Synthesis and Evaluation of Paclitaxel-Loaded Gold Nanoparticles for Tumor-Targeted Drug Delivery

Article abstract of DOI:10.1021/acs.bioconjchem.6b00405

The synthesis of a series of thiolated paclitaxel analogs is described as part of a novel nanomedicine program aimed at developing formulations of paclitaxel that will bind to gold nanoparticles for tumor targeted drug delivery. Preliminary evaluation of the new nanomedicine composed of 27 nm gold nanoparticles, tumor necrosis factor alpha (TNFα), thiolated polyethylene glycol (PEG-thiol), and one of several thiolated paclitaxel analogs is presented.

Full text of DOI:10.1021/acs.bioconjchem.6b00405

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Article
Synthesis and Evaluation of Paclitaxel-Loaded Gold
Nanoparticles for Tumor-Targeted Drug Delivery
Giulio Franco Paciotti, Jielu Zhao, Shugeng Cao, Peggy J Brodie, Lawrence Tamarkin,
Marja Huhta, Lonnie D Myer, Jay Friedman, and David George Ian Kingston
Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.6b00405 • Publication Date (Web): 30 Sep 2016
Downloaded from http://pubs.acs.org on October 3, 2016
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Synthesis and Evaluation of Paclitaxel-Loaded Gold
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Nanoparticles for Tumor-Targeted Drug Delivery
Giulio F. Paciotti,§ Jielu Zhao,† Shugeng Cao,† Peggy J. Brodie,† Lawrence Tamarkin,§ Marja
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Huhta,§ Lonnie D. Myer,§ Jay Friedman,§ and David G. I. Kingston*,†
†Department of Chemistry and the Virginia Tech Center for Drug Discovery, Virginia Tech, Blacksburg,
Virginia 24061, United States
§CytImmune Sciences Inc., 15010 Broschart Road, Rockville, MD 20850.
Supporting Information
ABSTRACT: The synthesis of a series of thiolated paclitaxel analogs is described as part of a
novel nanomedicine program aimed at developing formulations of paclitaxel that will bind to
gold nanoparticles for tumor targeted drug delivery. Preliminary evaluation of the new
nanomedicine comprised of 27 nm gold nanoparticles, tumor necrosis factor alpha (TNF),
thiolated polyethylene glycol (PEG-Thiol) and one of several thiolated paclitaxel analogs is
presented.
 INTRODUCTION
Chemotherapy has been and continues to be one of the most effective ways to treat
cancer, but many of the chemotherapeutic drugs used are cytotoxic and have significant side
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effects. Among chemotherapy drugs paclitaxel is one of the most widely used and effective
agents, and it is the first-line treatment for breast, ovarian, lung, and colon cancer. Its mechanism
of action involves binding to microtubules and stabilizing them, ultimately causing apoptosis.1, 2
It does, however have significant side effects, including alopecia, nausea and vomiting,
myelosuppression,3 and peripheral neuropathy.4 These side effects are caused in large part
because paclitaxel is given by intravenous infusion, and thus has the potential to be distributed
throughout the body and not just to the tumor site. In spite of these problems a 2008 review
stated “Paclitaxel and docetaxel are considered fundamental drugs in the treatment of breast
cancer,”5 and a 2012 review stated “Today, paclitaxel and docetaxel are widely prescribed
antineoplastic agents for a broad range of malignancies including lung cancer, breast cancer,
prostate cancer, Kaposi’s sarcoma, squamous cell carcinoma of the head and neck, gastric
cancer, esophageal cancer, bladder cancer, and other carcinomas. Although very active clinically,
paclitaxel and docetaxel are associated with many serious side effects, which often preclude
continued use of these agents in patients. A number of these side effects have been associated
with the solvents used for the dilution of these antineoplastic agents.”6 The major problem with
paclitaxel is thus a drug delivery problem; if it could be delivered specifically to the tumor, its
side effects would be minimized, potentially making it even more effective.
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This concept of targeting chemotherapeutic drugs in general and paclitaxel in particular
to tumors is not a new one, and numerous publications on different approaches have appeared in
the last few years. These include antibody-drug conjugates,7-9 and various nanoparticle-based
approaches.10 The advantage of the nanoparticle approach is that a measure of selectivity is
assured because of the enhanced permeability and retention (EPR) effect,11 which is primarily
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due to the preferential extravasation of nanoparticles through the leaky vasculature of tumors
over the tight vasculature of normal tissue.
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A fundamental issue in nanomedicine development is whether and to what extent the
EPR effect exists in human cancers, as over time it may become a self-limiting phenomenon.
One such limitation is that the combination of fluid leaking into the tumor coupled with impaired
lymphatic vessels, which drain fluid from the tumor, combine to pressurize the tumor. This
pressure, known as tumor interstitial fluid pressure (IFP), acts as a physical barrier that prevents
or limits the ability of cancer therapies, potentially including nanomedicines, from penetrating
the tumor. Thus it is not surprising that on average only 0.7% of the administered dose of
nanoparticle drugs is delivered to solid tumors.12 This number is however still ten to one
hundred-fold higher than that of naked drug molecules, and drug delivery efficiencies are closer
to 10% for nanomedicines in clinical trial.13
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Several nanomedicines that utilize the EPR effect to selectively deliver paclitaxel or its
analog docetaxel have been proposed. In the case of paclitaxel the albumin-bound formulation
Abraxane® and the polymeric micellar nanoparticle formulation Genexol-PM are in clinical use,
and the micellar nanoparticle formulation NK105 is in Phase III clinical trials.14, 15 However,
these formulations do not completely resolve the toxicity issues associated with paclitaxel, as
indicated by the fact that Abraxane® shows significant neuropathy.16
Various studies have also appeared of paclitaxel linked to iron oxide nanoparticles,17 to
gold nanoparticles,17-20 or to fullerenes,21 but none of these have resulted in any published
clinical or preclinical studies. Various other paclitaxel delivery systems are described in a recent
review.22 The use of nanoparticles for the delivery of docetaxel has also been reviewed recently
and thus will not be discussed further.23
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Given the many potential benefits of selective delivery of paclitaxel to tumors, we
initiated a research program to explore its delivery by nanoparticles. We selected gold
nanoparticles (AuNP) for this tumor-targeted delivery because they are readily prepared in a
range of sizes, they are chemically stable and biocompatible, and they can be functionalized with
a variety of ligands.24-26 As stated in a recent review “Because of their facile surface chemistry,
gold nanoparticles can act as artificial antibodies whose binding affinity can be precisely tuned
by varying the density of binding ligands on their surfaces.”27 In particular, they can be
functionalized with tumor necrosis factor (TNF) and polyethylene glycol to generate a potent and
biocompatible tumor-targeting drug delivery vehicle.28-30
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The cytokine TNF has several effects, including reduction of IFP31 and apoptosis of
cancer cells,32 but it is highly toxic, and cannot be used systemically. Its value has been
demonstrated by its clinical use in the isolated limb perfusion (ILP) surgical procedure involving
TNF and chemotherapy. In this technique, which has been approved in Europe since 1998, TNF
followed by a chemotherapy, such as melphalan or doxorubicin, are sequentially perfused via
complex surgery into the tumor-burdened limb, resulting in a 60-85% complete local response
rate in cancer patients with refractory tumors.33, 34 The localized delivery of TNF induces
vascular leak in the tumor, reduction in the tumor IFP,31 and enhanced tumor uptake of follow-on
chemotherapy.35 However, ILP is not applicable to most tumors, either primary or metastatic,
and the potential benefits of TNF are thus only available to a small subset of cancer patients, but
the success of ILP with TNF followed by chemotherapy suggests a strategy for developing
tumor-targeting nanomedicines for cancer treatment.
