Synthesis of water-soluble dendrimers based on melamine bearing 16 paclitaxel groups

Article abstract of DOI:10.1021/ol7024907

(Chemical Equation Presented) The design, synthesis, and characterization of triazine dendrimers derivatized with the anticancer agent paclitaxel are described. The precursor generation two dendrimer 1 is prepared in six linear steps in 64% overall yield

Full text of DOI:10.1021/ol7024907

ORGANIC
LETTERS
2008
Vol. 10, No. 2
201-204
Synthesis of Water-Soluble Dendrimers
Based on Melamine Bearing 16
Paclitaxel Groups
Jongdoo Lim and Eric E. Simanek*
Department of Chemistry, Texas A&M UniVersity, College Station, Texas 77843-3255
simanek@tamu.edu
Received October 11, 2007
ABSTRACT
The design, synthesis, and characterization of triazine dendrimers derivatized with the anticancer agent paclitaxel are described. The precursor
generation two dendrimer 1 is prepared in six linear steps in 64% overall yield and presents 16 amines and two groups for radioiodination.
This macromolecule is subsequently derivatized with a paclitaxel conjugate to yield a generation three dendrimer, 2, which is then pegylated
in two steps. The pegylated final products, 4a and 4b, with molecular weights of 46 and 77 kDa, respectively, solubilize paclitaxel in water.
Pegylated dendrimer 4a is 30 wt % paclitaxel, 52 wt % PEG, and 18 wt % dendrimer. Target 4b is 18 wt % paclitaxel, 71 wt % PEG, and 11 wt
% dendrimer.
To date, various types of polymers have been investigated
as drug delivery systems.1 Examples include grafts,2 stars,3
multivalent polymers,4 block copolymers,5 dendronized
polymers,6 and dendrimers.7 Dendrimers are promising as
drug delivery vehicles by virtue of their multivalency, well-
defined and large globular structure, mono- or low polydis-
persity, and amenability to postsynthetic manipulation. We
and others believe that dendrimers may surmount barriers
to chemotherapy, including poor solubility of some agents
in physiological conditions, high systemic toxicity, poor
bioavailability, and low stability. Through rational design,
dendrimers might shift in vivo pharmacokinetics of the drug,
accelerate cellular uptake, prolong plasma half-life, and even
specifically target the diseased cells either actively (via
ligands) or passively (via the enhanced permeability and
retention (EPR) effect8 resulting from a leaky vasculature
and the lack of lymphatic drainage in solid tumor tissues).
We have chosen paclitaxel (Taxol) as our illustrative drug
for four reasons. First, paclitaxel is a clinically relevant broad
spectrum anticancer agent useful for treating tumors of the
ovaries, breast, head and neck, lung, and AIDS-related
Kaposi’s sarcoma.9 Second, it displays low solubility in water
(<0.1 µg/mL). Third, it is amenable to synthetic manipula-
tions (through esterification of the 2′-hydroxyl group) that
provide a mechanism for conjugation and release. Finally,
the target molecules can be compared to a variety of de-
livery vehicles which have been described, including emul-
sions,10 micelles,11 liposomes,12 nanoparticles,13 and other
dendrimers.14
(1) Duncan, R. Nat. ReV. Drug DiscoVery 2003, 2, 347.
(2) (a) Simon, M.; Wittmar, M.; Bakowsky, U.; Kissel, T. Bioconjugate
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10.1021/ol7024907 CCC: $40.75
© 2008 American Chemical Society
Published on Web 12/19/2007

