# Synthesis and Anchoring of Antineoplastic Ferrocene and Phthalocyanine Derivatives on Water-Soluble Polymeric Drug Carriers Derived from Lysine and Aspartic Acid

Authors: M. David Maree; Eberhard W. Neuse; Elizabeth Erasmus; Jannie C. Swarts
Journal: Metal-Based Drugs
 (2008)

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## Abstract

The general synthetic strategy towards water-soluble biodegradable drug carriers and the properties that they must have are discussed. The syntheses of water-soluble biodegradable copolymers of lysine and aspartic acid as potential drug-delivering devices, having amine-functionalised side chains are then described. Covalent anchoring of carboxylic acid derivatives of the antineoplastic ferrocene and photodynamically active phthalocyanine moieties to the amine-containing drug carrier copolymers under mild coupling conditions has been achieved utilising the coupling reagent O-benzotriazolyl-N,N,N′,N′-tetramethyluronium hexafluorophosphate to promote formation of the biodegradable amide bond. Even though the parent antineoplastic ferrocene and phthalocyanine derivatives are themselves insoluble in water at pH
< 7, the new carrier-drug conjugates that were obtained are well water-soluble.


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## Body

## 1. Introduction

In accordance with the goals of this special issue of metal-based drugs, this article deems to
fulfil a teaching and guiding role in new anticancer drug research.  We hope it will spawn new ideas and new
research in the field of anticancer therapy. In what follows,
we first create an understanding of why new anticancer drugs are vigorously
searched for by highlighting some of the problems associated with a typical
chemotherapeutic agent, cisplatin. One
possible way to minimise the negative side effects associated with
chemotherapeutic agents is to make use of a synthetic polymeric drug delivery
system.  We explain how such systems may
significantly enhance the therapeutic effectiveness of chemotherapeutic agents
and highlight some of the structural features they must have. We then develop a general synthetic strategy
an experimental chemist may follow to actually synthesise a polymeric drug delivering
device and proceed to visualise this strategy utilising a literature
example. The need for certain
structural features in a delivering device is then motivated by a discussion of
the biological mechanism of cell uptake and drug release from the drug carrying
device.    In conclusion, the synthesis
and characterisation of a new example of a polymeric drug delivery system
derived from aspartic acid and lysine is presented.


## 2. Problems Associated with Chemotherapeutic Drugs

Many
potentially good pharmaceutical agents are either dose limited, or excluded
from use in clinical therapy due to insolubility in an aqueous medium, or
because of the many severe side effects that they may exhibit.   As far as cisplatin, the most successful
metal-containing chemotherapeutic drug of recent times [1], is concerned, these
side effects include inter alia:
(a)
poor aqueous solubility;(b)
intensive damage to the linings of the intestines, which leads to the loss of appetite (anorexia) and eventually
starving in the case of mice and rats [2];(c)
severe
nausea, vomiting, and audio toxicity [3];(d)
high
toxicity towards the kidneys and bone marrow [4];(e)
being a
moderate carcinogen itself.   It can
induce lung cancer, skin papillomas, and other carcinomas [5].
In general, most if not all chemotherapeutic drug side effects are due to their
general inability to differentiate between diseased and healthy cells [6].Even if a synthetic chemotherapeutic drug has few side effects, it does not imply
success in clinical use.  One must
realize that chemotherapeutic agents usually are poisons, or at the very least,
they are foreign to the body. The
defence mechanism of the body, the reticuloendothelial system, recognizes them
as such and tries to remove them as fast as possible. A fast excretion rate from the body often
proves detrimental to chemotherapy. Cisplatin, for example, is eliminated from the body in a biphasic
excretion mechanism in such a way that 50% of the initial administered dose is
excreted within 20 hours [7].   After 110
hours, 70% of the initial administered amount of drug is removed from the body
by the reticuloendothelial system. The
principle mode of excretion in mice appears primarily to be through the urine,
90% of the injected dose being excreted within five days after injection. This quick excretion rate causes large drug
concentration fluctuations in the body, and hence, also unpredictable
therapeutic activity, a highly undesirable situation. The negative impact of
such drug concentration fluctuations in the body becomes apparent when one
relates it to 50% lethal dosages (LD50 values), optimum dosages,
and a dosage having no influence on the diseased site at all.   For cisplatin [8], the LD50 dosage applicable to mice is 14 mg/kg mass of test animal, the optimum dose
is half of this, 7 mg/kg test animal, but 3 mg/kg mass of the test animal
has absolutely no influence on the diseased site at all.    With the excretion profile that cisplatin
shows, it is clear that it is extraordinary difficult to maintain drug levels
in a patient close to the optimum dose level, but still below the LD50 value for prolonged times. The initial
required overdose of drug that is required to maintain useful drug levels in
the body explains much
of the negative side effects of chemotherapeutic drugs.A
further point of consideration in developing and administering anticancer drugs
is the development of drug resistance by a tumour after prolonged use. A neoplastic population of cells is not a
static entity, but rather an ever-changing one. Tumours display an amazing ability to escape
or neutralize the actions of chemotherapeutic agents to which they were
initially sensitive [9]. Some common
examples of this evasive ability are as follows [10]:
(a)
the loss of specific receptors;(b)
down-regulation
of tumour-associated antigens; and(c)
shedding of
antigens into the body fluids.
The metastatic nature of cancer cells thus leads to the development of drug
resistance after continued drug dosage [11, 12].One
of the most fundamental barriers to selective drug delivery involves the
so-called targeting problem. The
central problem of cancer chemotherapy remains one of selective drug action. Ideally, one would like a toxic agent that
can discriminate between neoplastic and nonneoplastic cells. This approach presumes the existence of
something to aim at, that is, some molecular characteristic that differs
between target and nontarget cells. The
metastatic nature of cancer cells makes this a formidable task.  Even if a target can be identified, or a cell
need can be exploited, as has been done in
vitro, the problem remains of actually bringing the pharmaceutical
agent,in vivo, from the point of administering to the neoplastic
growth. To achieve this, many
physiological barriers including the reticuloendothelial system of the body
have to be bypassed.Consequently,
to improve the treatment of cancer, research today focusses on developing new
and better chemotherapeutic agents having less side effects (i.e., drugs that can zoom onto diseased
sites) [13], new methods of drug delivery are being researched [6], combination
therapy is being investigated in the hope of finding synergistic effects [14],
the scope of radiation therapy is broadened, and completely new methods of
combating cancer are sought.New
metal-based drugs include members of the titanocene and ferrocene family of
compounds [13, 15].  A promising example
of a new method of cancer treatment focusses on photodynamic therapy (PDT) [16, 17]. In PDT, the photo-active drugs in themselves
are inactive towards both healthy and cancer cells. However, when light of the correct
wavelength is shone on them, the drugs become photo-activated and give rise to conditions
that kill the cells
it is in contact
with, normally via the generation of singlet oxygen.   A good measure of selective cancer cell
killing is therefore induced into the treatment of a patient merely by
focussing light from a suitable laser source onto a tumourous growth.   Healthy cells will stay largely undamaged
provided that light
is not shone upon it.   If cancer cells
would selectively and preferentially absorb the PDT drug, this would lead to a
further enhancement of selective cancer cell destruction.   Phthalocyanines and related macrocycles are second-generation PDT agents.  Many phthalocyanine derivatives
have the advantage of being preferentially absorbed by cancer cells.   Some of them were found to be distributed upto
90% selectively in cancer cells, and only 10% in healthy cells after a certain
induction period [18].


