Paclitaxel succinate analogs: Anionic and amide introduction as a strategy to impart blood-brain barrier permeability

Article abstract of DOI:10.1016/j.bmcl.2008.09.103

A focused library of TX-67 (C10 hemi-succinate) analogs has been prepared, including C7 regioisomers, esters, amides, and one-carbon homologs. These were prepared to investigate whether the lack of TX-67 interaction with P-glycoprotein (Pgp) is due to the presence of the carboxylic acid moiety and whether this phenomenon was restricted to C10 analogs. Tubulin stabilization ability, cytotoxicity, and Pgp interactions were evaluated. All carboxylic acid analogs and several of the amides had no apparent interactions with Pgp at the concentrations used, whereas the ester variants displayed characteristics of Pgp substrates. Furthermore, it was demonstrated that hydrogen-bonding properties were significant with respect to Pgp interactions. Calculations of log D and cross-sectional areas revealed that these analogs are predicted to partition into the membrane and can compete for Pgp binding sites. The anionic and amide introduction strategy may allow for delivery of paclitaxel into the CNS and may be a potential approach for the delivery of other, structurally complex and lipophilic non-CNS permeable drugs.

Full text of DOI:10.1016/j.bmcl.2008.09.103

Bioorganic & Medicinal Chemistry Letters 18 (2008) 5971–5974
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Paclitaxel succinate analogs: Anionic and amide introduction as a strategy
to impart blood–brain barrier permeability
Brandon J. Turunen a, Haibo Ge a, Jariat Oyetunji a, Kelly E. Desino b, Veena Vasandani b, Sarah Güthe c,
Richard H. Himes d, Kenneth L. Audus b, Anna Seelig c, Gunda I. Georg a,
*
a Department of Medicinal Chemistry, University of Kansas, Malott Hall 4070, 1251 Wescoe Hall Drive, Lawrence, KS 66045, USA
b Department of Pharmaceutical Chemistry, University of Kansas, 226 Simons, 2095 Constant Avenue, Lawrence, KS 66047, USA
c Department of Biophysical Chemistry, Biocenter of the University of Basel, Klingelbergstrasse 70, 4065 Basel, Switzerland
d Department of Molecular Biosciences, University of Kansas, Haworth Hall, 1200 Sunnyside Avenue, Lawrence, KS 66045, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 28 June 2008
Revised 19 September 2008
Accepted 22 September 2008
Available online 2 October 2008
A focused library of TX-67 (C10 hemi-succinate) analogs has been prepared, including C7 regioisomers,
esters, amides, and one-carbon homologs. These were prepared to investigate whether the lack of TX-
67 interaction with P-glycoprotein (Pgp) is due to the presence of the carboxylic acid moiety and whether
this phenomenon was restricted to C10 analogs. Tubulin stabilization ability, cytotoxicity, and Pgp inter-
actions were evaluated. All carboxylic acid analogs and several of the amides had no apparent interac-
tions with Pgp at the concentrations used, whereas the ester variants displayed characteristics of Pgp
substrates. Furthermore, it was demonstrated that hydrogen-bonding properties were significant with
respect to Pgp interactions. Calculations of logD and cross-sectional areas revealed that these analogs
are predicted to partition into the membrane and can compete for Pgp binding sites. The anionic and
amide introduction strategy may allow for delivery of paclitaxel into the CNS and may be a potential
approach for the delivery of other, structurally complex and lipophilic non-CNS permeable drugs.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Paclitaxel
TX-67
Analogues
Succinates
Amides
P-glycoprotein
Blood–brain barrier
Brain permeability
Polar surface
logD
Tubulin assembly
Cytotoxicity
Paclitaxel (Taxol, 1, Fig. 1), a structurally complex natural
product derived from the bark of the Pacific Yew, is one of the most
studied and active anti-cancer agents known.1–3 Although its clin-
ical success is remarkable, the efficacy of the parent compound has
limitations.4,5
One such shortcoming is paclitaxel’s inability to cross the
blood–brain barrier (BBB).6,7 Accordingly, paclitaxel is not an effec-
tive treatment for primary or metastatic brain cancer despite its
potent anti-proliferative activity. In addition to paclitaxel’s well
known anti-tumor properties, it has been shown to protect pri-
mary cortical neurons from beta amyloid (Ab)-induced cell death
as well as being non-toxic to primary cortical neurons.8 Indeed, a
paclitaxel derivative that could permeate the CNS is highly desir-
able from both the standpoint of chemotherapy as well as an inves-
tigational therapy for neurofibrillary pathology.
