Hydroxypyridinone and 5-Aminolaevulinic Acid Conjugates for Photodynamic Therapy

Article abstract of DOI:10.1021/acs.jmedchem.7b00346

Photodynamic therapy (PDT) is a promising treatment strategy for malignant and nonmalignant lesions. 5-Aminolaevulinic acid (ALA) is used as a precursor of the photosensitizer, protoporphyrin IX (PpIX), in dermatology and urology. However, the effectiveness of ALA-PDT is limited by the relatively poor bioavailability of ALA and rapid conversion of PpIX to haem. The main goal of this study was to prepare and investigate a library of single conjugates designed to coadminister the bioactive agents ALA and hydroxypyridinone (HPO) iron chelators. A significant increase in intracellular PpIX levels was observed in all cell lines tested when compared to the administration of ALA alone. The higher PpIX levels observed using the conjugates correlated well with the observed phototoxicity following exposure of cells to light. Passive diffusion appears to be the main mechanism for the majority of ALA-HPOs investigated. This study demonstrates that ALA-HPOs significantly enhance phototherapeutic metabolite formation and phototoxicity.

Full text of DOI:10.1021/acs.jmedchem.7b00346

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Article
Hydroxypyridinone and 5-aminolaevulinic
acid conjugates for photodynamic therapy
Sinan Battah, Robert C. Hider, Alexander J. MacRobert, Paul S. Dobbin, and Tao Zhou
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 31 Mar 2017
Downloaded from http://pubs.acs.org on March 31, 2017
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Hydroxypyridinone and 5ꢀaminolaevulinic acid
conjugates for photodynamic therapy
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a,c
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d*
Sinan Battah, Robert C. Hider, Alexander J. MacRobert, Paul S. Dobbin, Tao Zhou
a. School of Biological Sciences, University of Essex, Colchester, CO4 3SQ, UK
b. Division of Pharmaceutical Sciences, King’s College London, 150 Stamford Street, London,
SE1 9NH, UK
c. Division of Surgery and Interventional Science, University College London, Charles Bell
House, 67ꢀ73 Riding House Street, London, W1W 7JE, UK
d. School of Food Science and Biotechnology, Zhejiang Gongshang University, Hangzhou,
Zhejiang 310018, P. R. China.
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ABSTRACT
Photodynamic therapy (PDT) is a promising treatment strategy for malignant and nonꢀmalignant
lesions. 5ꢀAminolevulinic acid (ALA) is used as a precursor of the photosensitizer,
protoporphyrin IX (PpIX) in dermatology and urology. However, the effectiveness of ALAꢀPDT
is limited by the relatively poor bioavailability of ALA and rapid conversion of PpIX to haem.
The main goal of this study was to prepare and investigate a library of single conjugates designed
to coꢀadminister the bioactive agents ALA and hydroxypyridinone (HPO) iron chelators. A
significant increase in intracellular PpIX levels was observed in all cell lines tested when
compared to the administration of ALA alone. The higher PpIX levels observed using the
conjugates correlated well with the observed phototoxicity following exposure of cells to light.
Passive diffusion appears to be the main mechanism for the majority of ALAꢀHPOs investigated.
This study demonstrates that ALAꢀHPOs significantly enhance phototherapeutic metabolite
formation and phototoxicity.
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KEYWORDS: Photodynamic Therapy (PDT); 5ꢀaminolaevulinic acid (ALA); iron chelator;
hydroxypyridinone (HPO); protoporphyrin IX (PpIX)
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1
. Introduction
Photodynamic therapy (PDT) is based on the activation of exogenously applied or
endogenously formed photosensitizers by visible light in the presence of molecular oxygen.
Photosensitization results in formation of singlet oxygen, oxidizing cellular macromolecules
photochemical reactions, leading to the damage of a variety of subcellular substrates and cell
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ꢀ4
death.