## 3. Synthetic Polymeric Drug Delivery Systems

Many detrimental properties and side effects of promising anticancer drugs may at
least be partially overcome if the pharmaceutical agent is covalently anchored
to a polymeric drug carrier.   Polymeric
or macromolecular drug-carrying devices should be distinguished from sustained drug-release
systems.   Sustained drug-release systems
are normally associated with polymers; frequently insoluble; and in the form of
powders, pellets, and capsules from which the slow release of an encapsulated
drug from the “container” interior is possible.   They normally do not represent systems that
may affect target-specific drug
delivery as much as they are devices for the continued release of drugs.   The term “polymeric drug carrier” describes
a polymer or macromolecule that contains covalentbinding sites that are available to anchor active pharmaceutical
units as terminal or pendent groups protruding from the side of the polymeric
chain.    Polymeric drug-carrying devices
may be synthetic or naturally occurring. The naturally occurring ones such as antibodies [19, 20] induce a high
mode of selective drug action onto the polymeric drug-containing device, but
usually suffer from a lack of large amounts of drug-anchoring sites. Synthetic polymers on the other hand are
more prone to suffer from immunogenic side effects [21], but this may be
overcome by copolymerisation with ethylene glycol fragments.   Their big advantage is that they can be tailor-made
to be suitable for almost any purposes and can be engineered to have a large
amount of drug-anchoring sites.The
clinical administration of a polymer bound drug, as compared to the free agent,
may significantly enhance the therapeutic effectiveness in terms of the
following:(a)
accelerated
and unencumbered distribution in the aqueous central circulation system of the
body (the blood), thereby reducing the risks of premature degradation and
excretion;(b)
they facilitate cell entry via
endocytosis—a cell membrane penetration mechanism
generally unavailable to nonpolymeric compounds but highly desired for drugs
operating intracellularly;(c)
more
precisely controlled drug serum levels (i.e., the restriction of the drug
concentration to the gap between toxic and minimum effective levels);(d)
an enhanced
depot effect through slow drug release from the polymer-drug conjugate.
The
structural features of the polymer-drug conjugate required to comply with these
attributes include the following:
(a)
a highly
flexible linear main chain comprising structural entities that can provide
water solubility;(b)
a sufficiently large molecular mass to prevent quick excretion from the body (the
threshold for elimination via the
kidneys is ca. 70 000 gmol−1);(c)
a
biodegradable carrier backbone prone to catabolic elimination of the “spent”
polymer main chain after the payload of drug has been delivered to the target
site;(d)
a large
amount of reactive functional groups as suitable binding sites for drug
attachment,   these sites should be
distanced from the main chain by short (5–15 constituent atoms) spacer segments
to diminish the steric bulk effect of the polymeric carrier backbone, and they
should be sufficiently separated from each other along the backbone to prevent
intramolecular multifunctional drug binding;(e)
the
separation of binding sites on the polymeric backbone requires insertion of
side chains on the main chain between active binding sites that lack drug-binding
capabilities, but enhance other desirable properties. These may include enhancing the hydrophilic
nature of the ultimate polymeric drug-carrying device, the incorporation of
moieties that enhance drug-targeting capability (i.e., a tumour homing device),
antispasmodic properties, or any other desirable physiological effect;(f)
one or more
biofissionable functions (amide, ester, urethane, disulphide, etc.) to be
inserted into the carrier-drug connecting link.   At least one of these must be sufficiently
remote from the main chain to permit enzyme approach and cleavage action to
result in effective drug release in a targeted biological compartment;(g)
carrier
backbones and spacers which are non toxic per se.   They should
especially possess absolute minimal immunogenicity so as to preclude
carrier-induced pathological effects.
This means polymeric drug-carrying devices should be completely
biocompatible.
Akey component towards the success of polymeric drug-carrying devices centres
around biodegradable bonds and their capability to home preferentially onto a neoplastic
growth. Apart from targeting fleeting
receptors on a collection of metastatic neoplastic cells, the nutritional needs
of such cells may be utilized to gain preferential neoplastic cell access.   Neoplastic cells grow much faster than
healthy cells, and have a much higher demand for food in the form of amino
acids and energy.   Hence copolymers
constructed at least partially from amino acids are very attractive as
polymeric drug delivery devices. They
also introduce the biodegradable amide (peptide) bond.   Other degradable bonds include nucleotide
and sugar bonds. Sugar groups as a
source of energy will especially enhance preferential uptake by neoplastic
cells.   Other biodegradable groups that
may be employed include disulphide bonds that may be reduced to thiol groups
[22], or, since cell interior has a lower pH than blood plasma, for example,
weakly acid-labile groups such as thio-ethers [23].


