Arginine-Based Molecular Transporters: The Synthesis and Chemical Evaluation of Releasable Taxol-Transporter Conjugates

Article abstract of DOI:10.1021/ol035234c

(Equation presented) A flexible and efficient procedure has been developed for the conjugation of taxol to various arginine-based molecular transporters via the taxol C2′ O-chloroacetyl derivative. The resultant taxol-transporter conjugates are highly wat

Full text of DOI:10.1021/ol035234c

ORGANIC
LETTERS
2003
Vol. 5, No. 19
3459-3462
Arginine-Based Molecular Transporters:
The Synthesis and Chemical Evaluation
of Releasable Taxol-Transporter
Conjugates
,†
Thorsten A. Kirschberg,* Chris L. VanDeusen,‡ Jonathan B. Rothbard,†
,‡
Michael Yang,† and Paul A. Wender*
CellGate, Inc., 552 Del Rey AVenue, SunnyVale, California 95085,
and Department of Chemistry, Stanford UniVersity, Stanford, California 94305
kirschberg@cellgateinc.com; wenderp@stanford.edu
Received July 3, 2003
ABSTRACT
A flexible and efficient procedure has been developed for the conjugation of taxol to various arginine-based molecular transporters via the
taxol C2′ O-chloroacetyl derivative. The resultant taxol-transporter conjugates are highly water soluble and release free taxol with half-lives
of minutes to hours depending on the pH and the linker structure.
Taxol’s1 unprecedented structure2 and novel mode of action3
have provided the basis for major advances in chemistry,4
biology,5 and medicine, leading to its use in the treatment
of human ovarian and breast cancer.6 Notwithstanding this
progress, taxol suffers from very low water solubility and
as a consequence must be formulated for therapeutic use with
Cremophor EL.7 Concerns about this vehicle have prompted
considerable interest in the development of water-soluble
derivatives of taxol.8 Many of these derivatives are func-
tionalized at the C2′ position of taxol with a water-
solubilizing group that must be cleaved to release free taxol
before cellular uptake.9,10
(6) (a) Paclitaxel (Taxol): Current Practices and Future Directions in
Breast Cancer Management; Seminars in Oncology; Yarboro, J. W.,
Bornstein, R. S., Mastrangelo, M. J., Eds.; W. B. Saunders: Philadelphia,
PA, 1996; Vol. 23 (1-Suppl. 14), pp 23-31. (b) Taxane Anticancer
Agents: Basic Science and Current Status; Georg, G. I., Chen, T. T., Ojima,
I., Vyas, D. M., Eds.; ACS Symposium Series 583; American Chemical
Society: Washington, DC, 1995. (c) Taxol Science and Applications;
Suffness, M., Ed.; CRC Press: Boca Raton, FL, 1995.
† CellGate, Inc.
‡ Stanford University.
(1) Taxol is the registered trademark for the molecule with the generic
name paclitaxel.
(2) Wani, M. C.; Taylor, H. L.; Wall, M. E.; Coggon, P.; McPhail, A.
T. J. Am. Chem. Soc. 1971, 93, 2325-2327.
(3) (a) Diaz, J. F.; Andreu, J. M. Biochem. 1993, 32, 2747-2755. (b)
Schiff, P. B.; Fant, J.; Horwitz, S. B. Nature 1979, 277, 665-667.
(4) For the most recent total synthesis, see: Kusama, H.; Hara, R.;
Kawahara, S.; Nishimori, T.; Kashima, H.; Nakamura, N.; Morihira, K.;
Kuwajima, I. J. Am. Chem. Soc. 2000, 122, 3811-3820. For earlier
syntheses from the groups of Holton, Nicolaou, Danishefsky, Wender, and
Mukaiyama, see ref 7 in the above-cited paper.
(7) Terwogt, J. M. M.; Nuijen, B.; Ten Bokkel Huinink, W. W.; Beijnen,
J. H. Cancer Treatment ReV. 1997, 23, 87-95.
(8) (a) Takahashi, T.; Tsukamoto, H.; Yamada, H. Bioorg. Med. Chem.
Lett. 1998, 8, 113-116. (b) Li, C.; Yu, D.-F.; Newman, R. A.; Cabral, F.;
Stephens, L. C.; Hunter, N.; Milas, L.; Wallace, S. Cancer Res. 1998, 58,
2404-2409. (c) Greenwald, R. B.; Pendri, A.; Bolikal, D. J. Org. Chem.