A primary mechanism limiting the distribution of paclitaxel and
other highly lipophilic substances into the brain is active efflux by
the multidrug resistance gene product 1 (MDR1) P-glycoprotein
(Pgp).9–11 We have recently described a series of recognition ele-
ments required for Pgp interactions based upon the analysis of
over one hundred known Pgp substrates.12–14 This analysis
revealed clusters of spatially distinct hydrogen bond acceptor
units, which correlated, in their relative frequency, with the
strength of Pgp interaction. We have demonstrated that deletion
or modification of these ‘‘recognition elements” in paclitaxel
reduces interaction with Pgp in bovine brain microvessel endothe-
lial cells.15,16 These studies also bolstered our ascertation that the
C10 region of paclitaxel is particularly important for Pgp affinity.
Thissameexamination12–14ofknownPgpsubstratesalsorevealed
thatcompoundsthatcarryanegativechargeatphysiologicalpH,such
as those that contain a carboxylic acid, sulphonate, or nitro group,
with few exceptions, are not substrates for Pgp efflux.12–14 One caveat
to this observation was that chemical structures that contain addi-
tional recognition elements, maintain their affinity for Pgp.
* Corresponding author. Tel.: +1 612 626 6320; fax: +1 612 626 6318.
E-mail address: georg239@umn.edu (G.I. Georg).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.09.103

5972
B. J. Turunen et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5971–5974
O
O
O
OAc
10
OAc
g (75%)
OH
O
O
18 (R2=CO(CH2)2CO2H)
19 (R2=CO(CH2)2CO2Me)
20 (R2=CO(CH2)2CONH2)
O
Ph
NH
3'
O
Ph
NH
O
t-BuO
NH
3'
O
OR2
c, g (79%)
OH
OH
10
O
13
O
O
13
7
7
d, g (80%)
OR1
OH
OH
16
17
HO
H
AcO
HO
BzO
H
AcO
HO
H
AcO
e, g (60%)
f, g (62%)
BzO
21 (R2=CO(CH2)2CONHMe)
22 (R2=CO(CH2)2CONMe2)
BzO
O
O
O
1 (paclitaxel)
2 (docetaxel)
b (90%)
a (75%)
15 (R1=TBS, R2=H)
g (74%)
23 (R2=CO(CH2)3CO2H)
24 (R2=CO(CH2)3CONH2)
25 (R2=CO(CH2)3CONHMe)
26 (R2=CO(CH2)3CONMe2)
16 (R1=TBS, R2=CO(CH2)2CO2H
Figure 1. Paclitaxel and docetaxel.
d, g (91%)
e, g (77%)
17 (R1=TBS, R2=CO(CH2)3CO2H
Compounds 18-26 R1=H
f, g (80%)
OH
Pgp repulsion
motif
O
Type I
weak
Type I
Scheme 2. Synthesis of C7 TX-67 analogs.19 Reagents and conditions: (a) succinic
anhydride, DMAP, toluene 85 °C; (b) glutaric anhydride, DMAP, toluene 85 °C; (c)
MeOH, EDCI, DMAP; (d) NH4HCO3, THF, BOC2O, pyridine; (e) NH2MeꢀHCl, EDCI,
NMM, CH2Cl2; (f) NHMe2ꢀHCl, EDCI, NMM, CH2Cl2; (g) HF–pyridine, pyridine.
O
O
Type II
O
O
NH
O
OH
10
Ph
1` O
13
3`
Type II
weak
7
2
OH
4
HO
BzO
H
protecting group removal. Compounds 11–14 were prepared in
the same manner from glutaric acid derivative 6.
AcO
O
Type I
3 (TX-67)
Figure 2. TX-67 recognition and repulsion elements.