AuNPs provide an attractive alternative to the use of ILP as a means of selective delivery
of TNF to tumors. In a key study of this approach, AuNPs were functionalized with TNF, bound
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through its cysteine residues, and with sulfhydryl-polyethylene glycol (PEG-Thiol) to make them
able to target tumors and to be undetectable by the immune system, respectively.36 The resulting
nanomedicine, termed CYT-6091, has to date demonstrated many of the anti-tumor effects of
TNF without inducing its toxic side effects. For example, by targeting the tumor vasculature,
CYT-6091 induces the selective disruption of the tumor vascular bed37 and increases the uptake
of contrast MRI contrast agents, a surrogate for the uptake of chemotherapy in solid tumors.38
Following vascular disruption CYT-6091 reduces tumor interstitial fluid pressure in some but not
all tumors tested,39 and increases the efficacy of energy based treatments such as radio and
thermal based therapies40 as well as chemotherapy.41 Evaluation of CYT-6091 in MC38 tumor-
bearing mice indicated that it selectively induced tumor neovascular permeability.37 Finally, in a
pivotal Phase I clinical study CYT-6091 selectively accumulated in patient tumors, and increased
overall systemic exposure to the cytokine without inducing the dose limiting toxicities of
hypotension and hepatotoxicity associated with systemic TNF administration.28 These findings
suggest that CYT-6091 may be replicating the first arm of the ILP procedure to effectively
deliver the benefits of TNF to a broad spectrum of cancers.
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Although these findings are highly significant, both CYT-6091 and the ILP protocol
require the use of a separate chemotherapeutic agent, which is not targeted specifically to the
tumor. Consequently, linking a chemotherapeutic agent directly to the TNF-PEGylated-AuNP
construct would target both TNF and the chemotherapeutic agent to the tumor and thus reduce
side effects associated with untargeted agents. We selected paclitaxel as the chemotherapeutic
agent of choice because of its premier place among cancer drugs. Portions of this work are the
subject of a US Patent.42
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The design of a paclitaxel analog for binding to gold nanoparticles for drug delivery must
meet three basic requirements: it must contain a thiol or substituted thiol group positioned to
bind to the gold nanoparticle, it must be stable in whole blood so that it does not release the drug
in the circulation, and it must be capable of release from the gold nanoparticle to regenerate
paclitaxel once it reaches the tumor. This last criterion is important if the objective is to deliver
native paclitaxel to the tumor. In earlier work we designed paclitaxel analogs such as 1 (Fig. 1)
that could be linked to gold nanoparticles,29 but did not readily regenerate paclitaxel from the
nanoparticle since the linkage was the relatively stable C-7 ester,43 and their efficacy was thus
limited. A similar concern applies to the well-characterized paclitaxel-linked AuNP (2) prepared
by Gibson et al.,18 since these were also linked to the nanoparticle through a relatively stable
ester linkage.
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Figure 1. Previous thiolated paclitaxel derivatives.
In this work we describe the synthesis and initial evaluation of four paclitaxel analogs
that meet the criteria desired above and are suitable for development as TNF-PEGylated-AuNP-
linked drugs for selective delivery to tumors, and provide preliminary biological data on two of
them.
 RESULTS
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Synthesis of New Thiolated Paclitaxel Analogs. The series of paclitaxel analogs 3 - 6
(Fig. 2) was designed with thiopyridyl or lipoic acid moieties at the C-7 and C-2' positions as the
linkers to the AuNP. By design each of these compounds is converted to paclitaxel at the tumor
site by either a reductive or a hydrolytic cleavage mechanism. The C-7 derivatives 3 and 4 were
designed with a C-7 acyloxyalkoxy carbonate group, since this group had been shown to be
readily hydrolyzed under mildly acidic conditions to regenerate paclitaxel,44 and would thus be
expected to hydrolyze in the acidic tumor environment.45 The C-2' lipoic acid derivative 6
likewise contained an acyloxyalkoxy carbonate linkage which would be expected to hydrolyze to
2'-O-succinylpaclitaxel, which would then be hydrolyzed to paclitaxel.46, 47 The 7-O-thiobenzyl
derivative 5 was designed to undergo reductive cleavage from the AuNP by glutathione in the
reducing tumor environment and to release native paclitaxel.48 Glutathione is the most abundant
intracellular thiol, with concentrations ranging from 0.2–10 mM,49 and with concentration in
tumors higher than in normal tissue.50 And, this range of concentrations is far higher than the
2.09 ± 1.14 M found in human plasma,51 indicating that a compound such as 5 should be
reasonably stable in plasma but should undergo reductive cleavage in the tumor.
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Figure 2. Paclitaxel derivatives 3 – 6.
Compound 3 was prepared from carboxylic acid 8 and paclitaxel derivative 11 (Scheme 1).
Compound 11 was prepared from 2'-(O-tbutyldimethylsilyl)paclitaxel46 by the reported
procedures.52 Acid 8 was prepared from commercially available acid 7 and 2,2'-dipyridyl
disulfide in one step. Compound 11 was then refluxed with NaI in acetone to carry out halogen
exchange, followed by subsequent SN2 reaction with acid 8 to produce the desired analog 3 in
60% yield.
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Scheme 1. Synthesis of paclitaxel derivative 3a
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a(a) 2,2'-dipyridyldisulfide, MeOH, AcOH, 55%; (b) chloromethyl chloroformate, pyridine, CH2Cl2, 12 h, 75%; (c)
HF-pyridine, THF, rt, 12 h, 90%; (d) NaI, acetone, reflux 10 h; (e) 8, K2CO3, 18-crown-6, benzene, 2 h, 76 °C, 60%.
Compound 4 was prepared by reaction of compound 11 with racemic lipoic acid using a
similar procedure to that used for the conversion of 11 to 3 (Scheme 2).
Scheme 2. Synthesis of paclitaxel derivative 4a
a(a) NaI, acetone, reflux 10 h; (b) Lipoic acid, K2CO3, 18-crown-6, benzene, 3.5 h, 65 °C, 78%.
Paclitaxel analog 5 was prepared (Scheme 3) by coupling precursor 1253 with the
corresponding alcohol 13,48 followed by deprotection of the resulting TBDMS ether 14.
Scheme 3. Synthesis of paclitaxel derivative 5a
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a(a) 13, DMAP, CH2Cl2, 18 h, 88%; (b) HF-pyridine, THF, rt, 16 h, 92%.
Compound 6 is the only analog in this series that possesses a C-2' linker. Reaction of 2'-
succinyl paclitaxel 1546 with chloromethyl chlorosulfate under phase-transfer conditions
generated the chloromethyl ester 16. Compound 16 was then treated with lipoic acid as
previously described to furnish analog 6 (Scheme 4).52
Scheme 4. Synthesis of paclitaxel derivative 6a
a(a) Chloromethyl chlorosulfate, NaHCO3, TBAH, H2O/CH2Cl2, 46%; (b) Lipoic acid, 18-crown-6, NaI, benzene,
reflux, 60%.
The gold nanoparticle-bound version of 5 consisting of compound 5, PEG-Thiol, and TNF
bound to gold nanoparticles was designated CYT-21625, and the similar nanoparticle
formulation of 3 was designated CYT-20203.
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Paclitaxel Release from Analogs in Buffer, Media, and Whole Blood. The stability
of the linkers used to connect paclitaxel with the gold surface is a key for the gold nanoparticle-
based system to achieve selective delivery of paclitaxel to tumors. Ideally the linkers should be
stable as the nanomedicine travels through the circulatory system, but should be cleaved after
entering the tumor to release paclitaxel. As noted earlier, analog 4 was designed to release
paclitaxel under acidic conditions, so an initial investigation of its stability in buffer solution at
the physiological pH 7.4 was carried out. Analog 4 was dissolved in a 3:1 mixture of methanol
and PBS buffer (pH 7.4), with the methanol used to increase the solubility of the analog.
