In situ blood-brain barrier permeability of a C-10 paclitaxel carbamate

Article abstract of DOI:10.1016/j.bmcl.2008.10.024

We report the synthesis and blood-brain barrier (BBB)-permeability of ¹⁴C-CNDR-29, a paclitaxel C-10 carbamate derivative shown to be devoid of P-glycoprotein (Pgp)-interactions, in an in situ mouse brain perfusion model, in comparison with ¹⁴C-paclitaxel. The results presented reveal a 3- to 4-fold higher BBB-permeability for the C-10 modified taxane compared to paclitaxel. These results support the notion that circumvention of Pgp-mediated efflux can lead to higher BBB-permeability. Further studies however are needed to evaluate the therapeutic potential of the C-10 carbamates paclitaxel derivatives for the treatment of CNS diseases.

Full text of DOI:10.1016/j.bmcl.2008.10.024

Bioorganic & Medicinal Chemistry Letters 18 (2008) 6119–6121
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
In situ blood–brain barrier permeability of a C-10 paclitaxel carbamate
a
a
a
b
Carlo Ballatore a,b, , Bin Zhang , John Q. Trojanowski , Virginia M.-Y. Lee , Amos B. Smith III
*
a Center for Neurodegenerative Diseases Research, Institute on Aging, University of Pennsylvania, 3600 Spruce Street, Philadelphia, PA 19104-6323, USA
b Department of Chemistry, University of Pennsylvania, 231 South 34th Street, Philadelphia, PA 19104-6323, USA
a r t i c l e i n f o
a b s t r a c t
We report the synthesis and blood–brain barrier (BBB)-permeability of 14C-CNDR-29, a paclitaxel C-10
carbamate derivative shown to be devoid of P-glycoprotein (Pgp)-interactions, in an in situ mouse brain
perfusion model, in comparison with 14C-paclitaxel. The results presented reveal a 3- to 4-fold higher
BBB-permeability for the C-10 modified taxane compared to paclitaxel. These results support the notion
that circumvention of Pgp-mediated efflux can lead to higher BBB-permeability. Further studies however
are needed to evaluate the therapeutic potential of the C-10 carbamates paclitaxel derivatives for the
treatment of CNS diseases.
Article history:
Received 17 September 2008
Revised 1 October 2008
Accepted 3 October 2008
Available online 10 October 2008
Keywords:
Taxane
Paclitaxel C-10 carbamate
Blood–brain barrier
Mouse brain perfusion
Neurodegenerative diseases
Ó 2008 Elsevier Ltd. All rights reserved.
Over the past several years, there has been growing interest in
the development of brain-penetrant microtubule stabilizing
(MT)-agents for the treatment of different central nervous system
(CNS) conditions, including cancer as well as non-proliferative dis-
eases such as Alzheimer’s disease (AD) and related disorders.1
Among the various classes of MT-stabilizing natural products, tax-
anes have been arguably one of the most intensely studied.
and suggest that circumvention of Pgp-mediated efflux can pro-
duce therapeutically relevant paclitaxel brain levels.6,8 However,
since Pgp-function comprises an important defense mechanism,
protecting the CNS from toxic substances, ideal candidate com-
pounds for brain diseases should be able to reach therapeutically
effective brain-concentrations without causing a significant dis-
ruption in Pgp-function. This is particularly important in the con-
text of chronic neurodegenerative diseases, where lack of Pgp-
function, and/or other BBB-dysfunctions, have been clearly linked
to the pathology of these diseases.9,10
Indeed, paclitaxel2 (Fig. 1), the first chemotherapeutic MT-stabi-
lizing agent to receive FDA approval for the treatment of cancer,
was found to be a potential clinical candidate for other disorders,
including neurodegenerative diseases.3 However, the issue of lim-
ited brain uptake due to poor blood–brain barrier (BBB)-perme-
ability prevented further development of this compound for CNS
indications. Paclitaxel, as well as several related compounds, is
known to be a good substrate for the ATP-binding cassette (ABC)
active transporter P-glycoprotein (Pgp),4 which is highly expressed
in the kidneys, liver, intestine and the BBB.5 Previous studies6,7
have demonstrated that Pgp-knockout mice exhibit higher paclit-
axel cerebral-uptake compared to wild type animals and that phar-
macological modulation of Pgp in wild type mice can increase
paclitaxel brain levels by 3- to 6-fold compared to animals non-
treated with Pgp-modulators. Furthermore, unlike control animals,
when Pgp-knockout mice or wild type mice treated with Pgp-
inhibitors were implanted with a tumor in the brain, peripheral
administration of paclitaxel was found to produce significant
anti-neoplastic effects. Taken together, these findings highlight
the importance of Pgp in limiting paclitaxel’s entry in the CNS,
Importantly, recent studies have shown that selected chemical
modifications at the C-10 position of taxanes can result in ana-
logues that are neither substrates nor inhibitors of Pgp.11,12 These
R
O
OH
7
O
O
10
O
O
13
2'
O
H
N
H
O
OH OBz OAc
OH
O
H
N
R = CH3 (Paclitaxel)
O
(CNDR-3)
R =
R =
OH
OH
H
N
(TX-67)
R =
OH
(CNDR-29)
Figure 1. Paclitaxel and representative structures of C-10 modified derivatives
* Corresponding author.
known to be devoid of Pgp-interactions.
E-mail address: bcarlo@sas.upenn.edu (C. Ballatore).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.10.024