1995, 60, 331-336. (d) Mathew, A. E.; Mejillano, M. R.; Nath, J. P.; Himes,
R. H.; Stella, V. J. J. Med. Chem. 1992, 35, 145-151.
(5) Rodi, D. J.; Janes, R. W.; Sanganee, H. J.; Holton, R. A.; Wallace,
B. A.; Makowski, L. J. Mol. Biol. 1999, 285, 197-203.
10.1021/ol035234c CCC: $25.00 © 2003 American Chemical Society
Published on Web 08/26/2003

Recently, several classes of peptide molecules have been
identified that are water-soluble and undergo facilitated
uptake into cells and tissues.11 Among these molecular
transporters, peptide sequences found in HIV-tat and in
Antennapedia have shown high membrane translocation
efficiencies.12 Extensive structure-function studies of the
HIV-tat transporter sequence (RKKRRQRRR) have led to
the finding that short oligomers of arginine- (R7-9) and, more
specifically, guanidinium-based oligomers often exhibit
superior membrane translocation activity.13 Importantly, these
homo-oligomers can be prepared in a more cost-efficient
manner than conventional hetero-oligomers by using a
segment-doubling strategy.14 Of special note, the arginine-
based transporters have also been shown to penetrate human
skin, leading to the entry of a releasable oligo-arginine
Cyclosporin A conjugate into human trials for the treatment
of dermatological disorders.15 In contrast to simple solubi-
lizing functionalities, a particularly significant property of
these transporters is that they both enhance water solubility
and facilitate uptake through the nonpolar bilayer of a cell.
This obviously represents a potentially useful strategy for
improving the formulation and bioavailability of drugs such
as taxol. To explore this strategy, an efficient and flexible
procedure for the conjugation of taxol to arginine-based
transporters is required. In addition to being compatible with
Scheme 1. Chloroacetylation of Taxola
a Reagents and conditions: (a) (ClAc)2O, DIEA, DCM, rt,
quantitative.
the sensitive functionality of taxol, this procedure would need
to be scalable and sufficiently flexible to produce taxol
conjugates with tunable half-lives for release under physi-
ological conditions. We report here a versatile procedure for
the preparation of taxol-transporter conjugates and an evalu-
ation of their ability to release free taxol in PBS buffer at
37 °C.
The strategy devised for release of taxol from the
transporter is based on the observation that taxol can be
esterified at C2′ without protection of other functional groups.
If the attached ester incorporated a suitably positioned and
protected amine (e.g., 3, Scheme 2), release of free taxol
(9) (a) Lee, J. W.; Fuchs, P. L. Org. Lett. 1999, 1, 179-181. (b) Golik,
J.; Wong, H. S. L.; Chen, S. H.; Doyle, T. W.; Wright, J. J. K.; Knipe, J.;
Rose, W. C.; Casazza, A. M.; Vyas, D. M. Bioorg. Med. Chem. Lett. 1996,
6, 1837-1842. (c) Vyas, D. M.; Wong, H.; Crosswell, A. R.; Casazza, A.
M.; Knipe, J. O.; Mamber, S. W.; Doyle, T. W. Bioorg. Med. Chem. Lett.
1993, 3, 1357-1360. (d) Guenard, D.; Gueritte-Voegelein, F.; Potier, P.
Acc. Chem. Res. 1993, 26, 160-167.
Scheme 2. Synthesis of Taxol Conjugates
(10) (a) De Bont, D. B. A.; Leenders, R. G. G.; Haisma, H. J.; van der
Meulen-Muileman, I.; Scheeren, H. W.; Bioorg. Med. Chem. Lett. 1997, 5,
4405-414. (b) Rodrigues, M. L.; Carter, P.; Wirth, C.; Mullins, S.; Lee,
A.; Blackburn, B. K. Chem. Biol. 1995, 2, 223-227. (c) Ueda, Y.; Wong,
H.; Matiskella, J. D.; Mikkikineni, A. B.; Farina, V.; Fairchild, C.; Rose,
W. C.; Mamber, S. W.; Long, B. H.; Kerns, E. H.; Casazza, A. M.; Vyas,
D. M. Bioorg. Med. Chem. Lett. 1994, 4, 1861-1864. (d) Nicolaou, K. C.;
Guy, R. K.; Pitsinos, E. N.; Wrasidlo, W. Angew. Chem., Int. Ed. Engl.