## 4. Synthetic Strategies Towards Polymeric Drug Delivery Systems

Although
models as those mentioned
above for synthetic polymeric drug carriers have been in existence for some time [24], until recently only a few efforts were made to actually prepare
such compounds.   A review by Neuse [25] describes some of the most recent researches in this
field.   Scheme 1 illustrates the
synthetic approach to actually obtain such polymeric drug-delivering
devices.   The centre of Scheme 1 serves
to visualise the overall strategy.   At
the left and right ends of Scheme 1 an example for an actual synthesis of a polymeric
drug delivery system [26] is highlighted next to each colour-coded step of the
general strategy. The use of the water-soluble
polyaspartic acid drug carrier not only allowed the water-insoluble parent
ferrocene-containing drug to be well soluble in water in concentrations
exceeding 100 g dm−3 but it also increased the cytotoxicity of the active drug as
tested on murine EMT-6 cancer cells from an LD90 value of 500 μg/cm−3 in the case of free
3-ferrocenylbutanoic acid to 80 μg/cm−3 drug content for the polymeric
drug delivery device [27].Scheme 1
Main
picture:  Colour-coded general
synthetic strategy towards the syntheses of polymeric drug carrier
devices.   Right, in blue:  functionalisation of the antineoplastic drug,
ferrocene, to 3-ferrocenylbutanoic acid.
Left:   The synthesis of a specific
ferrocene-containing drug delivery device derived from aspartic acid.   The colour codes in the side portions are
the same as those used in the central main picture.     The
main picture was reproduced with permission from copyright owner “International
Union of Pure and Applied Chemistry.” An adaption of the main part of this scheme
was previously published as follows: J. C. Swarts, in Macromol. Sym., Eds. K. Levon and A. Guiseppi-Elie), vol 186,
pages 123–128, 2002; Copyright Wiley-VCH Verlag GmbH & Co. KgaA, reproduced with
permission.At
least two factors probably contribute to the enhanced activity of the above-mentioned polymeric
drug delivery device.  The first must be
related to the enhanced aqueous solubility of the ferrocene drug. Free ferrocene itself, which is insoluble in
water, has been shown to be completely inactive as cytotoxic agent. The second factor centres around the use of
aspartic acid as monomeric subunit in the main chain of the polymeric drug carrier.   As the cancer cells have a large need for
nutrients such as amino acids, the use of a polyamino acid as drug carrier may
cause the drug carrying device to be absorbed faster and more efficiently by
cancer cells than would be the case for the free drug.A key factor should be observed in the synthetic sequence shown
in Scheme1.  The general strategy in the middle shows the use of difunctional monomers. Aspartic
acid is trifunctional. The use of
tri- or higher-functional polymerization monomers is useful in the synthesis of
polymeric drug carriers, as it provides a free arm where the drug may
eventually be anchored.   Only two of the
three or more monomer functionalities should be used to obtain a linear, highly
flexible, and soluble monomer.   The
other functional group(s) ultimately will provide drug attachment sites.   However, normally the use of tri- or higher-functionalised
monomers will lead to the formation of highly crosslinked, rigid, and
insoluble polymers. In the case of
aspartic acid, this did not happen because the formation of stable
five-membered succinimide rings is thermodynamically highly favoured.   It effectively masked the second carboxylic
acid functional group of aspartic acid from unwanted crosslinking
reactions.   For other tri- or higher-functionalized
amino acids, such as trifunctionalised lysine or tetrafunctionalised cystine
[22], protection of the additional functional groups is required to prevent
formation of pharmaceutically undesired crosslinked polymers.


