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naturally occurring canine cancers. Herein, we discuss the molec-
ular modeling studies, synthesis (Scheme 1), and reaction kinetics
of five-membered ring and six-membered ring formation of
cisplatin chelates with HA-Lys in water. A key feature of our
approach is generating the cisplatin chelates in water using a
photoremovable protecting group (PPG) [8,9]. These analogues
substantially sustain the release kinetics of cisplatin from the car-
rier and result in more sustained delivery of active drugs.
Ac-homo-Lys [mixture of 11 (~80%) and 12 (minor)] was reacted
with HA to afford HA-pHP-Ac-homo-Lys, 13. Photolysis followed by
dialysis was performed to generate HA-Ac-homo-Lys, 14. Resulting
conjugate, 14 was then reacted with cisplatin (diaqua) to produce
the final product of HA-Ac-homo-Lys-Pt conjugate, 15 as described
above.
In both cases, a change in color from yellow to beige and fall in
pH during the coupling of Pt-diaqua to HA-Lys were useful to
monitor the kinetics of the drug conjugate formation (7 vs. 15).
Noticeable beige coloration was observed in the reaction mixture
for the synthesis of drug conjugate 15 overnight, whereas it took
more than 24 h for drug conjugate 7 to develop a similar appear-
ance in the reaction mixture. A sharp initial drop in pH occurred for
both reactions, which was adjusted back to ca. pH 5 with NaOH. The
reaction mixture of conjugate 15 required several more additions of
NaOH over 48 h to maintain a pH of ca. 5, whereas the reaction pH
of conjugate 7 was nearly constant for 36 h. The Pt loading degrees
of conjugates 7 and 15 were determined to be 5% and 6%, respec-
tively, by inductively coupled plasma mass spectrometry (Agilent
7500a ICP-MS, method described in Ref. [6]).
In our design of new sustained release of drug conjugates using
a ringer linker chemistry, we attempted to avoid or circumvent the
inherent instability of direct conjugation of cisplatin onto the car-
boxylates of hyaluronic acid, as the resulting conjugate had a short
release half-life (10 h in PBS) in our previous studies [10]. Our ul-
timate goal was to develop a rapid and scalable synthetic protocol
to generate HA-Lys-Pt conjugates in water for drug delivery appli-
cations, in which the HA-Lys-Pt conjugates demonstrate favorable
pharmacokinetic properties and slower release rates in vivo. As far
as synthesis of these HA-based drug conjugates are concerned, it
was a prudent choice to select linker chemistry, which involves not
only milder and faster reaction conditions, but also potential for
scale-up to the clinical scale. Since the hydrophilic and biode-
gradable polymer HA is pH sensitive, acid and base labile protecting
groups could not be utilized effectively. In this study, we success-
fully demonstrated that the use of photoremovable protecting
groups (PPG; e.g., p-hydroxyphenacyl group, pHP) in the synthesis
of drug conjugates 7 and 15 [8,9]. In this process, light is considered
as a “traceless” reagent to release the substrate [11] (e.g., HA-Lys).
pHP is an emerging PPG with advantageous qualities for these
syntheses, mainly due to its properties of: 1) absorption at wave-
lengths near or above 400 nm; 2) enhanced chemical and photo-
chemical quantum yields; 3) improved rate of release, ideally in the
range of picoseconds to nanoseconds time constants; 4) good water
solubility; and 5) its clean photochemistry. In this study we choose
a derivative of pHP, methyl 5-acetylsalicylate, to induce intra-
molecular H-bonding between hydroxyl group and carbonyl of the
ester functionality (Fig. 2). Therefore, this H-bonding interaction
reduced the unwanted coupling of phenolic hydroxyl group with
the carboxylic acid functional group of HA during the amidation
reaction.
2. Results and discussion
2.1. Molecular modeling studies
Molecular modeling studies were initially performed to probe
the feasibility of five-membered and six-membered ring formation
with cisplatin using Spartan software (version 14) in vacuum
(Fig. 1). In silico calculations for the energy minimized structures
showed that heat of formation for the ring generation with cisplatin
is almost identical (five-membered ring; ꢀ703 hartrees vs ꢀ742
hartrees for the six-membered ring). Therefore, we performed the
synthesis of these second-generation ring-conjugates in plain wa-
ter to identify the more readily formed and sustained released
cisplatin conjugate for in vitro and in vivo studies.
2.2. Chemistry
The HA-Ac-Lys-Pt conjugate (with a five-membered ring) and
HA-Ac-homo-Lys-Pt conjugate (with a six-membered ring) were
prepared as illustrated in Schemes 2 and 3. Synthesis of the HA-Ac-
Lys-Pt conjugate 7 was accomplished by first amidation of the
caged Ac-Lys with HA in water using a p-hydroxyphenacyl (pHP)
group as the photoremovable protecting group, followed by reac-
tion of cisplatin (diaqua form) in water. The synthesis was begun
with readily available starting material, methyl 5-acetylsalicylate 1.
a
-Bromination of 1 with copper (II) bromide gave 2 (85%), which
was then reacted with Ac-Lys(Boc)-OH in MeCN to afford pHP caged
Ac-Lys(Boc), 3. Boc deprotection gave pHP-Ac-Lys 4 in good yield
(85%) which was reacted with HA (75 kDa) in water to afford HA-
pHP-Ac-Lys conjugate 5. The pHP group was removed by photol-
ysis in water at ~300e350 nm. The resulting photoproducts were
removed by dialysis to afford HA-Ac-Lys 6, which was then con-
verted to the HA-Ac-Lys-Pt conjugate 7 at 50 ꢁC, using cisplatin
(diaqua form) as reported previously [6].
Synthesis of HA-Ac-homo-Lys-Pt conjugate 15 was performed
using a similar approach as described above. Briefly, pHP caged
Fmoc-homo-Lys(Boc)-OH 8 was synthesized from the pHP a-bromo
analog 2 in good yields (86%). Fmoc deprotection with care gave 9,
which was reacted with acetyl chloride to generate the acetyl
protected analog 10 (92%). After removing the Boc protection, pHP-
Previously we reported that HA-Ac-Lys could be utilized to
conjugate cisplatin forming a robust five-membered ring with the
COOH group and nitrogen of N-acetyl group of lysine linker [6].
However, this method involved the reaction between HA-
tetrabutylammonium salt and N-Ac-Lys in DMSO, which is diffi-
cult to scale up and time-consuming to purify. Industrial scale
batches of polymer drug conjugates are typically produced under
aqueous condition and purified and concentrated using a tangential
flow filtration system. Therefore, we have developed the syntheses
discussed in this paper in water as an improved method, which
involves a PPG in the synthesis. To further improve the reaction
times and Pt loading degree, we have attempted to optimize the
synthesis using Ac-homo-Lys as the linker, which forms a six-
membered ring with cisplatin. Six-membered non-aromatic
Scheme 1. Synthesis of HA-Lys-Pt via five-membered and six-membered rings.
Senadheera, Sanjeewa N.