The chemistry that we describe here is outlined in Scheme
1. The benefits of triazine chemistry derive from the stepwise
fashion that the chlorine atoms on triazine rings are sub-
stituted with amine nucleophiles.7c,15 The results are products
with compositional diversity. Dendrimer 1, a second genera-
tion dendrimer, possesses Bolton-Hunter-type groups ame-
nable to radioiodination.16 Reaction of 1 with a dichlorotri-
azine decorated with paclitaxel yields a generation three
dendrimer 2. Pegylation is performed in two steps to convey
biocompatibility and solubility to the hydrophobic triazine
dendrimer while simultaneously allowing us to tailor its size
and, accordingly, its biodistribution kinetics and fates.
Scheme 2. Synthesis of 1
Scheme 1. Summary of the Chemistries Reported Including
Paclitaxel Conjugation and Pegylation
Dichlorotriazine 7 is obtained by reacting 6 with cyanuric
chloride. Dimerization of 7 with piperazine yields 8. The
radioiodination group (prepared as previously reported)15d,19
is incorporated to give 9. Finally, the Boc groups of 9 are
quantitatively removed using trifluoroacetic acid (TFA).
Overall, 1 is synthesized from the protected amine in 64%
yield in six linear steps.
Our strategy for the covalent attachment of paclitaxel to
dendrimer 1 follows the precedented esterification of the 2′-
hydroxyl group.20 While both glutaric and succinic esters
have been pursued, Safavy and co-workers20a reported that
the glutaric ester linked conjugates displayed better antitumor
activity compared to that of the succinic ester linked con-
jugate. Our strategy rests on the preparation of a dichloro-
triazine modified with paclitaxel that can be used to react
with the amines of 1, giving 16 monochlorotriazines to be
further manipulated. The chemistry employed is outlined in
Scheme 3.
Target 1 is synthesized using a convergent route (Scheme
2).17 Intermediate 5 is prepared in one pot by the reaction of
cyanuric chloride with 2 equiv of the Boc-protected tri-
amine18 and subsequent reaction with the secondary amine
of 4-aminomethyl piperidine (4-AMP). Elaboration of 5 to
6 proceeds in a similar way: cyanuric chloride is first reacted
with 2 equiv of 5 and is followed by the addition of 4-AMP.
(10) Stevens, P. J.; Lee, R. J. Anticancer Res. 2003, 23, 4927.
(11) (a) Lee, S. C.; Huh, K. M.; Lee, J.; Cho, Y. W.; Galinsky, R. E.;
Park, K. Biomacromolecules 2007, 8, 202. (b) Shuai, X.; Merdan, T.;
Schaper, A. K.; Xi, F.; Kissel, T. Bioconjugate Chem. 2004, 15, 441.
(12) Ceruti, M.; Crosasso, P.; Brusa, P.; Arpicco, S.; Dosio, F.; Cattel,
L. J. Controlled Release 2000, 63, 141.
(13) Zhang, Z.; Feng, S.-S. Biomaterials 2006, 27, 4025.
(14) (a) Ooya, T.; Lee, J.; Park, K. Bioconjugate Chem. 2004, 15, 1221.
(b) Khandare, J. J.; Jayant, S.; Singh, A.; Chandna, P.; Wang, Y.; Vorsa,
N.; Minko, T. Bioconjugate Chem. 2006, 17, 1464.
(15) (a) Zhang, W.; Tichy, S. E.; Pe´rez, L. M.; Maria, G.; Lindahl, P.
A.; Simanek, E. E. J. Am. Chem. Soc. 2003, 125, 5086. (b) Steffensen, M.
B.; Simanek, E. E. Org. Lett. 2003, 5, 2359. (c) Steffensen, M. B.; Simanek,
E. E. Angew. Chem., Int. Ed. 2004, 43, 5178. (d) Lim, J.; Simanek, E. E.
Mol. Pharm. 2005, 2, 273. (e) Hollink, E.; Simanek, E. E. Org. Lett. 2006,
8, 2293.
(16) Bolton, A. E.; Hunter, W. M. Biochem. J. 1973, 133, 529.
(17) Grayson, S. M.; Frechet, J. M. J. Chem. ReV. 2001, 101, 3819.
(18) Westerberg, D. A.; Carney, P. L.; Rogers, P. E.; Kline, S. J.; Johnson,
D. K. J. Med. Chem. 1989, 32, 236.
The conjugate, 13, is prepared in four steps in 60% overall
yield. Paclitaxel is reacted with glutaric anhydride in the
presence of pyridine to give 2′-glutarylpaclitaxel 10. NHS
ester 11 is obtained by reaction of 10 with N-succinimidyl
(19) Maeda, D. Y.; Berman, F.; Murray, T. F.; Aldrich, J. V. J. Med.
Chem. 2000, 43, 5044.
(20) (a) Safavy, A.; Georg, G. I.; Velde, D. V.; Raisch, K. P.; Safavy,
K.; Carpenter, M.; Wang, W.; Bonner, J. A.; Khazaeli, M. B.; Buchsbaum,
D. J. Bioconjugate Chem. 2004, 15, 1264. (b) Majoros, I. J.; Myc, A.;
Thomas, T.; Mehta, C. B.; Baker, J. R., Jr. Biomacromolecules 2006, 7,
572.
202
Org. Lett., Vol. 10, No. 2, 2008