Aliquots were taken at various time points and the concentrations of the analog and paclitaxel
were determined by quantitative HPLC analyses. The analog was gradually hydrolyzed within 24
hours in the buffer solution and paclitaxel was generated slowly as the only product (Figure S1).
A linear regression analysis indicated pseudo 1st order kinetics, and a half-life of analog 4 of 3.2
h at pH 7.4 was calculated. This study indicated that analog 4 was too labile at physiological pH
to be suitable as an AuNP drug candidate. Analogs 3 and 6 were not evaluated in this study. In
the case of analog 6, the relative ease of hydrolysis of 2' esters of paclitaxel43, 47 combined with
the acid-labile diacylmethylene linkage, indicated that this compound would most probably be
too labile for use as a drug candidate. Analog 3 was not evaluated for paclitaxel release in this
study, but it was evaluated in one experiment as the nanoparticle bound form CYT-20203 (see
below).
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Analog 5 was designed to release paclitaxel under reductive rather than hydrolytic
conditions, and so a time course study with analysis by HPLC was carried out for this compound
to assess the release of paclitaxel in the absence and in the presence of a 3-fold excess of
dithiothreitol (DTT). In bicarbonate buffer without DTT 5 released less than 10% paclitaxel after
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24 h, rising to 50% after 72 h (Table 1 and Figures S2 and S3). Treatment of 5 in buffer with a
threefold excess of DTT resulted in 90% conversion to paclitaxel within 60 min, comparable
with the results for the release of mitomycin from similar disulfides.48
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Table 1. Percent Conversion of 5 and CYT-21625 to Paclitaxel in
Buffer and in Rabbit Whole Blood
Incubation time (h)
Analyte
Medium
Buffer
0
1
4
4
-
24
48
72
0.3
4.1
4.2 14.4 50.4
5
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Buffer + DTT NDa 90
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Blood
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Rabbit Whole
Bloodb
CYT-
21625
0.1 0.1 1-2 NDa NDa
aNot determined.
bIn rabbit whole blood significant release of paclitaxel was only
achieved by pretreating the sample with BME
These results thus indicated that paclitaxel analog 5 was likely to be stable in blood but
would release paclitaxel in the reducing tumor environment.
Studies of Gold-Bound Analogs. The nanoparticle drug construct CYT-21625 was
prepared by binding analog 5, TNF, and PEG-Thiol to gold nanoparticles as described in the
experimental section. The construct CYT-20203 was prepared similarly from analog 3. CYT-
21625 was added to rabbit blood, and the paclitaxel released was analyzed by HPLC. No
conversion to paclitaxel was observed over a 24 h time period. Only by addition of excess -
mercaptoethanol (BME) to the blood sample was paclitaxel released from the nanomedicine,
although the amount was not quantified.
CYT-21625 was also treated in bicarbonate buffer with BME and various concentrations of
MeOH to assess the influence of solvent polarity on paclitaxel release. The amount of paclitaxel
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released from the nanomedicine was measured by quantitative RP-HPLC, while particle
agglomeration was measured by determining the decrease in absorbance of the particle
preparation at 525 nm. The results are shown in Table 2. Eliminating MeOH from the releasing
buffer significantly reduced the ability of BME to release paclitaxel from CYT-21625. These
data are consistent with the inability of BME to induce particle agglomeration and precipitation.
Adding 50% MeOH to the releasing buffer resulted in increased amounts of paclitaxel released
from CYT-21625 as well as MeOH-dependent particle agglomeration (Figure S2).
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Table 2. Effect of MeOH on BME-induced release of
compound 5 from CYT-21625 and on BME induced
particle agglomeration
Releasing Buffera
Compound Percent
5 released Loss in
(µg/mL)b,c OD 525 nm
MeOH (%)
BME (µM)
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-30
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-39
-54
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a Base buffer included Na2CO3 and NaHCO3
bConcentration of Compound 5 (detected as paclitaxel)
following release from the nanoparticle
cConcentration of paclitaxel when particles fully precipitated
= 40 µg/mL
The data presented in Table 2 are consistent with the hypothesis that the binding of the
paclitaxel analogs onto the particle surface orients them so that they form a hydrophobic layer on
the particle surface (Figure S3). The absence of MeOH from the releasing buffer is then
hypothesized to reduce the ability of the reducing agent BME from accessing the disulfide group
on the particle surface, thus preventing the release of the paclitaxel analogs as well as blocking
BME-induced particle agglomeration. Since blood is a highly buffered homeostatic environment
the hydrophobic layers serve to prevent release of the particle bound analog in the circulation
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(Table 1). However, we believe that the reductive tumor environment coupled with the potential
disruption of the tumor vasculature and tumor necrosis mediated by TNF leads to an eventual
breakdown of this layer and release of the particle bound chemotherapeutic.
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Biological Evaluation of the Thiolated Analogs. The antiproliferative activities of
analogs 3 - 6 were evaluated against the A2780 human ovarian cancer cell line (Table 3).
Analogs 3, 4, and 6 were slightly more potent than native paclitaxel, but analog 5 was
significantly less potent. These differences are presumably due to the lability of analogs 3, 4, and
6 as compared with the stability of analog 5 in buffer. The antiproliferative activities of
nanoconstructs CYT-20203 and CYT-21625 were also determined. As with the unbound
analogs, CYT-20203 had similar activity to native paclitaxel, but CYT-21625 was almost four-
fold less potent, consistent with its greater stability.
Table 3. Antiproliferative activities of paclitaxel and analogs against the A2780 cell line
Compound
IC50 (nM)
paclitaxel
15 ± 1a
3
4
5
6
CYT-20203 CYT-21625
6.7 ± 2.2 56 ± 30
2.8 ± 0.3
4.5 ± 0.4
34 ± 18
6.6 ± 1.7
aData from reference 54
These results taken together indicate that although analogs 3, 4, and 6 are most probably not
suitable for use as AuNP drug candidates, because of their potential lability at physiological pH,
analog 5 does have the right combination of stability in buffer and lability in the presence of
DTT to serve as a drug candidate. The nanoparticle formulation CYT-21625 was thus
investigated in more detail.
Pharmacokinetics and Tumor Accumulation of Paclitaxel, Compound 5 and
CYT-21625. The data reported above indicate that compound 5 is rapidly converted to
paclitaxel in rabbit whole blood, while CYT-21625 exhibits little to no release and conversion of
the particle bound compound 5 to paclitaxel. A similar study was then carried out in mice.
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B16/F10 tumor burdened mice were injected with 5 µg of compound 5 or CYT-21625 containing
the same amount of 5, and whole blood samples were collected at intervals and divided into 2
aliquots. The first aliquot was analyzed directly without any additional treatment while the
second aliquot was treated with BME. Both samples were analyzed for paclitaxel content by
ELISA. Consistent with the previous data compound 5 was rapidly degraded to the paclitaxel
parent in the circulation, but only marginal paclitaxel release from CYT-21625 was observed
(Figure 3A). In a second experiment (Figures 3B and 3C) either 50 µg of either paclitaxel or
CYT-21625 containing the same amount were intravenously injected into B16/F10 tumor
burdened C57Bl/6 mice. At 1, 3, 6, and 15 h a group of animals (n = 3 per time point) were
sacrificed and whole blood and tumors collected. The whole blood was processed as described
above while tumors were first homogenized and then treated identically to the whole blood
samples. Tumor measurements were normalized for protein content. The data (Figures 3B and
3C) show that CYT-21625 increased both the pharmacokinetic exposure and tumor uptake of
paclitaxel as compared to native drug. In addition, CYT-21625, by virtue of being a PEGylated
nanomedicine, significantly increased pharmacokinetic exposure, as measured by terminal half-
life and area under the curve, when compared to native paclitaxel (Figure 3B).