The desired C7 analogs were accessed in short synthetic
sequences from common intermediate 15 (Scheme 2). The C7 suc-
cinic acid analog 18 was prepared by acylating the C7 hydroxyl
group with succinic anhydride, giving 16, followed by removing
the 20OTBS protecting group. The methyl ester analog 19 was gen-
erated from a coupling between acid 16 and MeOH followed by the
same deprotection. The remaining compounds 21–26 were synthe-
sized as described for the C10 analogs.
All compounds were first evaluated for tubulin assembly ability
and their effectiveness against the MCF7 breast cancer cell line and
the drug resistant breast cancer cell line NCI/ADR-RES (Table 1).
In the tubulin assembly assay, all analogs maintained similar
activity to the parent compound (entries 2–19, Table 1). Against
the MCF7 breast cancer cell line most analogs maintained effec-
tiveness. The carboxylic acid analogs (entries 2–5), as a functional
group class, showed the largest drop in activity (approx. 20-fold).
The methyl ester variants (entries 6 and 7) maintained similar
potency as compared to paclitaxel while the amides (entries
8–19) generally experienced an approximate 5-fold reduction in
cytotoxicity. Most analogs were similar to paclitaxel against the
NCI/ADR-RES cancer cell line. We had originally hypothesized an
increase in effectiveness of these analogs against the Pgp over-
Type I
With this in mind we prepared a C10-modified taxane, TX-67
(3, Fig. 2), which only differs from paclitaxel by the addition of
an acetic acid unit to the terminus of the C10 acetyl ester.17
TX-67 (3) contains all of the recognition elements of paclitaxel,
however, our studies suggest Pgp efflux mechanisms are substan-
tially reduced or absent completely.17,18 Compound 3 demon-
strates improved distribution across the BBB without
co-administration of Pgp inhibitors both in vitro and in situ.17
Herein, we describe the synthesis and biological evaluation of a
focused library of TX-67 analogs that include C7 regioisomers,
esters, amides, and one-carbon homologs. These were made to
determine if the acid functionality was essential for Pgp evasion
and if this phenomenon was restricted to only C10 analogs.
The C10 functional group analogs were prepared from common
intermediate 4 (Scheme 1). TX-67 (3) was prepared as previously
described17 while compounds 7–10 were accessed via acid 5. Acid
5 is cleanly and quantitatively converted to the methyl ester via
treatment with diazomethane (Scheme 1), which, after treatment
with HF–pyridine solution provides methyl ester 7 in good yield.
The primary amide analog 8 is prepared via mixed anhydride for-
mation with BOC2O followed by amide formation via ammonium
bicarbonate (NH4CO3) under basic conditions. The N-methyl and
N,N-dimethyl, amides (9 and 10 are generated via standard cou-
pling procedures (Scheme 1). The described functional group trans-
formations were each followed by fluoride anion assisted
Table 1
ED50 ratios (compound/paclitaxel) for in vitro tubulin assembly and cytoxicity.
Entry
Compound
Tubulin assembly
MCF7
NCI/ADR-RES
1
2
3
4
5
Paclitaxel (1)
1.0
1.7
1.8
3.8
1.0
1.0
1.0
1.3
5.8
5.8
13.6
TX-67 (3 – C10 CO2H)
C10 CH2CO2H (11)
C7 CO2H (18)