## 5. Mechanism of Cell Uptake and Drug Release

Uptake by neoplastic (and healthy) cells
of the soluble polymeric device, shown in Scheme1, probably occurs by
endocytosis, in particular, by fluid-phase pinocytosis as described by Duncan
and Kopeček [6].   As shown in Figure 1,
polymers in solution are internalised by means of fluid-phase pinocytosis. Cell internalisation may also occur if the
polymeric drug delivery device binds to surface cell receptors by means of
adsorptive pinocytosis.   They are then
internalized as attachments to the infolding membrane.   Polymeric drug carriers with porphyrins or
phthalocyanines attached to them will probably gain access to neoplastic cells
by means of adsorptive pinocytosis as this would be consistent with the
selective uptake of these macrocycles by cancer cells.    Mixtures of fluid-phase and adsorptive
pinocytosis may also occur.   It was
found that pinocytotic uptake by cells of polymers bearing phenol-containing
side chains was greatly enhanced [28].
Once internalized, fusion of the pinocytotic vesicles with primary lysosomes
takes place to form secondary lysosomes.
Release of the drug from the polymeric drug carrier inside the secondary
lysosomes occurs by enzymatic hydrolysis of the biodegradable amide bonds that
link the drug, ferrocene in Scheme 1, with the polymeric drug carrier, a
polyaspartic acid derivative in Scheme 2.
Primary lysosomes may contain more than 50 hydrolytic enzymes that are
generated in the Golgi apparatus.  Next,
the drug and other useful low-molecular-mass degradation products like amino acids
are released from the secondary lysosomes for use inside the cell nucleous.   Finally, the “spent” polymer main chain and
any other nondegradable material leave the cell by exocytosis.Scheme 2
Syntheses of polymeric drug carrying devices11, 12 (M = Co), and 13.   For PDT applications, M = Zn or Cl–Al would
be more appropriate [18].   All polymers
have a random recurring unit arrangement in the backbone.   The indicated structures are meant to show
building block ratios rather than block polymers.   Opening of the succinimide rings of 6 with nucleophiles R1-NH2 lead to a mixture of α and β ring-opened aspartates.   For convenience, only the α-form is shown.Figure 1
Mechanism
of drug uptake and release inside cells.
Diagram reproduced with permission from copyright owner “International
Union of Pure and Applied Chemistry.”
It was previously published as follows: J. C. Swarts, inMacromol. Sym., Eds. K. Levon and A.
Guiseppi-Elie), vol 186, p123–128, 2002;
Copyright Wiley-VCH Verlag GmbH & Co. KgaA, reproduced with
permission.A key factor emerges from this
discussion. According to this
mechanism, no drug is released outside a living cell because these drugs are not in
contact with appropriate hydrolytic enzymes.
As the folding pattern of the polymeric drug carrier normally
inactivates the drug while polymer-bound, it means no negative side effects can
be induced by the drug prior to release from the polymeric main chain.   In addition, if the polymeric drug delivery
device could be designed in such a way that it is internalized selectively into
only neoplastic cells, the negative side effects associated with chemotherapy
could be circumvented completely.