5ꢀAminolaevulinic acid photodynamic therapy (ALAꢀPDT) utilizes the haem
biosynthesis pathway to transiently produce excess (and clinically useful) amounts of the natural
endogenous photosensitizer protoporphyrin IX (PpIX). This is achieved by the addition of
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,6
exogenous ALA, which enters the haem biosynthesis pathway. The main advantage of ALAꢀ
PDT is the short halfꢀlife of its photosensitizing effects, which reduces the duration of skin
7
photosensitivity and its efficacy using topical administration. However, several factors affect
ALAꢀPDT and limit its clinical potential. In particular, the hydrophilic nature of ALA limits its
8
rate of uptake into neoplastic cells and/or penetration into tissue. Considerable efforts have been
made recently to overcome the bioavailability limitations associated with ALA, which have
mainly been centred on the development of ALA prodrugs with higher lipophilicity, such as
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,9ꢀ15
16ꢀ18
esters
and peptide derivatives.
In simple ester conjugates, ALA is cleaved at the Cꢀ
terminus via interaction with esterases, but in some ALA conjugates cleavage at the Nꢀterminus
15,16
14
is also required via peptidases
and phosphatases. The methyl and hexyl esters of ALA have
been approved for topical treatment of basal cell carcinoma in both Europe and Australia. The
use of different delivery vehicles, such as microspheres, liposomes, conjugate antibodies, has
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also been investigated.
The stage following PpIX production in the haem pathway, is the insertion of ferrous iron
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(Fe ) under the action of the ferrochelatase to convert PpIX into haem. The presence of free
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haem acts as a negative feedback mechanism inhibiting ALA synthesis. The exogenous
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administration of large amounts of ALA bypasses this negative feedback signal. The resultant
accumulation of PpIX within the cells is the rateꢀlimiting step for conversion of PpIX to haem by
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ferrochelatase.
An additional approach to achieve a greater PpIX accumulation involves the
chelation of intracellular iron. As a result the conversion of PpIX to haem is decreased, further
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enhancing the accumulation of PpIX in the cell.
The nonspecific membraneꢀimpermeable
metal chelator ethylenediamine tetraacetic acid (EDTA) has been shown to moderately increase
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PpIX levels in epithelial skin tumors in combination with ALA.
Desferrioxamine (DFO) is
more effective than EDTA at elevating PpIX in cells due to its greater affinity for iron than
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EDTA.
In our studies using 3ꢀhydroxypyridinꢀ4ꢀone chelators (HPO), a combination of ALA and
1,2ꢀdiethylꢀ3ꢀhydroxypyridinꢀ4ꢀone (CP94, 26) in PDT proved to be an effective technique to
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increase the efficacy of ALAꢀPDT within cells in vitro and in vivo.
The concept of exploiting
high affinity iron(III) chelators for ferrochelatase inhibition in order to increase the ALAꢀinduced
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+
3+
PpIX levels is based upon the ability of these compounds to stimulate the oxidation Fe to Fe
at low concentration, with O acting as the oxidant. Compound 26 is a member of the
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hydroxypyridinone family of bidentate iron chelators. It is particularly effective at chelating
intracellular iron due to its lower molecular weight and higher lipophilicity than either DFO or
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EDTA.
However, coꢀadministration of ALA and iron chelator would be limited by the
differing pharmacological properties of the two agents. In contrast, the molecules in which ALA
and HPO agents are coupled via cleavable linkages would release the two agents following
cellular uptake. The enhanced porphyrin levels would therefore arise principally from
simultaneous delivery of the synergistically acting bioactive agents to cell. Indeed, in our
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previous work, it has also been demonstrated that some ALA/HPO conjugates are more efficient
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than ALA and ALA in combination with HPO for PpIX production in some tumor cell lines.
Since HPOs, such as 26, are more effective at chelating intracellular iron than
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desferrioxamine due to the superior cell membrane permeability of HPOs, in the present work,
a range of ALAꢀHPO conjugates were designed and synthesized in order to synergistically
promote intracellular phototoxicity. There is compelling evidence with regards to the important
role of the conjugation bonding, the spacers/linkers types, and length of the spacers for ALAꢀ
7,9ꢀ13,16,17,36,37
PDT, based on the literature and our previous work.
For example, Battah et al.
found that increasing the linker length in ALA dendritic derivatives led to an enhancement of
intracellular porphyrin generation which was ascribed to a reduction in steric hindrance to
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esterase access. However linkers can also affect the lipophilicity of the conjugate which will in
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turn affect its bioavailability.