1994, 33, 3, 1583-1587. (e) Nicolaou, K. C.; Riemer, C.; Kerr, M. A.;
Rideout, D.; Wrasidlo, W. Nature 1993, 364, 464-466. (f) Ueda, Y.;
Mikkilineni, A. B.; Knipe, J. O.; Rose, W. C.; Casazza, A. M.; Vyas, D.
M. Bioorg. Med. Chem. Lett. 1993, 3, 1761-1766. (g) Zhao, Z.; Kingston,
D. G. I. J. Nat. Prod. 1991, 54, 1607-1611.
(11) Langel, U., Ed. Cell Penetrating Peptides; CRC Press: Boca Raton,
FL, 2002.
(12) (a) Lindgren, M.; Haellbrink, M.; Prochiantz, A.; Langel, Ue. Trends
Pharm. Sci. 2000, 21, 99-103. (b) Frankel, A. D.; Pabo, C. O. Cell 1988,
55, 1189-1193. (c) Bhorade, R.; Weissleder, R.; Nakakoshi, T.; Moore,
A.; Tung, C.-H. Bioconjugate Chem. 2000, 11, 301-305. (d) Josephson,
L.; Tung, C.-H.; Moore, A.; Weissleder, R. Bioconjugate Chem. 1999, 10,
186-191. (e) Fawell, S.; Seery, J.; Daikh, Y.; Moore, C.; Chen, L. L.;
Pepinsky, B.; Barsoum, J. Proc. Natl. Aca. Sci. U.S.A. 1994, 91, 664-668.
(13) (a) Mitchell, D. J.; Kim, D. T.; Steinman, L.; Fathman, C. G.;
Rothbard, J. B. J. Peptide Res. 2000, 56, 318-325. (b) Wender, P. A.;
Mitchell, D. J.; Pattabiraman, K.; Pelkey, E. T.; Steinman, L.; Rothbard, J.
B. Proc. Natl. Acad. Science U.S.A. 2000, 97, 13003-13008. (c) Rothbard,
J. B.; Kreider, E.; VanDeusen, C. L.; Wright, L. R.; Wylie, B. L.; Wender,
P. A. J. Med. Chem. 2002, 45, 3612-3618. (d) Umezawa, N.; Gelman, M.
A.; Haigis, M. C.; Raines, R. T.; Gellmann, S. H. J. Am. Chem. Soc. 2002,
124, 368-369. (e) Wender, P. A.; Rothbard, J. B.; Jessop, T. C.; Kreider,
E. L.; Wylie, B. L. J. Am. Chem. Soc. 2002, 124, 13382-13383. (f) Rueping,
M.; Mahajan, Y.; Sauer, M.; Seebach, D. ChemBioChem 2002, 3, 257-
259. (g) Futaki, S.; Ohashi, W.; Suzuki, T.; Niwa, M.; Tanaka, S.; Ueda,
K.; Harashima, H.; Sugiura, Y. Bioconjugate Chem. 2001, 12, 1005-1011.
(14) Wender, P. A.; Jessop, T. C.; Pattabiraman, K.; Pelkey, E. T.;
VanDeusen, C. L. Org Lett. 2001, 3, 3229-3232.
could then be controlled by pH-dependent release of the free
amine for reaction with the C2′ ester carbonyl.16,17 The
participation of the amine in ester cleavage could also be
modulated by conversion to simple amide or other less
reactive derivatives. To explore this approach, taxol was
initially converted to the C2′ chloroacetyl derivative 2
(16) Testa, B.; Mayer, J. M. Drug Metab. ReV. 1998, 30, 787-807.
(17) Recently, a similar approach was used as a solid-phase linker
strategy: Ando, H.; Manabe, S.; Nakahara, Y.; Ito, Y. Angew. Chem., Int.
Ed. 2001, 40, 4725-4728.
(15) Rothbard, J. B.; Garlington, S.; Lin, Q.; Kirschberg, T.; Kreider,
E.; McGrane, P. L.; Wender, P. A.; Khavari, P. A. Nature Med. 2000, 6,
1253-1257.
