Tuning the pH sensitivities of orthoester based compounds for drug delivery applications by simple chemical modification

Article abstract of DOI:10.1016/j.bmcl.2010.02.035

Orthoesters are acid-sensitive moieties that allow substantial structural diversity for biological applications including drug delivery. Here, the pH-sensitivity of a range of novel orthoester based compounds was compared in the range 7.5-4.5 that is characteristic of the increased acidification during endocytosis. We find that simple modifications close to the orthoester had major effects on both the rate and extent of hydrolysis, suggesting this could be exploited for activating drug delivery systems on endocytic pathways.

Full text of DOI:10.1016/j.bmcl.2010.02.035

Bioorganic & Medicinal Chemistry Letters 20 (2010) 2200–2203
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Tuning the pH sensitivities of orthoester based compounds for drug
delivery applications by simple chemical modification
*
Hélène Bruyère, Andrew D. Westwell, Arwyn T. Jones
Welsh School of Pharmacy, Cardiff University, Redwood Building, Cardiff CF10 3NB, Wales, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
Orthoesters are acid-sensitive moieties that allow substantial structural diversity for biological applica-
tions including drug delivery. Here, the pH-sensitivity of a range of novel orthoester based compounds
was compared in the range 7.5–4.5 that is characteristic of the increased acidification during endocytosis.
We find that simple modifications close to the orthoester had major effects on both the rate and extent of
hydrolysis, suggesting this could be exploited for activating drug delivery systems on endocytic
pathways.
Received 16 December 2009
Revised 8 February 2010
Accepted 8 February 2010
Available online 13 February 2010
Keywords:
Orthoester
Ó 2010 Elsevier Ltd. All rights reserved.
pH sensitive linkers
Drug delivery
Polymer based architectures have been extensively studied as
backbones for drug delivery systems.1,2 The strategy is often based
on their conjugation to a therapeutic cargo that is then delivered to
the inside of cells by endocytosis.3–5 There is often a necessity for
the therapeutic to be released from the polymer backbone for it
to then reach the cytosol and possibly other intracellular targets.
An approach to this design is to use the considerable drop in pH
that molecules encounter as they are trafficked through endocytic
pathways.6 These are used by cells to internalize molecules that
will then encounter a drop from extracellular pH 7.3–7.5 to 4.5–
5.0 in lysosomes.7 These organelles and to a lesser extent late
endosomes constitute a strong hydrolytic environment, thus there
may be a requirement to release the therapeutic early during endo-
cytosis (pH 5–6) if it is labile to these conditions.
Several pH sensitive linkers have been studied in this context,
including hydrazones,8 cis-aconityl,9 vinyl ethers,10 and acetals.11
Also included here are orthoesters, and to date these are shown
to be one of the most acid-labile functionalities available.12 As
noted, drug delivery systems may need to be activated at a specific
pH, considerably higher than that encountered in lysosomes. A
slightly acidic pH has also been observed in tumours13 and inflam-
matory tissues14 thus there may also be an interest in designing pH
specific systems for mediating effects in these environments.
Orthoesters are of particular interest in attempting to achieve this
goal because they can be modified with respect to structural pat-
terns or stereochemical, stereoelectronic and steric factors.15–17
There are numerous reports describing the behaviour of these in
acidic conditions and examples include poly(orthoesters) and
other orthoester- or diorthoester–lipid conjugates, designed for
delivery of biologically active cargo through endocytic path-
ways.18–21 Pure chemical and mechanistic studies tend to be per-
formed in organic or mixed organic/aqueous solvents and rarely
consider the efficiency of the system in an aqueous environment
or with respects to time and temperature. Whereas those inter-
ested in drug delivery rarely focus on characterizing a range of dif-
ferent structures.
Specifically towards the design of a polymer-peptide based drug
delivery system that is sensitive to acidic pHs found on endocytic
pathways,22 we designed, synthesized and measured the rates of
hydrolysis of different and novel model compounds containing
an orthoester linker. For proof of concept towards the design of
polymer-peptide conjugates, selected structures were linked to
diethylene glycol as a template polymer for polyethylene glycol
(PEG) and phenylalanine as a peptide substitute. Our studies high-
light that simple modifications on the orthoester backbone have a
major influence on hydrolysis profiles that is then transferable to
more complete drug delivery systems. This may allow a high de-
gree of flexibility with respects to pH activation that may, depend-
ing on the biological application, be a requirement.
The synthetic template is based on a previous publication21 and
is outlined in Scheme 1. The amino groups of commercially avail-
able amino-diols 1, 4, 5 and 6 were protected as their trifluoroace-
tate derivatives using ethyltrifluoroacetate. Reaction of these
protected amino-diols 2 and 7–9 with trimethyl orthoformate un-
der p-toluene sulfonic acid (PTSA) catalysis provided the orthoester
building blocks (3 and 10–12) primed for diethylene glycol and
phenylalanine functionalisation. Transetherification using diethyl-
* Corresponding author. Tel.: +44 0 2920 876431.
E-mail address: jonesat@cardiff.ac.uk (A.T. Jones).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.02.035