## 6. The Syntheses of Lysine-Based Polymeric Drug Carriers

Lysine,1, introduces a new dimension into
our discussion of the design of polymeric drug carriers.   To prevent crosslinking reactions, one of
the three lysine functional groups has to be protected.   Here we choose to protect an amino
group.   One can either protect the α- or the ε-amino group, Scheme 2, to obtain lysine
derivatives 3 or 4, respectively.   Only α-amino trifluoroacetyl protected lysine, 3, is obtained by direct treatment of
lysine with trifluoroacetic anhydride in trifluoroacetic acid because the ε-amino group is much more basic than the α-amino group [29]. The
zwitter-ionic structure of lysine, 1,
implies that the ε-amino
group is mainly in the protonated form and thus well protected against
acylation reactions.   However, treatment
of 1 with ethyl
thioltrifluoroactetate under basic conditions [30] according to the method
described by Schallenberg gave Nε-trifluoroacetyllysine, 4, in 33% yield after
recrystallisation from hot water/ethanol in a 2 : 3 ratio. It is advantageous to use the ε-amino group rather than the α-amino group eventually as the drug-anchoring
site because it separates it far enough from the polymer main chain to allow
easy enzyme approach to initiate cleavage of amide bonds to facilitate drug
release from the eventual drug-carrying devices 11–13. If the α-amino group is protected for later drug-anchoring
purposes, the steric bulk effect of the polymeric main chain may be so large
that enzyme-induced drug release will become kinetically very slow.   In addition, it was also previously
demonstrated [26] that drug-anchoring reactions of the type shown in Scheme 2
to generate 11–13 via amide bond formation proceed more effectively with compounds
possessing longer side chains.We
describe here the syntheses, fromNε-trifluoroacetyllysine 4, aspartic acid 5, and polymeric precursors 6 and 7 of polymeric drug carriers 8 and 9, Scheme 2.   We also
describe how the ferrocenyl and phthalocyanine moieties may be covalently
anchored to the carrier polymers 8 and 9 utilising the coupling agent 10 to obtain the polymeric drug
delivery devices 11, 12, and 13.It
was not very easy to predetermine reaction conditions to obtain a specific
targetx/y ratio in polymer 6 because of a tendency towards homopolymerisation by aspartic acid.   In first experiments, 4 and 5 were thermally
copolymerised in a monomer mole ratio of 1:10 (4:5) at 180°C under nitrogen and atmospheric pressure in
the presence of a polyphosphoric acid (PPA) mass fraction of 0.8 for 8 hours. Two products were isolated. The first was extracted with water, and
after dialysis in an
8,000 molecular mass cutoff membrane tubing, 6 was isolated having an x/y = 2.6/1 ratio, and inherent viscosity ηinh = 0.06 dl g−1 in 3%
yield, m.p = 301°C (dec.).
The x/y ratio was established by comparing the 1H NMR signal
integrals at 5.1–5.5 ppm (CH of imide, one proton per closed imide unit),
4.4–4.6 ppm (CH of aspartic acid, the integral values showed only 65% ring
closure of the aspartic acid recurring unit occurred), with that of the
combined β, γ, and δ methylene protons of the lysine recurring unit
(6 protons per lysine recurring unit) at 0.9–1.8 ppm. The remaining water-insoluble residue was
dissolved in DMF and precipitated with ethanol to yield 56% polymer having ηinh(DMF) = 0.07 dl g−1,
m.p  = 286°C (dec.)   In this case, the x/y unit ratio was
established by 1H NMR as 22/1.
The percentage of
aspartic acid ring closure was found to be ca. 97%.   The remainder of material had molecular mass
<8,000 g mol−1, and was dialysed away.When4 and 5 were thermally co-polymerised in a monomer mole ratio of 1:6 = 4:5,
other conditions still being the same, the water-soluble fraction was isolated
in 8% possessing a monomer ratio of x/y = 1.4/1. The DMF-soluble fraction was isolated in 30% yield having a monomer ratio of x/y = 25/1. In another experiment, when the
PPA mass fraction was lowered from 0.8 to 0.5, the water-soluble fraction was very
small, but the DMF-soluble fraction was isolated in 50% yield having also a
monomer ratio of x/y = 25/1.The
above results showed that4 and 5 have a tendency to
homopolymerise. The low lysine content
in each recovered fraction was attributed to the tendency of α-amino acids to form 6-membered
diketopiperazine rings upon heating [31].
In the case of 4, this would
result in termination of any polymerisation process, and account also for the
large % of low-molecular-mass material that was lost upon dialyses. 13C NMR spectroscopy showed that
the trifluoroacetyl protective group did not survive the polymerisation
conditions.   However, loss of the
trifluoroacetyl protective group must have happened in the latter stages of
polymerisation, since no insoluble portion was found in the workup of 6.
Crosslinking was, therefore, ruled out as the cause of the relatively
moderate yields.Further
experiments focussed exclusively on a 1:2 =4:5 monomer mole ratio
in a PPA mass fraction of 0.5 at 180°C for only 2.5 hours at
pressures below 2 torr.   These
conditions resulted in almost no water-soluble fraction (<1%), and a
DMF-soluble fraction (18% yield, decomposition temperature = 269°C)
having a repeating unit ratio of x/y = 7/1 and 29% of aspartic acid fragments
still uncyclised. 13C NMR
showed that the trifluoroacetyl protective group remained intact when using
these shorter reaction times. By
substituting PPA with 85% ortho
H3PO4, repeated
experiments gave products having recurring unit ratios ranging 2/1 < x/y
< 5/1.   Yields were slightly higher
(25%).   The increase in yield and
better recurring unit ratios probably arose because it is easier to mix 4 and 5 to a homogeneous distribution in ortho H3PO4 than in PPA.    Lower reaction
temperatures (140°C) lowered yields substantially to only 6% and
virtually the entire product became water-soluble, even after 7 hours of
reaction time.   Dialyses were again
performed in 8,000
molecular mass membrane tubing.   The
absence of any 1H NMR signals at 5.25 ppm, as well as the presence
of the signal at 4.2–4.6 ppm showed that for this product, ring closure of the
aspartic acid fragment did not occur. It had a repeating unit ratio of x/y =
4/1 as compared to the target of 2/1. 13C
NMR showed that the trifluoroacetyl protective group remained intact under
these conditions of decreased temperatures even though the reaction time was
increased to 7 hours.Nucleophilic
attack of amines on the succinimide rings of polymeric precursor6 takes place with ease, and the
success rate is independent of x/y ratios.
By choosing the correct nucleophiles, the solubility properties of