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Consistent with previous results with CYT-609128, 36 we observed that TNF mediated
targeting of CYT-21625 to B16-F10 solid tumors. Figure 3C demonstrates that CYT-21625
delivered significantly more paclitaxel (both free and bound) to B16-F10 tumors when compared
to the native drug. Initially we observed a rapid uptake of CYT-21625 into B16-F10 solid
tumors and a clear latency between its arrival at the tumor and the release of compound 5 from
the particle and its conversion to paclitaxel.
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This observation supports the theory that CYT-21625 mimics the entire surgical
procedure of ILP on a single gold nanoparticle. As shown in Figure 3D, a control preparation of
5 bound to pegylated gold but without the TNF targeting molecule exhibits very similar uptake
into B16/F10 tumors as native paclitaxel. These data are consistent with those reported by
Paciotti et.al.36 who demonstrated that passive targeting of MC38 colon tumors, for example by
the EPR effect, is ineffective and active targeting was only achieved by incorporating TNF into
the gold nanoconstructs. Since CYT-21625 was engineered to deliver therapeutic doses of both
TNF and paclitaxel it is possible that TNF is not only targeting its receptor on the vascular
endothelium but also inducing many of mechanisms demonstrated for CYT-6091.28, 36
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10000
100000
75000
CYT-21625 Total
CYT-21625 Released
Compound 5 Total
1000
100
Compound 5 Converted
50000
25000
0
CYT-21625 Total
CYT-21625-Released
Paclitaxel
A.
B.
0
6
12
TIme (H)
18
24
0.0
0.5
1.0
1.5
Time (H)
2.0
2.5
3.0
3.6
80
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10
0
2.7
1.8
0.9
0.0
CYT-21625
JZ-21625 Bound to PEGylated Gold
Paclitaxel
CYT-21625 Total
CYT-21625 Released
Paclitaxel
C.
D.
0
1
2
3
4
5
0.0
6.0
12.0
TIme (H)
18.0
24.0
Time (H)
Figure 3. Pharmacokinetics and tumor accumulation profiles of compound 5, CYT-21625 and paclitaxel in tumor
bearing mice. Figure 3A: levels of CYT-21625 and 5 in B16/F10 tumor burdened mice after injection with 5 µg of
compound 5 or the same dose as CYT-21625. Figure 3B: Blood levels of CYT-21625, of paclitaxel released from
CYT-21625 after treatment with DTT, and of native paclitaxel after treatment of tumor burdened C57Bl/6 mice with
50 µg of either native paclitaxel or the equivalent paclitaxel dose on CYT-21625. Figure 3C: Tumor levels of CYT-
21625, of paclitaxel released from CYT-21625 after treatment with DTT, and of native paclitaxel from a similar
experiment as described for Figure 3B. Figure 3D: Tumor levels of CYT-21625, compound 5 bound to PEGylated
gold nanoparticles, and native paclitaxel; 5 g of paclitaxel or its equivalent dose on CYT-21625 were used.
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Treatment of tumor-burdened mice with CYT-21625 and CYT-20203. The
efficacy of CYT-21625 was then tested on tumor-burdened mice using significantly lower doses
of paclitaxel, based on the significant increases in overall system exposure to paclitaxel observed
for this construct (Figure 3B). B16/F10 melanoma was established in C57/Bl6 mice, and the
mice were treated with either paclitaxel (2.5 or 40 mg/kg), CYT-21625 (2.5 mg/kg), or CYT-
20203 (2.5 mg/kg) and tumor volumes were measured over time. In this initial experiment a
50% reduction in tumor volume after 9 days was selected as a significant anti-tumor response.
Consistent with the targeted delivery leading to increased uptake of paclitaxel in the B16F/10
tumors, CYT-21625 and CYT-20203 induced similar anti-tumor responses as paclitaxel but at 16
fold lower doses of drug (Table 4).
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Table 4. Anti-Tumor Responses of B16/F10 Tumor
Burdened C57Bl/6 Mice to Paclitaxel and CYT-21625
Response: Decrease in
Paclitaxel
Dose (mg/kg)
Treatment Group
Tumor Volume vs
Control: (%)
PBS control
Paclitaxel
0
0
2.5
40
2.5
2.5
-24
-68
-62
-60
Paclitaxel
CYT-21625
CYT-20203
 DISCUSSION
As part of the effort to develop a colloidal-gold-nanoparticle-based drug delivery system for
the targeted delivery of paclitaxel to tumors, paclitaxel derivatives 3 – 6 with various sulfur
containing linkers were synthesized. In studies of the hydrolytic stabilities of the analogs, analog
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4 was converted to paclitaxel in buffer solution with a half-life of 3.2 hours, but analog 5 was
stable in the absence of a reducing agent such as DTT or BME. Gold bound analog Au-3
released paclitaxel much more slowly than the corresponding native analog 3.
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Analogs 3 and 5 were converted to the nanoparticle constructs CYT-20203 and CYT-21625,
respectively, containing the analog, TNF, and PEG-Thiol in predetermined ratios. CYT-21625
was shown to deliver paclitaxel selectively to tumors and to serve as a slow-release reservoir of
paclitaxel, and both constructs were effective at reducing the volume of B16/F10 melanomas at
doses of 2.5 mg/Kg, with CYT-21625 showing slightly better efficacy.
CYT-21625 represents a completely novel nanomedicine with the capabilities of not only
targeting solid tumors (via particle bound TNF) but also providing a two-pronged attack on the
architecture of the solid tumor. Since the nanomedicine platform delivers therapeutically
relevant doses of TNF and paclitaxel we predict that the particle-bound TNF will induce vascular
leakage, similar to that observed with CYT-6091,38, 55 and a reduction in tumor interstitial fluid
pressure.39 As these events unfold CYT-21625 is expected to release paclitaxel that can then
attack the cancer cells.
Since the TNF-mediated events of targeting and creation of vascular leakage are early
stage events, coupled with the latency of the release of paclitaxel, it is reasonable to expect that
CYT-21625 could successfully mimic the ILP paradigm as a systemically deliverable
nanomedicine that can attack not only primary but also metastatic tumors. The full promise of
this approach to cancer treatment requires additional routine pharmacologic studies and a study
of the general applicability of TNF, since both Farma55 and Koonce39 report differential
sensitivities of solid tumors to TNF-mediated vascular effects. However, in tumors where TNF
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does induce vascular disruption CYT-21625 may provide a strong and synergistic attack on solid
tumors.
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 EXPERIMENTAL SECTION
The following standard conditions apply unless otherwise stated. All reactions were performed
under argon or nitrogen in oven-dried glassware using dry solvents and standard syringe
techniques. Tetrahydrofuran (THF) was distilled from the sodium benzophenone ketyl radical
ion. DCM was distilled from calcium hydride. HPLC grade methanol and water were purchased
from Fisher Scientific. All reagents were of commercial quality and used as received. 1-
Mercapto-3,6,9,12,15,18,21,24-octaoxaheptacosan-27-oic acid was purchased from Polypure
AS, Gaustadalleen 21, NO-0349 Oslo, Norway. PEG-Thiol (MW 20,000) was purchased from
SunBio, Inc., Seoul, South Korea, and TNF was purchased from Boehringer Ingelheim, GmbH,
Vienna, Austria. After workup, partitioned organic layers were washed with water and brine and
dried over Na2SO4. Reaction progress was monitored using aluminum-backed thin layer
chromatography (TLC) plates pre-coated with silica UV254. Purification by preparative thin
layer chromatography (PTLC) was performed using glass-backed plates pre-coated with silica
UV254. HPLC was conducted on a Shimadzu SCL-10AVP system using a column purchased
from Phenomenex (Luna 5μ C18 (2) 25 × 4.6 mm). 1H and 13C NMR spectra were recorded on a
400 MHz (400 MHz for 1H and 100 MHz for 13C) spectrometer or a 500 MHz (500 MHz for 1H
and 126 MHz for 13C) spectrometer in CDCl3 unless otherwise stated. All chemical shifts (δ)
were referenced to the solvent peaks of CDCl3 (7.26 ppm for 1H, 77.0 ppm for 13C).