The goal of this investigation was to optimize and determine the efficacy of several spacers
that control the PDT of ALAꢀHPO conjugates. The chosen iron chelators have similar chemical
structures to that of 2ꢀethylꢀ3ꢀhydroxypyridinꢀ4ꢀone (26) attached to ALA via a variety of links
(
Chart 1). The two moieties were covalently bonded through ester bonds, which were predicted
to hydrolyze enzymatically under intracellular conditions. The efficacy of these ALAꢀHPO
conjugates has been investigated in various cell lines and the kinetics of PpIX fluorescence and
phototoxicity monitored.
2. Results and Discussion
2.1 Chemistry
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The ability of ALAꢀHPO conjugates to penetrate the tumour cell membrane and ease of
liberating free ALA molecule intracellularly are important factors affecting the activity of the
prodrugs. In order to obtain ALA prodrugs with high PpIX generation efficiency and high
phototoxicity against tumour cells, a range of ALAꢀHPO conjugates were designed. (a) HPO has
a similar structure to 26, features a ethyl group at position 2, and is linked to ALA through
different lengths of hydrocarbon spacers (CH ) , n = 2–12) in compounds (7a–7f) via readily
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cleavable ester bond; (b) the same HPO with ethyl group at position 2 is linked to ALA through a
triethyleneglycol spacer in compound (23); (c) the ethyl group of HPO at position 2 was
substituted with 1ꢀhydroxylethyl in compound (12) and a methylamino carbonyl group in
compound (18), and both groups were linked to ALA via ester bonds in these two compounds
with a (CH ) hydrocarbon chain; (d) hexylꢀALA and octylꢀALA were chosen for comparison
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(compounds 24 and 25), respectively (Chart 1).
Preparation of ALAꢀHPO conjugates 7. The synthetic route of ALAꢀHPO conjugates 7
starting from ethyl maltol (1a) is outlined in Scheme 1. The benzylation of ethyl maltol with
benzyl chloride provided 2a in 66% yield. The amino alcohol spacers 3a, 3b and 3c (n=2, 4 and
6, respectively) are commercially available, whereas 3d, 3e and 3f (n=8, 10 and 12, respectively)
were synthesized by the Mitsunobu reaction using triphenylphosphine (Ph P), phthalimide and
3
diisopropyl azodicarboxylate (DIPAD), followed by hydrazination. Condensation of 2a and
amino alcohol 3 under alkaline condition provided the 2ꢀalkylꢀsubstituted 3ꢀhydroxypyridinꢀ4ꢀ
ones (4). The protected pyridinones 4 were coupled with NꢀCbz ALA, which was prepared in
aqueous solution at pH 8ꢀ10 at room temperature in reasonable yield, forming an ester link using
the standard coupling agents 1,3ꢀdicyclohexylcabodiimide (DCC) and 4ꢀdimethylaminopyridine
(DMAP), yielding the protected ALAꢀHPO conjugates 6. Deprotection of 6 was achieved by
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hydrogenation at 40 psi H atmosphere in methanol and ethyl acetate (1:3, by volume) in the
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presence of benzyl chloride to afford the hydrochlorides of ALAꢀHPO conjugates 7. The clogP
values of this series 7aꢀ7f range from ꢀ1.96 to 0.91 (Table 1).
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Preparation of ALAꢀHPO conjugates 12 and 18. The synthetic route of ALAꢀHPO
conjugate 12 is presented in Scheme 2. Conversion of the 2ꢀethyl group of 2a to 2ꢀhydroxyethyl
was accomplished using selenium oxide as a oxidizing agent in phenyl bromide with reflux,
providing compound 8 in 43 % yield; at the same time, another oxidized product 2ꢀacetylꢀ3ꢀ
(benzyloxy)ꢀ4Hꢀpyranꢀ4ꢀone was detected (11 % yield). The newly formed hydroxyl group in
compound 8 was subsequently protected using benzyl bromide in a twoꢀphase reaction using
tetrabutylammonium sulfate as phase transfer catalyst, yielding 9 in 62 % yield. Compound 9
was converted to the pyridinone 10 via condensation with 6ꢀaminoꢀhexanꢀ1ꢀol. Conversion of 10
to 12 was achieved using the similar procedures to that for ALAꢀHPO conjugates 7 (Scheme 2).