13.3
27.9
>30.0
8.8
C7 CH2CO2H (23)
O
6
7
C10 CO2Me (7)
C7 CO2Me (19)
1.0
1.2
2.5
0.54
0.4
0.5
OR2
g (88%)
3
7
8
9
(R2=CO(CH2)2CO2H)
(R2=CO(CH2)2CO2Me)
(R2=CO(CH2)2CONH2)
(R2=CO(CH2)2CONHMe)
O
Ph
NH
O
OR3
c, g (68%)
d, g (77%)
e, g (39%)
f, g (43%)
8
9
10
11
C10 CONH2 (8)
C10 CH2CONH2 (12)
C7 CONH2 (20)
0.4
1.8
0.6
0.6
10
10
3.2
5.0
6.0
5.3
0.6
6.5
O
OR1
5
HO
BzO
H
AcO
O
C7 CH2CONH2 (24)
10 (R2=CO(CH2)2CONMe2)
b (93%)
a (95%)
4 (R1=TBS, R2=H, R3=TES)
12
13
14
15
C10 CONHMe (9)
C10 CH2CONHMe (13)
C7 CONHMe (21)
0.8
1.8
0.8
0.8
7.9
2.3
2.2
3.7
6.0
>6.3
1.0
g (70%)
d, g (80%)
e, g (60%)
11 (R2=CO(CH2)3CO2H)
12 (R2=CO(CH2)3CONH2)
13 (R2=CO(CH2)3CONHMe)
5 (R1=TBS, R2=CO(CH2)2CO2H, R3=TES
C7 CH2CONHMe (25)
3.2
6 (R1=TBS, R2=CO(CH2)3CO2H, R3=TES
Compounds 3, 7-14 R1=R3=H
6
16
17
18
19
C10 CONMe2 (10)
C10 CH2CONMe2 (14)
C7 CONMe2 (22)
1.6
2.2
1.1
0.7
2.1
5.6
10.8
3.9
2.7
>6.3
0.8
f, g (64%)
14 (R2=CO(CH2)3CONMe2)
Scheme 1. Synthesis of C10-modified TX-67 analogs. Reagents and conditions: (a)
succinic anhydride, DMAP, toluene 85 °C; (b) glutaric anhydride, DMAP, toluene
85 °C; (c) CH2N2, THF; (d) NH4HCO3, THF, BOC2O, pyridine; (e) NH2MeꢀHCl, EDCI,
NMM, CH2Cl2; (f) NHMe2ꢀHCl, EDCI, NMM, CH2Cl2; (g) HF–pyridine, pyridine.
C7 CH2CONMe2 (26)
3.0
Paclitaxel has a mean ED50 of 3.2 nM 1.8 and 1.5
ADR-RES lines, respectively.
lM
1.3 in the MCF7 and NCI/

B. J. Turunen et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5971–5974
5973
4.50
4.00
3.50
3.00
2.50
2.00
1.50
1.00
0.50
0.00
C
1
3
11 18 23
7
19
8
12 20 24
9
13 21 25
10 14 22 26
COOH
COOCH3
CONH2
CONHMe
CONMe2
Figure 3. Rhodamine uptake results for compounds 1 (paclitaxel), C10 and C7 acids (3, 11, 18, and 23), C10 and C7 esters (7 and 19), C10 and C7 primary amides (8, 12, 20,
and 24), C10 and C7 secondary amides (9, 13, 21, and 25), and C10 and C7 tertiary amides (10, 14, 22, and 26) in BMEC’s. Paclitaxel and the derivatives were present at a
concentration of 10 lM. The concentration of rhodamine was 5 lM. C, control. Compounds that significantly increase rhodamine 123 uptake are show as red bars.
Compounds causing decreased or limited rhodamine 123 uptake are shown in blue.
expressing cancer cell line MCF7-ADR. However, this was not the
case as none of the analogues in Table 1 had superior potency
compared to paclitaxel.
5
4
3
2
19
26
25
7
22
Our next screen was the rhodamine 123 uptake assay
(Fig. 3).20,21 This assay is a preliminary screen to evaluate a com-
pound’s interaction with Pgp in bovine brain microvessel endothe-
lial cells (BMECs). In this assay, rhodamine 123 is used as a
surrogate Pgp substrate. The effect of the test compound on rhoda-
mine 123 is determined by monitoring intracellular fluorescence. If
the test compound has higher affinity to Pgp than rhodamine 123,
then uptake of the latter will increase relative to the negative
control.
21
20
1
24
14
13
10
12
9
8
23
1
18
11
0
3
All analogs containing a carboxylic acid functionality (3, 11, 18,
and 23) showed no apparent interaction with Pgp. When the
carboxylic acid is capped with a methyl group (methyl esters 7
and 19), a type I Pgp recognition element, in both the C7 and C10
series, a marked increase in rhodamine accumulation is observed.