6120
C. Ballatore et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6119–6121
compounds are typified by the C-10 succinimidyl ester TX-67
(Fig. 1),13 which notably was found to have an apparent permeabil-
ity coefficient 6–10 times higher than paclitaxel, in a rat brain per-
fusion model.11 Other examples of C-10 modified taxanes lacking
Pgp-interactions include a series of C-10 carbamates (e.g., CNDR-
3 and CNDR-29, Fig. 1) which were found to be comparatively more
stable than C-10 esters like TX-67, and therefore potentially more
desirable for further clinical development.12 Preliminary SAR for
the C-10 carbamate series suggested that efficient circumvention
of Pgp-mediated efflux in vitro could generally be achieved by
derivatives bearing a polar moiety, typically a carboxylic acid moi-
ety, at the C-7 or C-10 position. Interestingly, among the C-10 car-
bamates found to be devoid of Pgp-interactions, CNDR-29 was the
only example that lacked the carboxylic acid residue. This rela-
tively more lipophilic compound was found to be more cell-perme-
able than the corresponding carboxylic acid containing analogues,
as suggested by cell-cytotoxicity studies.12 Thus, because of the
more favorable combination of lipophilicity and lack of Pgp-inter-
actions, CNDR-29 was selected for BBB-permeability studies in a
mouse brain perfusion model. Towards this end [14C]-labeled
CNDR-29 was prepared and tested in comparison with [14C]-
paclitaxel.
Figure 3. CPMs in [14C]-CNDR-29 and [14C]-paclitaxel infused mice brain.
The results, summarized in Figure 3, reveal that the total counts
observed in brain samples of [14C]-CNDR-29-treated mice was
approximately 3–4 times higher than that found in the corre-
sponding paclitaxel-treated animals, demonstrating that CNDR-
29 exhibits higher BBB-permeability than paclitaxel, and thus sug-
gesting that CNDR-29 may be able to reach comparatively higher
brain levels than paclitaxel, after peripheral administration. It
should be noted, however, that brain perfusion models are rela-
tively controlled experimental systems that do not consider other
potentially limiting factors such as metabolism and plasma protein
binding, which may greatly influence the overall brain uptake.
Thus, although these results clearly suggest a higher permeability
for CNDR-29, further studies are needed to evaluate whether the
observed relative increase of 3- to 4-fold in BBB-permeability in
the in situ mouse brain perfusion model may translate into thera-
[
14C]-CNDR-29 was prepared by reacting an appropriately pro-
tected paclitaxel-10-deacetyl-10-carbonyl-N-methylimidazol-ini-
um iodide salt A14 with [1,2 14C]-ethanolamine (5–10 mCi/
mmol), to furnish carbamate B, which was directly treated with
HF/Py obtaining [14C]-CNDR-29 (8.5 mCi/mmol; Fig. 2).15 The brain
perfusion experiment16 was conducted in triplicate by infusing
0.1 l lL of buf-
Ci of the test compound (or [14C]-paclitaxel), in 500
fer, into the carotid artery of an appropriately prepared mouse over
a 60-s time period. Following the infusion, the vasculature was
washed for an additional 60 s with buffer containing non-radiola-
beled compound and finally the animal was sacrificed and the
brain collected for quantification of radioactivity in a scintillation
counter. A brain perfusion experiment was also conducted with
[
14C]-sucrose to verify the physical integrity of the BBB.
I
OH
*
N
1) [1,2 14C]ethanolamine hydrochloride 5-10
mCi(185-370 MBq)/mmol (250 μCi of a 100
μCi/mL solution in EtOH)
2) DIEA (9.2 μL)
*
HN
N
Si
Si
O
O
O
O
O
O
O
O
3) 30 min. stirring, room temperature
O
O
O
O
O
O
O
O
H
H
N
H
N
H
OAc
OAc
OH OBz
OH OBz
O
O
Si
Si
B
A
0.5 ml Py,
0 °C to room temp.
HF/Py (100 μl)
16 hrs
OH
*
HN
*
O
O
O
OH
O
O
O
O
H
N
H
OAc
OH OBz
OH
[
14C]-CNDR-29
Figure 2. Synthesis of [14C]-CNDR-29. [14C]-labels are indicated with an asterix.