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(Scheme 1) in essentially quantitative yield by reaction with
chloroacetic anhydride. This process can be easily scaled
[∼10 mg to over 2 g], and the product can be obtained after
two aqueous washings (0.1 M HCl, bicarbonate). Derivative
2 obtained according to this procedure is sufficiently pure
for all further transformations.18
headgroup. All final products were isolated as white powders
after RP-HPLC purification and lyophilization. Conjugate 6
was efficiently converted to the acetate counterion form (8)
with the aid of an ion-exchange resin.23 Using the conditions
described for the synthesis of conjugate 6 allowed the
corresponding octa-(L)-arginine-based conjugate 9 to be
synthesized (63% yield) and converted to the acetate form
10 as described above. The coupling procedure was found
to be general for a variety of transporters.
While conjugates 3-10 incorporate different linker fea-
tures, they are all C2′ taxol derivatives. Therefore, a
conjugate was prepared that utilized the C7 hydroxyl group
as the point of attachment. The synthesis is depicted in
Scheme 3. Employing a TBS protecting group at the C2′
Attachment of 2 to a transporter was conducted in two
ways. Method A, preferred in cases when an N-terminal
cysteine residue of the transporter can be incorporated into
a solid-phase peptide synthesis, entails thioether formation
by halogen displacement with a cysteinyl sulfur. Alterna-
tively, method B involves a stepwise assembly. A sulfhydryl-
containing free acid is used in the thioether formation step,
and the resultant acid, following in situ activation, is coupled
to the N-terminal free amine of octa-arginine. Method B is
generally more flexible, since it allows for the use of any
sulfhydryl-containing acid.19 Method B is favored in a
process that relies on octa-arginine synthesis via segment-
doubling methodology. Under optimized conditions, both
methods are high-yielding and reliable.
Scheme 3. Synthesis of Conjugate 13a
Illustrative of method A is the synthesis of conjugate 3.
The efficiency of this single-step process is dependent on
the concentration of reactants, molar equivalents of tertiary
amine base, and the temperature. High concentrations [∼0.1
M] and low temperatures [0 °C] favor the intermolecular
coupling and suppress the intramolecular release of taxol.
An excess of amine base (beyond 1.5 equiv) leads to
premature taxol release as expected from the pH-dependent
mechanism of release. The conjugates 4 and 5 were
synthesized in the presence of larger DIEA amounts, since
no release of parent drug under the conjugation conditions
was observed (method A). The chemoselectivity of this
approach presumably results from the higher acidity of the
thiol relative to the ammonium group and the greater
nucleophilicity of the resultant thiolate.20
a Reagents and conditions: (a) (ClAc)2O, DIEA, DCM, rt, 94%;
(b) HF/pyr, THF, rt, 74%; (c) NH2-(L)-Cys-(D)-Arg8-CONH2
(9TFA), DIEA, DMF, rt, 24%.
alcohol,24 the chloroacetylation at the C7 hydroxyl was
performed in high yield by using an excess of acylating agent
and amine base. Deprotection of the TBS group with HF/
pyr yielded the coupling precursor 12. The conjugation to
the thiol of NH2-(L)-Cys-(D)-Arg8-CONH2 was considerably
slower and yielded the product in 24% along with unreacted
starting material.
In all, nine conjugates were prepared. As expected from
the solubilizing effect of the arginine-based transporters, all
were readily water soluble. In the case of 6, for which
solubility was measured, the conjugate was >1000 times
more soluble in water than taxol.25 The half-life for release
of free taxol was determined for each conjugate under
physiological conditions (e.g., PBS-buffer, pH ) 7.4, T )
37 °C). Taxol was released from all conjugates without
detectable intermediates and characterized by MS analysis
and reversed-phase HPLC retention time in comparison to
an authentic sample. The results of this study are summarized
in Table 1.
The synthesis of conjugate 6 is representative of method
B, which is a three-step one-flask operation. The initial
displacement was conducted with 1 molar equiv of N-Boc-
Cys-OH and 2 equiv of DIEA. This ratio accelerated the
thioether formation and allowed for the consecutive car-
boxylate activation with isobutylchloro formate. This cou-
pling reagent was well suited for our study and allowed for
a selective amide bond formation to the primary N-terminal
amine of the octa-arginine.21 No interference of the guani-
dinium headgroups of arginine was observed during the
conjugate synthesis and purification, as expected from the
strongly basic character and tight association of the guani-
dinium ions with TFA counterions.22 The tertiary amine
DIEA is not sufficiently basic to deprotonate the guanidinium
As desired for in vitro and in vivo evaluation, the half-
lives of these conjugates vary over a range of times from 1
min up to almost 4 h under the described conditions.