Scheme 3
diphenylphosphate (SDPP).21 This ester is treated with an
excess of 1,3-diaminopropane at low temperature to afford
the amine 12. The linear, flexible diamine was chosen to
reduce steric hindrance. Finally, the reaction of amine 12
with cyanuric chloride using DIPEA as a base yields the
desired dichlorotriazine 13.
The reaction of dendrimer 1 with 13 gives 2 (Scheme 1).
The resulting poly(monochlorotriazine) dendrimer undergoes
nucleophilic aromatic substitution with 4-AMP to afford 3.
Steric hindrance prevents complete pegylation of 3. Pegy-
lation with NHS-mPEG 2 and 5 kDa gives 4a (MW ∼ 46
kDa for 14 PEG chains) and 4b (MW ∼ 77 kDa for 12 PEG
chains), respectively.
With the exception of reactions involving 1-4, most
reactions are readily monitored by thin layer chromatography.
NMR spectroscopy and mass spectrometry are useful over
the entire range of targets. The MALDI-TOF MS data of
intermediates 5-13 were consistent with single chemical
entities: these spectra showed peaks derived from a single
Figure 2. MALDI-TOF spectrum of 2. Peaks appearing at lower
m/z reflect either incomplete reaction (inconsistent with NMR data)
or decomposition during ionization.
compound corresponding to [M + H]+, [M + Na]+, and [M
+ K]+. A ladder of peaks from loss of Boc groups during
the ionization was observed for 5-9.
Targets 2-4 are more difficult to characterize. Figure 1
shows the assignment and integration of the 1H NMR
spectrum of 2 which suggests 16 molecules of paclitaxel are
conjugated. Obtaining consistent MALDI-TOF MS from the
hydrophobic dendrimer 2 was challenging. The spectrum in
Figure 2 shows a reasonable agreement between the calcu-
lated exact mass 21 038 and the observed mass 21 114 (m/
z). Due to resolution in this mass range, the ladder of peaks
occurring at lower mass cannot be uniquely attributed to
incomplete reaction with 13 or loss of paclitaxel due to the
labile ester link during ionization.
1
Both 4a and 4b were analyzed with GPC, H NMR, and
MALDI-TOF MS. The pegylated dendrimers are readily
soluble (>20 mg/mL) in aqueous media, allowing purifica-
tion by dialysis. Separately, we find that pegylation with 0.6
Figure 3. The analytical GPC chromatogram obtained using 0.1
M NaNO3 (aq) as an eluent with a RI detector: (a) before (dotted
line) and after (solid line) dialysis purification of 4a; (b) GPC traces
of 4a and 4b.
Figure 1. 1H NMR assignment and integration of 2 in the expanded
region of 4.8-8.2 ppm. The spectrum was obtained in CDCl3/
MeOH-d4 (10:1).
Org. Lett., Vol. 10, No. 2, 2008
203

and 1.2 kDa PEG groups leads to products that are not water-
soluble. The dendrimers were analyzed using GPC in 0.1 M
NaNO3 (aq) with a refractive index (RI) detector (Figure 3).
1H NMR spectra of 4a and 4b shown in Figure 4 display
in the intervening steps including dialysis. The molecular
weight was determined by MALDI-TOF MS: 46 100 Da
for 4a and 77 400 Da for 4b. This corresponds to macro-
molecules that are 30 wt % (4a) and 18 wt % (4b) paclitaxel.
In summary, we have described a drug delivery vehicle,
2, that has Bolton-Hunter-type groups to report biodistri-
bution and multiple paclitaxel groups attached by labile ester
linkages. The precursor dendrimer 1 was prepared at 64%
overall yield from the bisprotected amine, and the modifica-
tion of paclitaxel proceeded in four steps with 60% overall
yield. Both mass spectrometry and NMR spectroscopy of 2
show that the drug conjugate contains 16 molecules of
paclitaxel, indicating full coupling of 1 with the modified
drug. After pegylation, we obtained two dendrimers 4a and
4b with a molecular weight of about 46 and 77 kDa,
respectively. The degree of drug loading in 4a at 20 mg/mL
corresponds to the concentrations achieved in Cremophor
EL (the clinically relevant castor oil derivative used for
solubilization), namely, 6 mg/mL (before the requisite
dilution with saline for intravenous delivery).22
unique lines for the aromatic protons of paclitaxel (δ 7.25-
Acknowledgment. Support from the National Institutes
of Health (NIGMS 65460) and the Robert A. Welch
Foundation (A 1439) is acknowledged.
Figure 4. 1H NMR of the pegylated paclitaxel conjugates 4a and
4b purified from dialysis. The spectra were obtained in CDCl3/
MeOH-d4 (10:1).
Supporting Information Available: Detailed experi-
mental procedures, NMR, and mass spectrometry data. This
material is available free of charge via the Internet at
http://pubs.acs.org.
8.09 ppm), the radioiodination group (δ 6.68 and 6.96 ppm
for ortho and meta protons, respectively), and the PEG chains
(δ ∼3.6 ppm). The integration values are similar to those of
dendrimer 2, suggesting little hydrolysis of paclitaxel occurs
OL7024907
(21) Ogura, H.; Nagai, S.; Takeda, K. Tetrahedron Lett. 1980, 21, 1467.
(22) Goldspiel, B. R. Ann. Pharmacother. 1994, 28, S23-S26.
204
Org. Lett., Vol. 10, No. 2, 2008