This is in agreement with our previous results suggesting that
the carboxylic acid functionality is advantageous for Pgp evasion.17
Many members of the amide series (8–10, 12–14, 20, 21, and
24) did not significantly increase rhodamine accumulation. The
weak interaction of secondary and primary amides with Pgp had
already been suggested by us in an analysis of Pgp substrates
and non-substrates.12 As the secondary amides are converted from
hydrogen bond donors (8, 9, 12, 13, 20, 21, and 25) to tertiary
amides, hydrogen bond acceptors (10, 14, 22, and 26), increased
rhodamine uptake is noted indicating that the molecules are inter-
acting more strongly with Pgp. This is in accord with our hypoth-
esis that H-bond donors will not interact significantly with Pgp
and that H-bond acceptors will serve as substrates.
It is also of note that the succinic amides 8, 20, 9, 21, and 10
showed less rhodamine uptake than the corresponding one carbon
homologs, the glutaric amides 12, 24, 13, 25, and 14, indicating
that the length of the tether plays a role in binding to Pgp.
Our data further suggest that groups added to decrease Pgp inter-
actions are generally more effective at the C10 position on the paclit-
axel structure than on the C7 position. For example, the 10-linked
methylamides 9 and 13 showed less rhodamine uptake than the
O7-linked methylamides 21 and 25. This implies that the C10 region
on these analogs has a very intimate relationship with Pgp.
Our results suggest that Pgp evasion may not only be achieved
by the introduction of a carboxylic acid moiety but also by the
introduction of a primary or secondary amide into Pgp substrates.
To compare the different analogs with respect to their ability to
partition into the membrane and bind to Pgp we modeled the
membrane-binding conformations and calculated the respective
cross-sectional areas, AD. In addition we calculated the logD values
at pH 7.4 using an ionization constant pKa 4.2 for the carboxylic
acid group (Fig. 4).11 The former varied between logD 0 and 4
-1
120
130
140
150
A
[Å2]
D, calc
Figure 4. Calculations of cross-sectional areas, AD and logD values at pH 7.4 for
paclitaxel and analogues. ꢂ = paclitaxel,
D
= CO2H, . = CO2Me, } = CONH2, s = CON-
HMe, d = CONHMe, h = CONMe2, j CONMe2 (filled symbols = Pgp substrates).
and the latter between AD 125 and 143 Å.2 The analogs of paclitaxel
are thus clearly more hydrophobic and larger than rhodamine 123
with a logD ꢁ 3.13 and an AD 69 Å2 and can therefore be predicted
to compete for Pgp binding sites provided they carry the relevant
recognition elements.
In summary, we have prepared a focused library of paclitaxel
analogs in short synthetic sequences from the parent molecule.
Biological evaluation in tubulin stabilization and cytotoxicity
assays demonstrated that the designed analogs maintained the
desired properties of paclitaxel. The rhodamine assay indicated
that the introduction of esters at C10 and C7 increased rhodamine
123 uptake, whereas the placement of a carboxylic acid functional-
ity on either the C7 or C10 position resulted in decreased uptake.
Since carboxylic acid transporters are known to exist in the BBB,
it should also be considered that the carboxylic acid functionality
may be a substrate for an influx pump, in a similar fashion that
paclitaxel acts as a substrate for cellular efflux. It is possible that
this influx transport system is shuttling TX-67 into the cell, past
the Pgp efflux system, allowing for the observed increase in BBB
permeation. Furthermore, we have illustrated that positioning an
acid or amide at C10 of paclitaxel is generally more effective than
C7 attachment and that succinic acid and succinic amides lead to
less rhodamine 123 uptake than the corresponding glutaric acid
and amide analogs. We have also discovered that the hydrogen-
bonding character of the amide analogs plays a significant role
and that the primary amides were generally superior compared
to the secondary and tertiary amides analogs in evading interac-
tion with Pgp.

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The anionic introduction strategy and the introduction of a
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paclitaxel into the CNS. This strategy could also hold promise for
the delivery of other, highly complex, non-CNS permeable drugs,
provided they are highly lipophilic.
6. Schinkel, A. H. Adv. Drug Delivery Rev. 1999, 36, 179.
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Acknowledgments
This work was supported by a grant from the National Cancer
Institute (CA82801). B.J.T. would like to acknowledge the Depart-
ment of Defense Breast Cancer Research Program for a predoctoral
fellowship (DAMD17-02-1-0435). Paclitaxel was generously do-
nated by Tapestry Pharmaceuticals, Boulder, CO. We thank Jacque-
lyn Huff for her excellent technical assistance.
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