choice may be introduced to the carrier polymer. Here, the 3-aminopropyl
morpholine unit was used to generate water-soluble 8 in 83% yield after stirring the intermediate product 7 with 2% NaOH solution to remove the
trifluoroacetyl protecting group and dialysis in 8,000 molecular mass cutoff
membrane tubing. Although intermediate 7 can be isolated en route to 8, this is not necessary. One can go directly from 6 to 8 without isolation of 7. 2-Phenylethyl amine was used to generate white, water-insoluble 9 in 90% yield after removal of the
protective group and slow precipitation with water from a DMF solution.To
demonstrate the carrier capabilities of8 and 9, the antineoplastic ferrocenyl
moiety [15] and an example of the family of drugs that may be used in
photodynamic cancer therapy [17], a phthalocyanine, have been anchored on these
polymeric drug carriers. Both ferrocene and phthalocyanine are insoluble in
water when not functionalised, or as carboxylic acid derivatives. Even as
sodium carboxylates, they are poorly soluble in water.   However, once anchored on the polymeric drug
carrier 8, the ferrocene-containing
derivative 11 was soluble in excess
of 30 g/dm3, while the phthalocyanine derivative exceeded an aqueous
solubility of ca. 5 g dm−3 at pH’s more extreme than those found in
the gastrointestinal system (1–8.5) [32, 33].
However, prior to drug anchoring, the ferrocene and phthalocyanine
fragments needed to be modified in such a way that coupling with the available amine
active sites on the polymeric drug carriers 8 and 9 is possible. To
generate a biodegradable bond between drug and polymeric carrier, formation of
a peptide (i.e., amide), saccharide, or nucleotide bonds [34] is desirable.  Here, the amide bond was aimed for as a
biodegradable bond linking carrier to drug.
Thus, 3-ferrocenylbutanoic acid [35] and cobaltotetracarboxyphthalocyanine
[36] were prepared according to known procedures for later reaction with the
amine-containing polymeric carrier devices 8 and 9.Although
polymers8 and 9, the ferrocene-containing derivatives 11 and 13, and the
phthalocyanine-containing derivative 12 are robust, frequently the drug moiety of interest (as opposed to the drug
carrier) may be very labile.   It is advantageous,
therefore, to be able to anchor the drug onto the polymeric carrying device
under very mild conditions in order to preserve labile moieties.   Also, coupling procedures need to be
developed that will allow coupling in media in which both the water-soluble
drug carrier, here 8 and 9, and poorly soluble or
aqueous-insoluble drug may be partially dissolved or suspended.   Coupling with the aid of
O-benzotriazolyl-N,N,N′,N′-tetramethyluronium
hexafluorophosphate, 10, at room temperaturein DMF or DMSO, sometimes with THF or
water as cosolvent, satisfies this requirement [26].    24-hour reactions between
tetracarboxyphthalocyanine and 8 with a x/y ratio of 3/1 in the presence of this coupling agent led to only ca.
10% phthalocyanine binding in 12 as
ascertained by 1H NMR utilising the phthalocyanine phenyl aromatic
proton signal (12 hours per phthalocyanine group) at ca. 7.3 ppm and the –CH2–O–CH2 morpholine signals (4 hours per morpholine group) at 3.6 ppm. This result means z=0.1y.The
closeness of the COOH functional group in the phthalocyanine moiety had two
effects, both associated with an extremely slow coupling rate due to the
closeness of the bulky phthalocyanine core (slow coupling reactions because of
steric crowding have already been commented on, and are described in reference
25). Firstly, it explains the low
binding efficiency (z=0.1y) of cobaltotetracarboxyphthalocyanine to polymeric
drug carrier 8. Secondly, it explains why crosslinking
reactions involving cobaltotetracarboxyphthalocyanine apparently did not take
place to a noticeable extent.   As
ascertained by 1H NMR, no precipitate from the dialysis tubing other
than free cobaltotetracarboxyphthalocyanine (i.e., unbound) could be found.In
contrast, 3-ferrocenylbutanoic acid was anchored to a derivative of8 having a x/y ratio of 2/1 ca. 70–80% effectively (i.e., zaverage=0.75y) within 1 hour at room
temperature to give 11 as
product.   This result was obtained by
comparing 1H NMR signals (obtained in D2O) of the –CH2–O–CH2 morpholine signals (4 hours per morpholine group) at 3.53 ppm with those of the
CH3 substituent in 3-ferrocenylbutanoic acid at 1.1 ppm (it is a
well-defined peak that can be uniquely identified next to the lysine signals),
the combined β, γ, and δ methylene protons of the lysine recurring unit
(6 protons per lysine recurring unit) at 1.2–1.6 ppm, and the aspartate CH
protons at 4.2–4.5 ppm.   An atomic
absorption iron elemental analysis for 11 with x/y = 2/1 showed an iron content of 4.8% which corresponds to z=0.67y.
CHN elemental analyses were not
performed.Coupling
of 3-ferrocenylbutanoic acid to a derivative of9 with x/y ratio 3/1 to obtain 13 in DMF as solvent was more successful, probably because there was no solvent
water present during the coupling reaction, see experimental.   By comparing the integral values of the
phenyl group 1H NMR signals (in DMSO-d6−x) at 7.15 ppm
with those of the CH3 substituent in 3-ferrocenylbutanoic acid at
1.2 ppm (it is a well-defined peak that can be uniquely identified between the
lysine signals) and the combined β, γ, and δ methylene protons of the lysine recurring unit
(6 protons per lysine recurring unit) at 0.9–1.6 ppm, it was ascertained that
the ferrocenyl group was anchored 94% efficiently, that is, z=0.94y.  An atomic absorption iron elemental analysis
for 13 with x/y = 3/1 showed an iron
content of 5.08% which corresponds to z=0.87y.   CHN elemental analyses were not performed.The
water-insoluble polymer12 is of
particular interest because it demonstrates two separate functional moieties
can be supported on the same polymer drug carrier, here phenyl and
ferrocenyl. This begs the
question:  “can more than one
antineoplastic group be anchored on the same polymeric drug carrier?”    If this would be possible, combination
therapy would be possible without administering more than one type of drug in
separate doses to a patient. The synergistic effect usually associated with
combination therapy [36, 37] would then be potentially obtainable in a single
drug dose by binding to the carrier polymer all the drugs required.   This topic is at present being
researched.   Biological studies are at
the moment in progress to determine if present lysine-containing polymeric drug-carrying
devices 11 and 12 can compare in efficiency with previously described polyaspartic
acid drug-delivering systems [26].