The synthesis of ALAꢀHPO conjugate 18 using maltol (1b) as a starting material is shown in
Scheme 3. The methyl group in benzyl protected maltol was selectively oxidized by selenium
oxide to form an aldehyde 13, which was further oxidized to a carboxylic acid 14 in the presence
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of sodium hypochlorite and sulfamic acid. The carboxyl group was activated by DCC/Nꢀ
hydroxysuccimide (NHS) to form an activated ester, which was subsequently coupled to
methylamine, providing a pyranone derivative 15. Insertion of the amino alcohol spacers into the
pyranone ring was achieved by refluxing the corresponding spacer with 15 in an alcoholic
solution of sodium hydroxide at pH 12 to afford the pyridinone 16, which was coupled to NꢀCbz
ALA, providing 17, the latter was subjected to hydrogenation, generating the ALAꢀHPO
conjugate 18 (Scheme 3). The two conjugates 12 and 18 are relatively hydrophilic, the clogP
values being ꢀ1.28 and ꢀ1.65 respectively (Table 1).
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Preparation of the triethylene glycolꢀlinked ALAꢀHPO conjugate 23. The amino alcohol
0 was prepared from triethylene glycol using the Mitsunobu reaction (Scheme 4). Condensation
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of 20 with the benzyl protected ethyl maltol (2a) yielded the derivative 21. Conjugation with
protected ALA using similar methods to that described for the hydrocarbon linked molecule (4)
yielded the desired ester (22), which upon hydrogenation yielded the ALAꢀHPO conjugate (23)
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2.2 Biological evaluation
MCFꢀ7 cells (human breast adenocarcinoma) and MCFꢀ7R cells (doxorubicin resistant
subline, MCFꢀ7/DXR) were chosen to assess the efficacy of the newly synthesized ALA–HPO
conjugates to generate the photoactive, fluorescent photosensitizer PpIX. PDT has been proposed
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in several studies as an alternative in overcoming multidrug resistance (MDR) phenotype.
Therefore, the goal was to compare the accumulation and photosensitization of PpIX in
MCFꢀ7 human breast adenocarcinoma cells and its doxorubicin MDR resistant counterpart,
which is characterized by the overexpression of Pꢀgp. Human KB cells were also selected, which
are derived from an oral epidermal squamous cell carcinoma of the mouth. ALA has been used
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for head and neck cancer, both topically and with oral administration.
2.2.1 Concentration dependence profile
The fluorescence intensity of PpIX generated in MCFꢀ7R cells after exposure of ALA or
HPOꢀALA conjugates for 6 h is presented in Figure 1. Fluorescence spectra were consistent
with the production of PpIX, and the peak emission at 635 nm was recorded for each compound.
The protophyrin PpIX fluorescence induced by exposure to the majority of ALAꢀHPO
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conjugates indicated an efficient internalization and metabolism within all the investigated cell
lines. PpIX production exhibited a dose dependent response for all ALAꢀHPO single conjugates.
Porphyrin generation was enhanced over a range of concentrations between (1–100 ꢁM) after 6 h
incubation for the compounds with long hydrocarbon spacers between ALA and HPO with the
ethyl group at position 2 (Figure 1). PpIX generation was elevated significantly from 7b (n = 4
clogP ꢀ1.42) to 7f (n = 12, clogP 0.91) by factor of about 2ꢀ25, reaching the maximum level with
compound 7e (n = 10), while compound 7a failed to increase PpIX greatly as compared to that
produced with ALA itself at the highest concentration (100 ꢁM). This effect is attributed to the
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graduation of lipophilicity and the changes in steric hindrance around the ester hydrolysis sites.
The presence of the HPO moiety within the conjugates enhanced the fluorescence intensities for
7c, 7d, and 7e compounds when compared with those of compounds 24 and 25, having same
43
esters (hexyl and octyl ALA esters). The ALAꢀHPO featuring the triethylene glycol spacer 23
with a clogP value of ꢀ2.37 elevated PpIX production levels to over 20 fold higher than ALA at
100ꢁM. PpIX accumulation induced by HPOs with the side chain, 2ꢀethyl (compound 7c) was
superior to those HPOs with the modified side groups (hydroxyethyl and amido ꢀCONHCH ) of
3
compounds 12 and 18, respectively. Although, compounds 7c, 12, and 18 were linked to ALA
via the same spacers (CH ) , compound 7c produced PpIX fluorescence >15 fold that of ALA,
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compound 12 yielded >10 fold, while compound 18 failed to enhance the level of PpIX
production with respect to PpIXꢀALA.