1-(Pyridin-2-yldisulfanyl)-3,6,9,12,15,18,21,24-octaoxaheptacosan-27-oic acid
(9). A 25 mL round bottom flask was dried in the oven overnight, cooled to room temperature
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(rt) and equipped with a magnetic stirring bar and a rubber septum. The flask was evacuated for
5 min and flushed with argon. A solution of 1-mercapto-3,6,9,12,15,18,21,24-
7
8
9
octaoxaheptacosan-27-oic acid 8(4.31) (194 mg, 0.423 mmol) in dry tetrahydrofuran (2 mL) and
excess 2,2'-dithio-dipyridine56 was added into the flask via syringe in the presence of an argon
atmosphere (balloon). The solution was stirred vigorously at rt and 30 mL of glacial acetic acid
was added via syringe. The resulting solution was stirred at rt for 48 h. The resulting light yellow
solution was concentrated by rotary evaporation (35 °C) to yield a yellow oil. This sample was
applied to column chromatography (silica gel, 50 g) eluting with a mixture of hexane and EtOAc
(1:3, 60 mL) and then MeOH. The fraction eluted by MeOH was concentrated by rotary
evaporation (35 °C) to give a yellow oil, which was then purified by preparative TLC (developed
with CH2Cl2:MeOH = 10:1) to produce (9)4.32 (200 mg, 0.35 mmol) as a colorless oil (82 %).
(2'-tert-Butyldimethylsilyloxy)-7-chloromethyloxycarbonylpaclitaxel 10. To a 25
mL round bottom flask containing a magnetic stirring bar was charged compound 957 (399 mg,
0.41 mmol). The flask was then equipped with a rubber septum, and evacuated and flushed with
argon. Dry CH2Cl2 (5 mL) was added to dissolve the solid and the resulting solution was cooled
to 0 °C in an ice bath. To the mixture was added chloromethyl chloroformate (40.3 μL, 0.45
mmol) dropwise via syringe. After the solution was stirred for 5 min at 0 °C, pyridine (40.3 μL,
0.49 mmol) was added over a period of 5 min via syringe. The resulting mixture was allowed to
warm to rt and stir for 15 h under an argon atmosphere (balloon). The reaction was diluted with
EtOAc (100 mL), washed with water (2 × 5 mL) and brine (2 × 5 mL) and dried with anhydrous
Na2SO4. Rotary evaporation (35 °C) of the organic extract gave 10 (445 mg, crude) as a white
powder, which was used in the next step without further purification. 1H NMR (CDCl3, 400
MHz) δ 8.12 (2H, d, J = 8.5 Hz), 7.74 (2H, d, J = 8.5 Hz), 7.25-7.65 (11H, m), 7.08 (1H, d, J =
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9.0 Hz), 6.32 (1H, s), 6.26 (1H, t, J = 8.5 Hz), 5.98 (1H, d, J = 6.5 Hz), 5.73 (1H, d, J = 8.5 Hz),
5.69 (1H, d, J = 6.5 Hz), 5.57, 5.51 (1H, d, J = 6.5 Hz), 4.98 (1H, d, J = 9.5 Hz), 4.67 (1H, d, J =
2.5 Hz), 4.35 (1H, d, J = 8.0 Hz), 4.20 (1H, d, J = 8.5 Hz), 3.96 (1H, d, J = 7.0 Hz), 2.66 (1H,
m), 2.58 (3H, s), 2.41 (1H, dd, J = 15, 8.5 Hz), 2.14 (3H, s), 2.04 (3H, s), 1.99 (3H, s), 1.82 (3H,
s), 1.21 (3H, s), 1.15 (3H, s), 0.80 (9H, s), -0.03 (3H, s), -0.30 (3H, s); HRFABMS m/z
1060.3853 [M + H] + (calcd for C55H67ClNO16Si, 1060.3918).
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7-Chloromethyloxycarbonyl paclitaxel 11. A plastic vial (25 mL) equipped with a
magnetic stirring bar was charged with a solution of 10 (445 mg, crude) in tetrahydrofuran (10
mL). To the solution was added HF-pyridine (0.6 mL) dropwise at 0 °C. The resulting mixture
was allowed to warm up to rt and stir for 10 h. The reaction mixture was diluted with EtOAc
(100 mL), washed with saturated aqueous sodium bicarbonate (2 × 5 mL), water (2 × 5 mL) and
brine (2 × 5 mL) and dried with anhydrous Na2SO4. Rotary evaporation (35 °C) gave a light
brown residue, which was then purified by column chromatography (silica gel, 150 g, eluted
with hexane:EtOAc, 3:2~2:3) to yield 12 (298 mg, 0.32 mmol) as a white powder (77 % for two
steps from 10. 1H NMR (500 MHz) δ 8.11 (d, J = 7.4 Hz, 2H), 7.76 (d, J = 7.4 Hz, 2H), 7.62 (t, J
= 7.4 Hz, 1H), 7.55 – 7.46 (m, 6H), 7.41 (m, 4H), 7.35 (t, J = 7.3 Hz, 1H), 7.04 (d, J = 8.9 Hz,
1H), 6.28 (s, 1H), 6.18 (t, J = 8.5 Hz, 1H), 5.97 (d, J = 6.5 Hz, 1H), 5.80 (dd, J = 8.9, 2.4 Hz,
1H), 5.67 (d, J = 6.9 Hz, 1H), 5.51 (q, J = 5.7 Hz, 2H), 4.95 (d, J = 8.4 Hz, 1H), 4.80 (dd, J =
4.7, 2.5 Hz, 1H), 4.32 (d, J = 8.5 Hz, 1H), 4.18 (d, J = 8.5 Hz, 1H), 3.92 (d, J = 6.9 Hz, 1H), 3.60
(d, J = 4.9 Hz, 1H), 2.65 (ddd, J = 14.5, 9.5, 7.3 Hz, 1H), 2.38 (s, 3H), 2.36 – 2.30 (m, 2H), 2.15
(s, 3H), 2.04 – 1.97 (m, 1H), 1.85 (s, 3H), 1.81 (s, 3H), 1.21 (s, 3H), 1.15 (s, 3H); 13C NMR (100
MHz) δ 201.54, 172.59, 170.53, 169.29, 167.12, 166.92, 152.71, 140.80, 138.10, 133.91, 133.74,
132.96, 132.04, 130.26, 129.09, 128.84, 128.80, 128.43, 127.16, 127.15, 83.78, 80.92, 78.62,
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76.64, 76.47, 75.49, 74.26, 73.29, 73.06, 72.23, 56.25, 55.04, 47.00, 43.29, 35.63, 33.32, 26.61,
22.62, 20.98, 20.86, 14.72, 10.72.
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Paclitaxel analog 3. A dry 25 mL round bottom flask was charged with a magnetic
stirring bar, compound 11 (140 mg, 0.15 mmol) and sodium iodide (33.3 mg, 0.22 mmol).