C. Ballatore et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6119–6121
6121
12. Ballatore, C.; Hyde, E.; Deiches, R. F.; Lee, V. M.-Y.; Trojanowski, J. Q.; Huryn, D.;
Smith, A. B., III Bioorg. Med. Chem. Lett. 2007, 17, 3642.
peutically useful brain concentrations after peripheral administra-
tion. Nonetheless it is interesting to note that, bearing in mind the
experimental differences, these brain perfusion data appear to be
in agreement with reports that co-administration (ip) of paclitaxel
with a range of Pgp-modulators produce a relative increase of 3–6
times in paclitaxel mouse brain concentrations.6,8 These results are
also consistent with the notion that circumvention of Pgp-medi-
ated efflux can lead to higher BBB-permeability. Finally, it should
be noted that the prototype compound TX-67, under similar brain
perfusion experimental conditions, was reported to have an even
more pronounced relative enhancement in BBB-permeability (6-
to 10-fold) compared to paclitaxel. The relatively higher BBB-per-
meability of TX-67 may support the hypothesis of the involvement
of an active transport mechanism (e.g., monocarboxylic acid trans-
porters).11 To evaluate this possibility, and in order to select the
most plausible candidate compound(s) for efficacy studies in a
transgenic animal model of neurodegenerative taoupathies, a com-
parative evaluation in mouse PK models of C-10 modified paclit-
axel analogues, including TX-67, as well as other MT-stabilizing
agents from different classes of natural products is ongoing in
our laboratories.
13. Yanbin Liu, F.; Ali, S. M.; Boge, T. C.; Georg, G. I.; Victory, S.; Zygmunt, J.;
Marquez, R. T.; Himes, R. H. Comb. Chem. High Throughput Screen. 2002, 5, 39.
14. Ballatore, C.; Aspland, S. E.; Castillo, R.; Desharnais, J.; Eustaquio, T.; Sun, C.;
Castellino, A. J.; Smith, A. B., III Bioorg. Med. Chem. Lett. 2005, 15, 2477.
15. Detailed experimental procedure for the synthesis of [14C]-CNDR-29: to a
previously prepared imidazolinium iodide salt A (14.4 mg, 0.011 mmol) in a
20 mL scintillation vial (screw cap) fitted with a magnetic stir bar, an addition
of [1,2 14C]-ethanolamine hydrochloride (250
lCi; 5–10 mCi/mmol;
0.0333 mmol in 2.5 mL of ethyl alcohol; Moravek Biochemicals and
Radiochemicals) is made using a disposable 5 mL syringe, followed by an
addition of diisopropylethyl amine (DIEA; 9.2
lL; ꢀ0.066 mmol). The reaction
vessel is then capped and stirred for 30 min at room temperature to give
compound B, which is directly used for the next step without any purification
or work-up. Thus, the crude reaction mixture is diluted with 0.5 mL of pyridine,
cooled to 0 °C, and finally added with 100 lL of HF/pyridine. The reaction
mixture is then allowed to stir for 16 h (overnight) allowing the temperature to
rise to room temperature. After 18 h the reaction is quenched by addition of
100 lL of a saturated solution of CuSO4, then the mixture is concentrated via a
stream of nitrogen (3–4 h). The residue is re-dissolved in ꢀ5 mL of ethyl
acetate and the resulting mixture is washed with 1 mL of a saturated solution
of CuSO4 directly in the same scintillation vial used for the synthesis. The top
organic layer is then collected with a Pasteur and transferred into a new
scintillation vial. After removal of the volatiles via a stream of nitrogen the
residue is re-dissolved in the minimal amount of dichloromethane/methyl
alcohol 9:1 (ca. 100 lL). Finally, the compound is purified by preparative TLC
(dichloromethane/methyl alcohol, 9:1) obtaining the desired compound
(4.2 mg; 42% yield; 8.5 mCi/mmol).
16. The brain perfusion experiment was adapted from previously described
procedures17: brain perfusion experiments were conducted using B6C3 mice.
A 0.5-in. incision was made in the skin of anesthetized animals in order to
expose the carotid triangle. After separation of superficial structures, such as
thyroid and salivary glands, non-blood vessel soft tissue and fat, the carotid
sheath was exposed and carefully dissected to expose the internal and external
branches of the carotid artery. A single suture was then tied to the external
carotid branch, while two additional sutures were set loosely in the proximal
and distal region of the common carotid artery, respectively. The proximal
ligature was then gently tighten and the distal portion of the artery was
clamped off. One end of the P10 tubing was then inserted near the proximal
ligature to a depth of ꢀ2 mm, while the other end was connected to a 1-mL
syringe filled with saline. The distal clamp was then released and a small
amount of saline was injected into the internal branch of the carotid artery to
check for signs of leaking or bleeding. The P10 tubing was then connected to a
perfusion pump with 1 mL syringe filled with the test compound. The heart
was then exposed, stopped and the right atrium cut to allow blood outflow and
finally infusion of the test compound was made at a 0.5 mL/min for a total
Acknowledgments
Financial support for this work was provided by the NIH (U01
AG029213). We thank Professor Donna Huryn and Dr. Hugo Geerts
for helpful discussions.
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a
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