H. Bruyère et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2200–2203
2201
O
OH
OH
O
H
N
H
N
(ii)
(i)
(i)
OH
O
F3C
OH
F3C
H2N
H2N
OH
O
O
3
2
1
O
OH
O
O
O
(ii)
O
OH
R1
OH
R1
F3C
N
F3C
N
R
R1
10-12
H
R
R
H
4 (R,R1=H)
7 (R,R1=H)
5 (R=H,R1=Ph)
6 (R=Me,R1=H)
8 (R=H,R1=Ph)
9 (R=Me,R1=H)
O
O
O
O
NHAc
NH
O
O
O
O
(iv), (vi)
(iii)
H
N
O
3
F3C
O
O
O
O
O
14
13
O
O
O
O
10 (R,R1=H)
(iii)
(iv), (v) or (vi)
O
R1
O
11 (R=H,R1=Ph)
F3C
N
N
H
O
O
R
R
12 (R=Me,R1=H)
H
R1
O
O
NHAc
15 (R,R1=H)
18 (R,R1=H)
16 (R=H,R1=Ph)
17 (R=Me,R1=H)
19 (R=H,R1=Ph)
20 (R=Me,R1=H)
Scheme 1. Synthesis of orthoester model compounds 14 and 18–20. Reagents and conditions: (i) ethyltrifluoroacetate, THF, room temperature; (ii) trimethylorthoformate,
PTSA, CH2Cl2, room temperature; (iii) diethylene glycol monomethyl ether, PPTS, toluene, reflux; (iv) 1 M NaOH, THF, room temperature; (v) N-Ac-
DMAP, CH2CL2, THF, room temperature; (vi) N-Ac- -phenylalanine, EDCI, CH2Cl2, THF, room temperature.
L
-phenylalanine, EDCI,
L
ene glycol monomethyl ether gave the intermediate orthoesters 13
and 15–17. The trifluoroacetyl protecting group was then removed
by the action of aqueous NaOH in THF, and pseudo-peptidic cou-
pling with N-acetyl phenylalanine provided the necessary orthoes-
ters 14 and 18–20. The specified conditions for synthesizing
compound 18 were not suitable for generating the other model
compounds unless DMAP was omitted as a catalyst. Due to acid-
sensitivity and the fact that no starting material was detectable
by 1H NMR after work-up and concentration, the intermediate
compounds were not purified. Indeed, attempts at purification
after trans-etherification did not lead to significant improvements
in yield of the final compound. Similarly, purification of the final
compounds (14 and 18–20) by flash chromatography on silica gels
was difficult thus neutral aluminum oxide was used as an alterna-
tive. Of note for NMR analysis is that several isomers were identi-
fied at different steps as the orthoester cycle is formed. From this,
four different orthoester model compounds were prepared (14, 18,
19, 20).
Exocyclic protonation
O
H2O
-H+
O
OH
R'
H
R'
H
O
O
H+
-ROH
O
O
O
H
O
R
H
R'
O
H2O
-ROH
R'
OH
-H+
O
H
R
H+
R'
O
H2O
-ROCHO
OH
HO
R'
OH
Endocyclic protonation
Scheme 2. Orthoester hydrolytic pathways.
Two different hydrolysis pathways have been described for
orthoesters depending on whether the first protonation occurs on
an exocyclic or endocyclic oxygen atom.23 The stages are detailed
in Scheme 2 using a six-membered ring orthoester as an example.
It is expected that any stereoelectronic or stereochemical/confor-
mational effect on a model molecule would influence the nature
of the hydrolysis products, their stability and their endocyclic or
exocyclic pathway ratio.16,24
In order to directly measure the hydrolysis ratio between the
parent orthoester and the hydrolysis products by NMR, we used
deuterated solvents to prepare all buffers. These were prepared
from a mixture of 0.5 M citric acid and 0.5 M Na2HPO4 solutions
in deuterium oxide. Two similar 1H NMR methods were then used
to evaluate the stability of the orthoester-containing products un-
der a range of pH values from 7.5 to 4.5. Initially, hydrolysis studies
were carried out for 90 min at 37 °C in single tubes inside the NMR
spectrometer that was previously equilibrated to this temperature.
Alternatively the reactions were performed in a 37 °C water bath
and aliquots were removed at different time periods for NMR anal-
ysis. To stop further hydrolysis these samples were quenched with
0.5 M Na2HPO4 in deuterium oxide (pH 8.5). We confirmed that
this completely inhibited any further hydrolysis irrespective of
the pH of the initial solution and experiments performed in the
laboratory and on the NMR instrument yielded identical data
(not shown). Some experiments were performed at room temper-
ature whereby the same reaction tubes were periodically placed on
the NMR instrument.
The extent of hydrolysis was determined by relative integration
of specific orthoester and newly formed hydrolysis peaks (Fig. 1).
Several peaks were visible in each specific area because of the cre-
ation of different diastereoisomers as mentioned above. The pH of
the hydrolysis mixtures did not change during the reaction