Acetone (5 mL) was added via syringe to dissolve the solid. The flask was then equipped with a
water condenser and the reaction mixture was allowed to reflux (~62 °C, oil bath) for 10 h. The
resulting yellow solution was concentrated by rotary evaporation (35 °C) to give a yellow
residue. To the flask was introduced a solution of 8 (128 mg, 0.22 mmol) in benzene (5 mL).
After K2CO3 (61.3 mg, 0.44 mmol) and 18-crown-6 (235 mg, 0.89 mmol) were added, the flask
was capped with a water condenser, warmed up to 75 °C and stirred for 2 h. The reaction
solution was allowed to cool to rt and diluted with EtOAc (100 mL), washed with saturated
aqueous sodium bicarbonate (2 × 5 mL), water (2 × 5 mL) and brine (2 × 5 mL) and dried with
anhydrous Na2SO4. Rotary evaporation (35 °C) gave a yellowish oil. This was applied to
preparative TLC (developed with 5 % MeOH in CH2Cl2) to yield 3 (136 mg, 0.092 mmol, 62 %).
1H NMR (500 MHz) δ 8.44 (1H, d, J = 4.8 Hz), 8.11 (2H, d, J = 7.6 Hz), 7.25-7.80 (15H, m),
7.10 (2H, m), 6.30 (1H, s), 6.18 (1H, dd, J = 8.0, 8.0 Hz), 5.90 (1H, d, J = 6.0 Hz), 5.79 (1H, dd,
J = 9.2, 2.4 Hz), 5.71 (1H, d, J = 6.0 Hz), 5.67 (1H, d, J = 6.8 Hz), 5.48 (1H, dd, J = 10.8, 6.8
Hz), 5.30 (1H, s), 4.94 (1H, d, J = 8.4 Hz), 4.80 (1H, dd, J = 6.4, 4.0 Hz), 4.31 (1H, d, J = 8.4
Hz), 4.19 (1H, d, J = 7.6 Hz), 3.91 (1H, d, J = 6.8 Hz), 3.55-3.80 (33H, m), 2.99 (2H, dd, J = 6.4,
6.4 Hz), 2.67 (2H, dd, J = 6.4, 6.4 Hz), 2.64 (1H, m), 2.38 (3H, s), 2.32 (2H, d, J = 9.2 Hz), 2.16
(3H, s), 2.01 (1H, m), 1.85 (3H, s), 1.80 (3H, s), 1.21 (3H, s), 1.16 (3H, s); 13C NMR (126 MHz)
δ 201.4, 172.6, 170.5, 170.1, 169.1, 167.0, 166.8, 153.3, 149.6, 140.7, 138.1, 137.2, 133.9, 133.0,
132.0, 130.3, 129.1, 129.1, 128.8, 128.8, 128.4, 127.1, 127.1, 120.7, 119.7, 84.5, 83.8, 82.6, 81.2,
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81.0, 79.2, 78.7, 76.5, 76.2, 75.7, 75.4, 74.3, 73.2, 72.5, 72.2, 70.6, 70.5, 69.0, 66.1, 65.5, 56.2,
54.9, 47.0, 43.3 , 38.5, 35.6, 34.8, 33.4, 29.8, 27.0, 26.6, 22.6, 21.0, 20.8, 14.8, 10.7; HRFABMS
m/z 1477.5421 [M + H] + (calcd for C73H93N2O26S2, 1477.5458).
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(7-O-Paclitaxelcarbonyloxy)methyl 5-(1,2-dithiolan-3-yl)pentanoate 4. A dry 25
mL round bottom flask was charged with a magnetic stirring bar, compound 11 (40 mg, 42.3
μmol) and sodium iodide (9.5 mg, 63.5 μmol). Acetone (2 mL) was added via syringe to dissolve
the solid. The flask was then equipped with a water condenser and the reaction mixture was
allowed to reflux (~62 °C, oil bath) for 10 h. The resulting yellow solution was concentrated by
rotary evaporation (35 °C) to give a yellow residue. To the flask was added lipoic acid (17.0 mg,
82.6 μmol) and benzene (2 mL). After K2CO3 (17.5 mg, 126.9 μmol) and 18-crown-6 (67.1 mg,
253.8 μmol) was added, the flask was capped with a water condenser, warmed to 65 °C and
stirred for 3.5 h. TLC was used to examine the reaction progress. Decomposed product could be
detected with prolonged reaction times. The reaction solution was allowed to cool down to rt and
diluted with EtOAc (75 mL), washed with saturated aqueous sodium bicarbonate (2 × 4 mL),
water (2 × 4 mL) and brine (2 × 4 mL) and dried with anhydrous Na2SO4. Rotary evaporation
(35 °C) gave a yellowish oily residue, which was purified by preparative TLC (developed with
hexane:EtOAc, 1:1) to yield 4 (36 mg, 32.2 μmol, 76 %). 1H NMR (400 MHz) δ 8.10 (2H, dd, J
= 7.5, 1.5 Hz), 7.76 (2H, d, J = 7.0, 1.5 Hz), 7.62 (1H, m), 7.47-7.52 (5H, m), 7.33-7.43 (4H, m),
7.04 (1H, d, J = 9.0 Hz), 6.30 (1H, s), 6.18 (1H, dd, J = 8.0, 8.0 Hz), 5.90 (1H, d, J = 6.0 Hz),
5.79 (1H, dd, J = 9.0, 2.0 Hz), 5.70 (1H, d, J = 6.0 Hz), 5.66 (1H, d, J = 7.0 Hz), 5.48 (1H, dd, J
= 10.0, 7.0 Hz), 4.94 (1H, d, J = 8.0 Hz), 4.79 (1H, d, J = 2.5 Hz), 4.31 (1H, d, J = 8.5 Hz), 4.17
(1H, d, J = 8.5 Hz), 3.91 (1H, d, J = 7.5 Hz), 3.56 (2H, m), 3.08-3.18 (2H, m), 2.62 (1H, m), 2.45
(1H, m), 2.38 (3H, s), 2.31 (2H, dd, J = 9.0, 4.0 Hz), 2.15 (3H, s), 1.96 (1H, m), 1.89 (1H, m),
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1.84 (3H, s), 1.80 (3H, s), 1.67 (6H, m), 1.46 (2H, m), 1.21 (3H, s), 1.18 (3H, s); 13C NMR (126
MHz) δ 201.44, 172.59, 172.00, 170.52, 169.11, 167.10, 166.92, 153.28, 140.66, 138.08, 133.90,
133.73, 133.05, 132.06, 130.26, 129.10, 128.84, 128.81, 128.44, 127.17, 127.14, 83.81, 82.65,
80.97, 78.62, 76.49, 76.22, 75.40, 74.27, 73.27, 72.29, 56.35, 56.34, 56.19, 55.01, 47.02, 43.31,
40.28, 38.57, 35.61, 34.64, 33.74, 33.38, 28.71, 28.70, 26.64, 24.29, 22.64, 20.98, 20.87, 14.75,
10.73.