## 7. Experimental

Equipment and materials
All organic solvents were distilled prior to use, water was double-distilled. Chemicals were from Merck & CO., Inc. (NJ,
USA)
or Sigma-Aldrich and used without further
purification.   Melting points are
uncorrected. NMR measurements, at 298 K, were recorded on a Bruker Advance DPX 300 NMR spectrometer.   Chemical shifts are reported asδ values
relative to SiMe4 at 0 ppm. Infrared spectra were recorded on a Hitachi
270–50 infrared spectrometer in a KBr
matrix.   Viscosity measurements were
made in a Cannon-Fenske tube at 35.2°C in water or DMF. Dialysis was performed in 8,000 molecular mass cutoff
cellulose membrane tubing. Iron
analyses were performed on a Varian Techtron atomic absorption spectrometer at
272 nm.Syntheses
Nε-trifluoroacetyllysine, 4, [30] and cobalto
tetracarboxyphthalocynanine [36] were prepared as described before.A representative example of the synthesis of polymer6:   Aspartic acid (1.331 g; 10 mmol), Nε-trifluoroacetyllysine (1.211 g; 5 mmol),
and polyphosphoric acid (2.542 g) were thoroughly mixed
in a 250 cm3 round-bottom flask.   The flask was
mounted on a rotating evaporator, and lowered (while rotating slowly) into an
oil bath preheated to 180°.
The pressure was reduced after 5 minutes to below 2 torr with the aid of
a mechanical pump. After 2.5 hours of
slow rotation the reaction mixture was allowed to cool and thoroughly extracted
with water. Dialyses of the water
extract for 16 hours gave after freeze drying only a negligible quantity of
product. The remaining residue after
water extraction was dissolved in 20 cm3 DMF. Precipitation by ethanol afforded 0.38 g (18%) of 6; ηinh(DMF) = 0.08 dl g−1,
x/y = 7/1 (see discussion text for conditions to obtain other x/y ratios); m.p.
295°C (dec.). 1H
NMR (DMSO-d6-x):  0.9–1.8 (6H;
β, γ and δ CH2’s of lysine); 2.1 (3.8 H; CH2 of open aspartic acid fragment);  2.6–2.9
(4.8 H; CH of succinimide); 3.0–3.4 (4.8 H; CH of succinimide);  4.4–4.6 (1.9 H; CH of aspartic acid
fragment); 5.1–5.5 (4.8 H; CH of succinimide). 13C NMR (DMSO-d6-x): 110–122, q, CF3;
155-156, q, CO̲CF3.
IR:   1798, 1720 (C=O); 1219,1189
(CF3).Polymers7, and 9: A solution of 0.21 g [0.5 mmol,
ηinh = 0.08 dl g−1, x/y =
3/1] of 6 and 2-phenylethyl amine
(0.202 g; 2 mmol) in 5 cm3 DMF was stirred for 5 hours at ambient
temperature.   The newly formed 7 was not isolated (although it can be
by precipitation with ethanol or water), but stirred for 5 hours with 20 cm3 of a 2% NaOH solution.   The resulting
clear reaction mixture was dialysed for 16 hours, and freeze dried to give 0.24 g (90% yield) of 9 as a white solid
which would not redissolve in pure water, but was soluble in DMSO and DMF; ηinh(DMF) = 0.07 dl g−1;
x/y = 3; m.p 275°C (dec.).Polymer8:  This polymer was obtained
in exactly the same way as polymer 9,
but by replacing 2-phenylethyl amine with 3-aminopropyl morpholine (0.289 g; 2 mmol), and the x/y ratio of 6 was
2/1.  Characterisation data for 8 : yield = 83%; ηinh = 0.06 dl g−1; x/y =
2; m.p. 196°C (dec.).
Unlike 9, this polymer
remained water-soluble after dialysis and freeze drying.Polymer11:   To a solution of 0.2g
(0.24 mmol; ηinh = 0.06 dl g−1; x/y = 2) of 8 in 1 cm3 water was added (in the correct order) 0.035 g (0.35 mmol)
triethylamine, a solution of 0.068 g (0.25 mmol) 3-ferrocenylbutanoic acid in 1 cm3 THF (the THF may be replaced with DMF) and 0.114 g (0.3 mmol) of