2202
H. Bruyère et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2200–2203
100
80
60
40
20
0
Hydrolysis products
peaks
Orthoester peaks
95 min
85
75
14 pH 4.5
18 pH 4.5
14 pH 6.5
18 pH 6.5
65
55
45
35
25
15
0
20
40
60
80
100
5
time (min)
Figure 3. Hydrolysis profiles of compounds 14 and 18 at 37 °C.
Figure 1. Labeled NMR profiles for orthoester hydrolysis of compound 18 at pH 5.5.
periods. For substituted compounds 19 and 20 that suffered from
low aqueous solubility, it was necessary to use DMSO as a co-sol-
vent for hydrolysis studies. For this, the orthoester compound was
dissolved in DMSO, prior to adding deuterated buffer solutions to
achieve the necessary pH. Using water soluble compound 14 as a
reference we confirmed that DMSO by itself did not influence
hydrolysis (see Supplementary Fig. S1).
Initially, we compared, at a range of pH, the time dependent
hydrolysis of the six-membered ring 18 at ambient temperature.
As seen in Figure 2, these results confirmed that the rate and extent
of hydrolysis were pH dependent. At pH 2.3, 100% hydrolysis oc-
curred in less than 10 min and the same extent of hydrolysis was
observed after 60 min at pH 4.1. We were unable to see greater
than 20% hydrolysis at pH 5.2 and no hydrolysis was observed at
pH >6.4.
100
14
18
19
20
80
60
40
20
0
0
20
40
60
80
100
time (min)
Figure 4. Time-dependent hydrolysis of 14, 18–20 at pH 5.5.
These first results allowed us to progress to performing real
time NMR studies at 37 °C with this compound and we also inves-
tigated the effect of the ring size on the hydrolysis rates at different
pHs by comparing model compounds 14 and 18. Within the exper-
iment time we observed complete stability for both compounds at
pH 7.5. Complete hydrolysis itself was achieved for both com-
pounds at pH 4.5 but while compound 14 was completely hydroly-
sed in 15 min, the six-membered ring (18) required incubation for
55 min to achieve this (Fig. 3). Similarly, at pH 6.5 compound 18
was considerably less sensitive to hydrolysis than its five-mem-
bered ring counterpart. This does not appear to be a universal phe-
nomenon as previous studies have shown a relative insensitivity to
hydrolysis of a five-membered ring orthoester at pH 4 and 5.21
However the immediate environment surrounding the six-mem-
bered ring orthoester was different in that study thus highlighting
the dramatic change in sensitivity that may be mediated by minor
structural modifications.
In order to focus on pH values representing early and late endo-
somes (pH 6–5), the hydrolytic profiles of all model orthoesters
were analyzed at pH 5.5 and at 37 °C. Results in Figure 4 showed
that when comparing substitution patterns (compounds 18–20),
the addition of substituents on two different positions dramatically
decreased the pH-sensitivity of the orthoester. At pH 5.5, com-
pound 19 (R1 = Ph) was insensitive and compound 20 (R = Me)
had a maximum hydrolysis of 20% after 90 min at 37 °C compared
with 45% hydrolysis for reference compound 18. This may be due
to stereoelectronic effects on the orthoester ring or decreased sol-
ubility in aqueous buffers. All six-membered ring compounds 18–
20 were less sensitive to hydrolysis compared with compound 14.
In this study, we showed that structurally diverse orthoester
compounds bearing a protected amino acid and diethylene glycol
(as PEG mimic) show favourable hydrolysis profiles for drug deliv-
ery applications. Our results in particular emphasise that the five-
membered orthoester 14 is a promising model compound showing
complete stability at neutral pH but complete hydrolysis at pH 4.5–
5.5 over a relatively short time period. Overall it may be possible to
design linkers that are fine tuned around the orthoester moiety to
influence the pH sensitivities for a range of biological applications
including drug delivery.
100
80
pH 2.3
60
pH 4.1
pH 5.2
pH 6.4
pH 7.3
40
20
0
Acknowledgements
0
20
40
60
The work was funded by BBSRC Grant D013038 awarded to
A.T.J. We thank the EPSRC National Mass Spectrometry centre
(Swansea, UK).
time (min)
Figure 2. Hydrolysis profiles for compound 18 at ambient temperature.

H. Bruyère et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2200–2203
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