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Synthesis of 2΄-(tert-butyldimethylsiloxy)-7-(((4-(pyridin-2-yldisulfanyl)-
benzyloxy)-carbonyloxy)-paclitaxel 14. To a stirred solution of compounds 1253 (48.5 mg,
0.043 mmol) and 1348 (16 mg, 0.064 mmol) in dry DCM (2 mL) was added 4-(dimethylamino)
pyridine (15.6 mg, 0.128 mmol), and the mixture was stirred at rt for 18 h. The resulting mixture
was diluted with DCM (40 mL) and washed with water (2 × 2 mL) and brine (2 × 2 mL). The
solution was dried with anhydrous Na2SO4 and concentrated in vacuo. The crude product was
purified by preparative TLC (50% EtOAc in hexanes) to afford compound 14 (47 mg, 88%): 1H
NMR (500 MHz) δ 8.46 (1H, d, J = 4.9 Hz), 8.11 (2H, d, J = 7.3 Hz), 7.76 (2H, d, J = 7.6 Hz),
7.25-7.55 (17 H, m), 7.10 (1H, d, J = 8.7 Hz), 6.39 (1H, s), 6.26 (1H, dd, J = 9.2, 8.1 Hz), 5.73
(1H, d, J = 8.7 Hz), 5.70 (1H, br d, J = 6.7 Hz), 5.54 (1H, dd, J = 10.1, 6.9), 5.16 (2H, br s), 4.97
(1H, d, J = 9.2 Hz), 4.67 (1H, br s), 4.34 (1H, d, J = 8.5 Hz), 4.20 (1H, d, J = 8.5 Hz), 3.97 (1H,
d, J = 6.7 Hz), 2.60 (1H, m), 2.58 (3H, s), 2.42 (1H, dd, J = 15.1, 9.4 Hz), 2.15 (3H, s), 2.15 (1H,
dd, J = 15.4, 8.9 Hz), 2.00 (1H, m), 2.00 (3H, s), 1.80 (3H, s), 1.22 (3H, s), 1.16 (3H, s), 0.80
(9H, s), -0.03 (3H, s), -0.30 (3H, s); 13C NMR (126 MHz) δ 201.6, 171.4, 170.0, 169.1, 167.1,
166.9, 159.0, 154.0, 149.0, 141.1, 138.2, 136.6, 135.7, 134.0, 133.7, 132.7, 131.8, 130.2, 129.7,
129.0, 128.8, 128.3, 128.0, 127.0, 126.3, 126.0, 121.5, 120.5, 115.7, 83.8, 80.9, 78.7, 76.3, 75.5,
75.3, 75.0, 74.4, 71.3, 69.2, 56.0, 55.7, 46.8, 43.3, 35.5, 33.3, 26.4, 25.5, 23.0, 21.4, 20.8, 18.1,
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14.6, 10.7, -5.2, -5.8; HRFABMS m/z 1243.4370 [M + H]+ (calcd for C66H75N2O16S2Si,
1243.4327).
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Synthesis of 7-(((4-(pyridin-2-yldisulfanyl)benzyloxy)carbonyloxy)-paclitaxel 5.
A plastic vial was charged with a solution of compound 14 (45 mg, 0.036 mmol) in 4.5 mL of
dried THF and cooled to 0 °C. To this solution was added 0.15 mL of HF-pyridine. The mixture
was allowed to warm to rt and stirred for 16 h. The reaction was quenched by careful addition of
saturated aqueous NaHCO3 until no bubbles were formed. The resulting solution was extracted
with EtOAc (3 × 15 mL). The organic solution was washed with water (2 × 2 mL) and brine (2 ×
2 mL), dried with anhydrous Na2SO4 and concentrated in vacuo. The residue was purified by
preparative TLC (50% EtOAc/hexane) to give 5 (35 mg, 92 % based on unreacted starting
material), together with 3 mg unreacted compound 14. 1H NMR (500 MHz) δ 8.46 (1H, d, J =
4.9 Hz), 8.11 (2H, d, J = 7.2 Hz), 7.76 (2H, d, J = 8.0 Hz), 7.25-7.65 (17H, m), 7.10 (1H, br dd, J
= 7.0, 4.1 Hz), 7.04 (1H, d, J = 8.8 Hz), 6.36 (1H, s), 6.19 (1H, t, J = 6.9 Hz), 5.80 (1H, dd, J =
8.8, 2.5 Hz), 5.67 (1H, d, J = 6.8 Hz), 5.47 (1H, dd, J = 10.7, 7.5), 5.17 (1H, d, J = 12.3 Hz), 5.13
(1H, d, J = 12.3 Hz), 4.93 (1H, d, J = 8.8 Hz), 4.80 (1H, dd, J = 5.0, 2.5 Hz), 4.31 (1H, d, J = 8.7
Hz), 4.18 (1H, d, J = 8.7 Hz), 3.93 (1H, d, J = 6.8 Hz), 3.85 (1H, d, J = 5.0 Hz), 2.58 (1H, m),
2.38 (3H, s), 2.33 (2H, d, J = 6.9 Hz), 2.15 (3H, s), 1.95 (1H, m), 1.85 (3H, s), 1.80 (3H, s), 1.22
(3H, s), 1.17 (3H, s); 13C NMR (126 MHz) δ 201.5, 172.5, 170.4, 169.0, 167.0, 166.8, 159.0,
153.8, 149.0, 140.5, 138.0, 137.8, 133.8, 133.8, 133.6, 133.0, 131.9, 130.1, 129.6, 129.0, 128.7,
128.7, 128.4, 127.7, 127.0, 127.0, 127.0, 121.5, 120.5, 83.8, 80.9, 78.5, 76.4, 75.6, 75.3, 74.2,
73.1, 72.2, 69.3, 56.1, 54.9, 46.9, 43.2 , 35.5, 33.4, 26.5, 22.5, 20.9, 20.8, 14.6, 10.6; HRFABMS
m/z 1129.3464 [M + H]+ (calcd for C60H61N2O16S2, 1129.3463).
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Synthesis of paclitaxel-2'-yl (chloromethyl)succinate (16). A mixture of CH2Cl2 (2
mL) and 2 mL of an aqueous solution containing compound 1546 (20.8 mg, 0.022 mmol),
NaHCO3 (9 mg, 0.11 mmol), and tetrabutylammonium bisulfate (1 mg, 0.003 mmol) was stirred
for 10 min at rt. Chloromethyl chlorosulfate (4.6 mg, 0.028 mmol) was then added and the
reaction mixture vigorously stirred at rt for 2 h. The reaction mixture was diluted with 10 mL
DCM, washed with brine (2 × 1 mL), dried over anhydrous Na2SO4, filtered, and evaporated
under reduced pressure. The residue was separated by PTLC with hexane and EtOAc (1:2) as
developing solvent to yield compound 19 (10 mg, 46% yield). 1H NMR (500 MHz):1.12 (s, 3H),
1.23 (s, 3H), 1.67 (s, 3H), 1.87 (m, 1H), 1.92 (s, 3H), 2.15 (m, 1H), 2.22 (s, 3H), 2.37 (m, 1H),
2.44 (s, 3H), 2.55 (m, 1H), 2.68 (m, 2H), 2.78 (m, 2H), 3.80 (d, J = 7.0 Hz, 1H), 4.19 (d, J = 8.5
Hz, 1H), 4.31 (d, J = 8.5 Hz, 1H), 4.43 (m, 1H), 4.96 (d, J = 7.5 Hz, 1H) 5.50 (d, J = 3 .0 Hz),
5.57 (s, 2H), 5.68 (d, J = 7.0 Hz, 1H), 5.98 (dd, J = 6.5, 3.0 Hz, 1H), 6.23 (t, 9.0 Hz, 1H) 6.28 (s,
1H), 6.94 (d, J = 9.0 Hz, 1H), 7.34-7.45 (m, 7H), 7.52 (m, 3H), 7.60 (m 1H), 7.77 (m, 2H), 8.14
(m, 2H); 13C NMR (126 MHz) δ 203.91, 171.37, 170.83, 170.38, 169.91, 169.17, 167.89, 167.16,
167.15, 153.29, 142.85, 138.91, 136.93, 133.79, 133.59, 132.85, 132.15, 130.33, 129.24, 129.21,
128.84, 128.80, 128.60, 127.28, 127.27, 126.59, 84.52, 81.15, 79.27, 77.30, 76.80, 75.68, 75.16,
74.45, 72.24, 72.00, 68.94, 62.97, 58.61, 52.74, 45.64, 43.25, 35.58, 29.09, 28.71, 26.89, 22.78,
22.20, 20.91, 14.91, 9.67.