O-benzotriazolyl-N,N,N′,N′-tetramethyluronium
hexafluorophosphate, 10.   The mixture was stirred for one hour at room
temperature during which time the heterogeneous mixture homogenized.  Water (10 cm3) was added to the
reaction mixture before it was centrifuged, dialysed for 16 hours in an 8,000
molecular mas cutoff membrane tubing and freeze dried to give 0.11 g  (44%) of 11; ηinh = 0.04 dl g−1; x:y:z = 2.00:0.35:0.65;  m.p. 88°C, dec. at 105°C;
Fe anal. 4.8% (req. 4.8% for z=0.65y). 1H NMR (D2O):
1.0–1.2 (0.65 of 3H, CH3); 1.2–1.6 (6H;
β, γ and δ CH2’s of lysine); 3.53 (8H, CH2–O–CH2 of morpholine), 3.9–4.2 (0.65 of 9H, C10H9Fe); 4.2–4.6
(3H, CH of aspartate). IR/cm−1:
1660 (C=O); 1550 (NH).Polymer12: The same procedure was used as for 11 except that the solvents were all DMF, 3-ferrocenylbutanoic acid
was replaced with an equivalent amount of cobalto tetracarboxyphthalocyanine
and the reaction time was increased to 24 hours.  Characterisation data: 44% yield; ηinh = 0.04 dl g−1; x:y:z
= 2.0:0.9:0.1;  m.p. 194°C
(dec.); 1H NMR (D2O): 1.2–1.6 (6H; β, γ and δ CH2’s of lysine); 3.53 (8H, CH2–O–CH2 of morpholine); 4.2–4.6 (3H, CH of aspartate) 7.3–8.3 (0.1 of 12H, phthalocyanine
aromatics); IR/cm−1: 1720, 1660 (C=O); 1550 (NH).Polymer13: The same procedure was used as for 12, except that 8 was
replaced by an equivalent amount of 9, tetracarboxyphthalocyanine was
replaced with a solution of 0.068 g (0.25 mmol) 3-ferrocenylbutanoic acid
dissolved in 1 cm3 DMF and the reaction was allowed to proceed for 1
hour only.   Characterisation data: 64%
yield; ηinh = 0.04 dl g−1; x:y:z = 3.00:0.13:0.87; m.p. 180°C
(dec.); Fe anal. 5.1% (req. 5.1% for z=0.87y. 1H NMR (DMSO-d6-x): 1.2
(0.87 of 3H, CH3); 0.9–1.6 (6H; β, γ and δ CH2’s of lysine); 4.1-4.2 (0.87 of
9H, C10H9Fe); 4.2–4.6 (4H, CH of aspartate). 7.1–7.3 (15
H, C6H5). IR/cm−1:   1655 (C=O); 1553 (NH).


## 8. Conclusions

In summary, it was shown that upon using the thermal polymerisation technique,
aspartic acid andNε-trifluoroacetyllysine
both have a tendency to homopolymerise.
This tendency becomes stronger as reaction conditions become harsher,
such as higher temperatures, longer reaction times, or lowering of pressures.   Less harsh conditions diminish the tendency
to homopolymerisation but also reduce yields.
Ring closure of the aspartic acid fragment becomes progressively less
efficient when milder reaction conditions are employed.  It was also shown that the trifluoroacetyl
protective group is less stable at elevated temperatures for prolonged periods
of time.   Chemical removal of the
trifluoroacetyl protective group was achieved under mild alkaline conditions to
liberate free amino groups on side chains of lysine-containing potential
polymeric drug carriers.   Coupling of a
carboxylic acid-functionalised ferrocene with the amine-containing polymeric
drug carrier was effectively achieved utilising O-benzotriazolyl-N,N,N′,N′-tetramethyluronium
hexafluorophosphate as coupling agent.
Coupling reactions were more efficient in the absence of water as cosolvent.   Coupling of cobaltotetracarboxyphthalocyanine
was much less successful.   This was
presumably due to the poor solubility of cobaltotetracarboxyphthalocyanine in
the required solvents.



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*Source: 217573-2007-12-06.xml*