)
Analogue
Paclitaxel (1)
7
174 (200)
190 (100)
118 (120)
174 (60)
197 (60)
165 (60)
13
14
Furthermore, compounds 7 and 13 were shown to pos-
sess activity in vivo against the ip/ip M109 Madison
murine lung carcinoma screen (Table 2).19 The in vivo
activity of these analogues was equivalent to that ob-
served for a reference dose of paclitaxel. Compound 14,
the gem-dimethyl macrocyclic analogue, which dis-
played reduced in vitro potency, was inactive in the in
vivo screen. These results suggest that the presence of an
unsubstituted macrocycle can be tolerated at the tubulin
binding site. Analogues 7 and 13 have the potential to
demonstrate greater in vivo efficacy against tumors
exhibiting an MDR phenotype and are worthy of fur-
ther evaluation.
paclitaxel, and exhibit reduced cross-resistance to a
paclitaxel resistant cell line. Analogues 7 and 13 also
displayed in vivo activity comparable to paclitaxel. The
data are consistent with the hypothesis that these ana-
logues are more potent than paclitaxel versus MDR
resistant cell lines because of their recalcitrance to P-
glycoprotein mediated efflux. This study serves to fur-
ther define the SAR of the northern hemisphere of
paclitaxel.
References and notes
In conclusion, a series of novel C7/C10 macrocyclic
taxane analogues has been prepared. Several com-
pounds in this series possess in vitro potency superior to
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d 8.04 (d, J ¼ 7:09 Hz, 2H), 7.60 (d, J ¼ 7:07 Hz, 2H),
7.55–7.28 (bm, 11H), 7.04 (d, J ¼ 8:93 Hz, 1H), 6.17 (m,
1H), 5.93 (s, 1H), 5.72 (dd, J ¼ 2:43 Hz, J ¼ 8:94 Hz, 1H),
5.59 (d, J ¼ 6:88 Hz, 1H), 4.87 (d, J ¼ 7:92 Hz, 1H), 4.77
(d, J ¼ 6:77 Hz, 1H), 4.71–4.65 (m, 3H), 4.42 (d, J ¼
7:72 Hz, 1H), 4.24–3.95 (bm, 6H), 3.77 (d, J ¼ 6:84 Hz,
1H), 3.65 (bs, 1H), 3.54 (d, J ¼ 10:88 Hz, 1H), 3.05 (d,
J ¼ 7:71 Hz, 1H), 2.47 (m, 1H), 2.30 (s, 3H), 2.23 (dd,
J ¼ 4:39 Hz, J ¼ 8:93 Hz, 1H), 1.96 (s, 1H), 1.92 (s, 3H),
1.80 (m, 1H), 1.69 (s, 3H), 1.11 (s, 3H), 1.08 (s, 3H);
LRMS (ESI): 896 [(M)1)ꢀ, 100%]; HRMS (ESI) calcd for
C49H55NO15 Na: 920.3469, found: 920.3497. Spectral
characteristics for 13: 1H NMR (CDCl3, 300 MHz) d
8.10 (d, J ¼ 7:2 Hz, 2H), 7.60 (m, 1H), 7.48 (m, 2H), 7.42–
7.25 (bm, 6H), 6.25 (m, 1H), 6.03 (s, 1H), 5.66 (d,
J ¼ 6:9 Hz, 1H), 5.35 (d, J ¼ 9:4 Hz, 1H), 5.29 (m, 1H),
4.95 (d, J ¼ 7:9 Hz, 1H), 4.86 (d, J ¼ 6:7 Hz, 1H), 4.79 (d,
J ¼ 6:7 Hz, 1H), 4.75 (d, J ¼ 7:7 Hz, 1H), 4.62 (bs, 1H),
4.50 (d, J ¼ 7:7 Hz, 1H), 4.30 (d, J ¼ 8:4 Hz, 1H), 4.24
(dd, J ¼ 7:2 Hz, J ¼ 10:5 Hz, 1H), 4.17 (d, J ¼ 8:3 Hz,
1H), 4.10 (m, 2H), 3.85 (d, J ¼ 6:7 Hz, 1H), 3.64 (d,
J ¼ 11:0 Hz, 1H), 3.32 (d, J ¼ 4:9 Hz, 1H), 3.14 (d,
J ¼ 8:0 Hz, 1H), 2.56 (m, 1H), 2.36 (s, 3H), 2.28 (d,
J ¼ 8:9 Hz, 2H), 2.04 (s, 3H), 1.86 (m, 1H), 1.76 (s, 3H),
1.34 (s, 9H), 1.21 (s, 3H), 1.19 (s, 3H); LRMS (ESIþ):
911.5 [(M+NH4)þ, 100%], 894.5 [(M+1)þ, 95%]; LRMS
(ESIꢀ): 892.6 [(M)1)ꢀ, 100%]. Spectral data for analogues
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