Compound 3 released taxol almost instantaneously. A facile
hydrolytic cleavage at the C2′ ester was observed for
(18) This compound was mentioned in ref 8c but was described to be
too unstable for synthetic application in an aqueous environment.
(19) Free primary and secondary amines are excluded.
(20) Textbook values for Cys: RNH3+, 10.3; SH, 8.3. For a recent study
about the influence of charge environment on the pKa of cysteine thiols,
see: Lutlof, M. P.; Tirelli, N.; Cerritelli, S.; Cavalli, L.; Hubbell, J. A.
Bioconjugate Chem. 2001, 12, 1051-1056.
(21) In general, the use of DCC gave reduced yields in these coupling
reactions.
(23) For most biological applications TFA counterions are undesirable.
(24) Magri, N. F.; Kingston, D. G. I. J. Nat. Prod. 1988, 51, 298-306.
(25) Aqueous solubility: Taxol < 4 µg/mL; conjugate 6 > 100 mg/mL.
For the taxol value, see ref 10e.
(22) Plapinger, R. E.; Nachlas, M. M.; Seligman, M. L.; Seligman, A.
M. J. Org. Chem. 1965, 30, 1781-1785.
Org. Lett., Vol. 5, No. 19, 2003
3461

Table 1. Half-Lives of Selected Conjugatesa
compound
half-life
compound
half-life
3
4
5
6
1 min
61 min
103 min
107 min
7
8
9
140 min
104 min
107 min
214 min
13
a Measurements were performed in PBS buffer at pH ) 7.4 and T ) 37
°C. Only starting material, released peptide, and taxol were observed under
these conditions.
Figure 2. Dependence of the half-life of conjugate 6 on the pH.
Half-lives are given in minutes.
conjugates 4-8, with half-lives ranging from 1 to 2.5 h. The
incorporation of a sulfur heteroatom at the R-position of the
C2′ ester led to increased hydrolysis rates. No effect of the
stereochemistry of the octa-arginine on the half-life was
detected. Compound 13 showed the longest half-life despite
the similarity in release mechanism to 3, indicating that the
position of cargo attachment also influences release rates.
The dependence of half-life on solution pH (adjusted PBS
buffer, 37 °C) was measured for compounds 3 and 6. The
results are given in Figures 1 and 2, respectively. The graphs
In conclusion, we have shown that conjugation of taxol
to arginine-based transporters provides conjugates with
greatly enhanced water solubility. A therapeutically mean-
ingful dose of taxol (300 mg) as its conjugate can be
dissolved in 10 mL of water. A significant aspect of this
work is the development of procedures for the conjugation
of water-soluble transporters with a complex drug cargo
possessing poor water solubility, a procedure that can be used
for related problems involving transporter-drug conjugate
synthesis. Additionally, a novel and general prodrug strategy
for the release of drugs incorporating a hydroxyl group has
been described. The rate of release of the free drug can be
tuned and controlled by the structural features of the linker.
Studies of these and related conjugates and their use in tissue
and animal uptake assays will be reported shortly.
Acknowledgment. Support for this work by grants
(P.A.W.) from the National Institutes of Health (CA31841,
CA31845) is gratefully acknowledged. The authors thank
D. Davalian, T. Flaherty, E. Kreider, S. Naganathan, L. Sista,
and L. Wright for valuable discussions.
Figure 1. Dependence of the half-life of conjugate 3 on the pH.
Half-lives are given in minutes.
clearly show a correlation of conjugate stability and pH. For
conjugate 3 the effect is very pronounced, since the proto-
nation state of the N-terminal amine is crucial for the
cyclization and taxol release. A steep increase of stability is
observed when adjusting the pH to 5 and below. A similar
trend is seen for conjugate 6.
Supporting Information Available: Experimental pro-
cedures for the preparation of compounds 2-13 and their
respective characterization data. This material is available
free of charge via the Internet at http://pubs.acs.org.
OL035234C
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