2.2.2 Fluorescence kinetics
ALAꢀHPOs single conjugates were clearly more efficient than ALA with respect to PpIX
generation over an incubation period up to 24 h in KB cell lines (Figures 2). PpIX fluorescence
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kinetics of ALAꢀHPOs with long hydrocarbon spacers, (7d, 7e and 7f) continued to exhibit
significant enhancement in PpIX generation over time.
The compounds with longer hydrocarbon spacers, demonstrated efficient PpIX production
which was further enhanced by the presence of HPO (with a similar structure to 26) over
incubation time ranging between 2 h and 24 h (Figure 2). The existence of HPO at the other end
of the spacers (n =6, n =8, n =10) probably led to a reduction of the toxicity of the spacers on
ester formation with ALA. On the other hand HPO could boost the penetration of conjugates
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through the cell membrane. ALA esters with hydrocarbons over n = 6 have reduced
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bioavailabilities as a result of being trapped within bilayers.
There have been many reports
detailing the mechanistic uptake of ALA and ALA esters, which showed that the mechanism of
7
,9ꢀ12
uptake of ALA is via active transport and its esters adopt a passive diffusion pathway.
We have compared the production of PpIX in the KB cell line at 37 °C and 4 °C to
investigate whether ALA and ALAꢀHPOs are transported into the cell primarily by active and
passive transport mechanisms, respectively. An active transport system would be significantly
inhibited by lowering the temperature to 4 °C, whereas involvement of passive transport would
still enable uptake at lower temperatures. The results presented in Figure 3 show that
incorporation of ALA and ALAꢀHPOs into KB cells is temperature dependent: i.e. when ALAꢀ
HPOs uptake was measured at 4 °C, a marginal reduction of PpIX fluorescence was observed, up
to ≈ 5% for most of ALAꢀHPOs, except PpIX generated by 12 which impaired by ca. ≈ 50%. In
contrast PpIX fluorescence produced by ALA was found to be significantly reduced up to 90%,
4
5
which is in good agreement with previous studies. Thus, we can conclude that an incorporation
of ALA and ALAꢀHPOs into the KB cells (and by extrapolation the MCFꢀ7 and MCF7R cell
lines also) involves two different mechanistic pathways, with passive diffusion predominating as
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the main pathway for ALAꢀHPOs single conjugates.
A comparison of PpIX fluorescence kinetics produced upon a 24 h timeꢀcourse treatment of
compounds 7c, 12 and 18 highlighted the significant role of the HPO in PpIX production (each
compound contained a hexyl linker to ALA but different structures of HPO). Compound 12
exhibited higher porphyrin fluorescence levels than 7c, while compound 18 was not effective at
enhancing PpIX accumulation. Surprisingly, the hydrophilic conjugate 23, with a
triethylenglycol spacer, was found to be highly effective in all cell lines, particularly at longer
time points (Figures 1, 2 and 4).
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For compounds 7, there is a clear relationship between the length of the hydrocarbon linkers
and PpIX accumulation (Figures 1, 2 and 4), the effect increasing over the range n=2 to n=10,
whereas with n=12 a relative decline was found in PpIX accumulation probably due to the
relatively low solubility of this compound. The optimum compound (7e) has a clogP value of
0.73. The compounds 7d, 7e, 7f and 23 are all more effective than the hexylꢀ and octyl esters of
ALA (24 and 25) (Figure 1).
The length of the hydrocarbon link is also directly correlated with the clogP values, and in
view of their molecular size and range of clogP values, it is most probable that they gain access
1
2
to the cytoplasm of cells by nonꢀfacilitated diffusion. However the high efficiency of 23 (clogP
= ꢀ2.37) would indicate that this molecule is a substrate for transport, a possibility, which is
currently under investigation.