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Synthesis of ((5-(1,2-dithiolan-3-yl)pentanoyl)oxy)methyl paclitaxel-2'-yl
succinate (6). A mixture of 16 (10 mg, 0.01 mmol), lipoic acid (4.4 mg, 0.02 mmol), K2CO3
(4.4 mg, 0.03 mmol), 18-crown-6 (16.8 mg, 0.06 mmol) and NaI (cat.) was refluxed in benzene
for 5 hr. The reaction mixture was diluted with ether (10 mL) and washed with water (2 × 1 mL)
and brine (2 × 1 mL). The resulting crude product was purified by PTLC (1:2 hexane/EtOAc) to
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give compound 6 as a white solid (7 mg, 60% yield). 1H NMR (500 MHz):1.12 (s, 3H), 1.23 (s,
3H), 1.40-1.50 (m, 2H), 1.64 (m, 2H), 1.67 (s, 3H), 1.87 (m, 1H), 1.92 (s, 3H), 2.15 (m, 1H),
2.22 (s, 3H), 2.30-2.40 (m, 3H), 2.44 (s, 3H), 2.45 (m, 2H), 2.55 (m, 1H), 2.68 (m, 2H), 2.78 (m,
2H), 3.06-3.18 (m, 2H), 3.54 (m, 1H), 3.80 (d, J = 7.0 Hz, 1H), 4.19 (d, J = 8.5 Hz, 1H), 4.31 (d,
J = 8.5 Hz, 1H), 4.43 (m, 1H), 4.96 (d, J = 7.5 Hz, 1H) 5.50 (d, J = 3 .0 Hz), 5.64 (m, 2H), 5.68
(d, J = 7.0 Hz, 1H), 5.98 (dd, J = 6.5, 3.0 Hz, 1H), 6.23 (t, 9.0 Hz, 1H) 6.28 (s, 1H), 6.98 (d, J =
9.0 Hz, 1H), 7.34-7.45 (m, 7H), 7.52 (m, 3H), 7.60 (m 1H), 7.77 (m, 2H), 8.14 (m, 2H).
Preparation of CYT-21625 and CYT-20203. CYT-21625 is the designation of gold
nanoparticles bound to TNF, PEG-Thiol, and compound 5, and CYT-20203 is the corresponding
construct formed using analog 3 in place of analog 5. The procedure used was adapted from that
described previously.36 For both constructs the gold nanoparticles were made by bringing
deionized H2O (16 L) to a boil under reflux, and then adding 32 mL of a high purity 4% gold
chloride solution to the rapidly mixed boiling water. Nanoparticle initiation was induced by the
rapid addition of 600 mL of a 1% sodium citrate solution. The resulting gold nanoparticles were
filtered through a 0.22 µ sterilization filter and stored until needed. Two glass containers
equipped with bottom ports were connected via Tygon tubing to the side ports of a single T-
connector. One of the containers was filled with 800 mL of colloidal gold nanoparticles while
the second container was filled with 800 mL of a solution containing TNF, PEG-Thiol and
paclitaxel analog 5 in the concentrations of 0.1, 15, and 2.5 g/mL, respectively. The exit port of
the T-connector was similarly fitted with tubing containing in line mixers. This section of tubing
was threaded through a single peristaltic pump which when turned on drew both the gold
particles and the solution of TNF, PEG-Thiol, and 5 into the T-Connector, through the pump and
into a central collection vessel. After binding, a series of stabilizers were added to the solution,
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which was then concentrated by ultrafiltration and lyophilized to yield CYT-21625. The same
procedure using analog 3 in place of analog 5 gave CYT-20203. Each batch of nanodrug
produced was interrogated for TNF content using a cross antibody ELISA as previously
described.29 Paclitaxel content was determined by diluting the final drug product to a 50%
MeOH solution containing BME and NaHCO3 and Na2CO3 (10 mM each). The solution was
then incubated at 37°C until particle precipitation was evident (~ 4h). The precipitated material
was centrifuged and the supernatant containing paclitaxel released from compound 5 was
analyzed by quantitative HPLC using authentic paclitaxel as a standard. To determine the
amount of free paclitaxel an aliquot of the final drug product was diluted in 1% PEG 1450 and
centrifuged. The resultant supernatant was diluted and analyzed as described above. The
composition of CYT-21625 could be tuned by varying the amounts of each active
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pharmaceutical ingredient that was bound to the nanoparticle surface. Since both TNF and the
thiolated paclitaxel analogs were bound at the same time we observed that as the concentration
of one API (i.e., the paclitaxel analog) in the binding vessel increased as the amount of the
second API (i.e., TNF) detected on the particle decreased. These data support the concept that
both TNF and the thiolated paclitaxel analogs shared the surface of the nanoparticles. These
conditions yielded nanoparticles with up to 5000 molecules of paclitaxel analog and 25
molecules of TNF bound to each nanoparticle, as calculated from the weights of the starting
materials and confirmed by the lack of significant amounts of free analog in the final product.
Hydrolytic release of paclitaxel by compound 4 in buffer. Concentrations of 4 and
paclitaxel were determined by quantitative HPLC with a Shimadzu SCL-10AVP system. Reverse
phase C18 column was purchased from Phenomenex (Luna 5μ C18 (2) 25 × 4.6 mm). HPLC
analyses were run with a linear gradient elution from MeOH:H2O 1:1 to 100% MeOH in 20 min,
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followed by 100% MeOH for 10 min, at a flow rate of 1 mL/min. A fresh stock solution of 4 was
prepared by dissolving 5.20 mg 4 in 25 mL MeOH. A small vial equipped with a cap and
magnetic stirring bar was charged with 3 mL of the above solution. To this solution was added 1
mL 1 M PBS buffer (pH 7.4) quickly in one portion. A 50 μL aliquot of this solution
immediately injected onto the HPLC system. The vial was then capped and sealed with parafilm.
Aliquots (50 μL) were taken at 30 min, 60 min, 120 min, 180 min, 240 min, 360 min, 540 min,
690 min and 1440 min, and injected onto the HPLC system. Concentrations of paclitaxel and 4 in
each aliquot were determined based on previously prepared standard curves of both compounds.
The resulting concentration vs time plot is shown in Figure S1.
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Release of paclitaxel by compound 5 and by CYT-21625 in bicarbonate buffer.
Analog 5 (0.25 mg) was dissolved in 10 mL 90% CH3OH containing 5 mg NaHCO3, and the
solution was analyzed by HPLC. Less than 5% of paclitaxel was released over 24 h, but this rose
to 50% release after 72 h. A repeat of this study using CYT-21625 showed that paclitaxel release
was reduced, with less than 10% of paclitaxel released over 72 h (Figure S3).
Release of paclitaxel from compound 5 in bicarbonate buffer plus
dithiothreitol. The previous experiment was repeated to assess the release of paclitaxel in the
presence of a 3-fold excess of dithiothreitol (DTT). It was found that 90% of 5 was converted to
paclitaxel within 60 min under these conditions.
Generation of paclitaxel from compound 5 in rabbit whole blood. Analog 5 or
CYT-21625 were spiked into rabbit whole blood. At t = 0, 1, 2, and 24 h samples were collected
and processed. Rabbit whole blood (1 mL) was extracted with 2 mL of ice cold acetonitrile
(ACN), and the samples were centrifuged at 3500 rpm to pellet the precipitated protein extract.
In the case of 5 the supernatant was analyzed directly by HPLC as described below. In the case
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