Although coꢀadministration of ALA and HPOs separately (ie without conjugation) was
found to enhance PpIX levels when compared to ALA alone, the additive effect was significantly
weaker than when the related conjugates were investigated as shown in Figure 4. This
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corresponds well with our previous studies in cells using coꢀincubation of ALA and 26. The
use of 26 in combination with ALA and methyl ALA ester also produced significant
enhancements of PpIX fluorescence in human glioma cells. At the highest concentrations of each
prodrug, 26 enhanced PpIX fluorescence significantly at 3 h for ALA and by more than 50% at 6
h for ALA methyl ester. In our study, an increase in PpIX levels by approximately a factor of
two was observed in both MCFꢀ7 and MCFꢀ7R cell lines in the presence of the conjugates when
compared to the equivalent separate additions after 6 h incubation (Figure 4A). The synergistic
effect on PpIX enhancement was also confirmed in KB cells over a 24 h time course study,
employing ALAꢀHPO conjugates compared to coꢀadministered ALA and HPOs. There were 2ꢀ3
fold increases in PpIX production as a result of ALAꢀHPO conjugate incubation over the coꢀ
administered ALA and HPOs as a mixture, particularly at the longer incubation times of 6 and 24
h (Figure 4B).
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ALAꢀHPO conjugates developed in the present investigation were found to be more effective
in PpIX production than our previously reported compounds, in which ALA moiety was coupled
37
to HPO moiety via an amide linkage. For instance, after incubation with KB cells for 24h,
conjugates 7d, 7e and 7f (50ꢁM) could produce PpIX level with 8, 10.2 and 9.2 fold higher than
ALA; whereas the best compound in the previous study could only generate 2.9 fold higher
3
7
porphyrin levels than ALA. This is probably due to the fact that esterase activity in cells is
relatively high, thus rapidly generating free ALA and HPO, while peptidase activity in cells is at
a relatively lower level. The relative solubility of the compounds in the cell culture medium with
the ester linkage was much higher than with the amide linkage, which may also have adversely
affected the efficiency of the amide compounds.
Uehlinger et al. investigated ALA and a range of ALA esters (methyl, ethyl, butyl, hexyl,
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octyl and cycloꢀhexyl esters) for the efficacy in producing PpIX in some tumour cell lines (A549,
T24, J82 and BEASꢀ2B cells), and found that in most cell lines ALA hexyl ester showed the
11
most efficient PpIX formation. Wu et al. also demonstrates that the cellular PpIX content in 1
mM ALA incubated nasopharyngeal carcinoma (NPC) cells is similar to that in 0.03 mM ALA
hexyl ester incubated cells, reflecting that ALA hexyl ester is more efficient than ALA for
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1
inducing PpIX production. However in the present study, compounds 7c, 7d, 7e and 7f were
demonstrated to be even more efficient in PpIX formation than the ALA hexyl ester (24) (Figure
1).
Intracellular PpIX fluorescence of PAM212 keratinocyte cells has been reported to be
enhanced by a factor of 5 after 6 h incubation with 0.01 mM of an ALAꢀpeptide conjugate, Acꢀ
LꢀPheꢀALAꢀOMe, compared with ALA at the same concentration, where the amino acid is
16
conjugated at the Nꢀterminus of ALA. More extensive investigation of peptide prodrugs of
ALA (AcꢀXaaꢀALAꢀOR, where Xaa is an αꢀamino acid) indicated that some of the peptide
prodrugs are more efficient in inducing PpIX accumulation than ALA in PAM212 and also A549
17
and Cacoꢀ2 cell lines. For instance, at 0.1 mM, after incubation with PAM212 keratinocyte
t
cells for 6h, peptide prodrugs AcꢀLꢀLeuꢀALAꢀOMe, AcꢀLꢀPheꢀALAꢀOMe, AcꢀLꢀGluOBuꢀ ꢀ
ALAꢀOMe and AcꢀLꢀPheꢀMetꢀOMe could produce PpIX level with 7.5, 4.4, 6.5 and 5.9 fold
1
7
higher than ALA.
Recently, Babič et al. have reported that two ALA hexyl ester analogues, PꢀALAꢀHex and
PSIꢀALAꢀHex, which were obtained by modification of the Nꢀterminus amino group, displayed
reduced acute toxicity compared to the ALA hexyl ester with superior dose response profiles of
1
4
protoporphyrin IX synthesis and fluorescence intensity in human glioblastoma cells in vitro.
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2
.2.3 Phototoxicity studies
The phototoxicity of ALA and ALAꢀHPO conjugates, incubated with tumor cells for 4h and
2
irradiated with blue light (2.5 J/cm ), was investigated over a range of concentrations between 2ꢀ
0
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100 ꢁM. The data for KB cells are presented in Figure 5. The percentages of cell survival with
respect to control cells (without compounds) were calculated for various concentrations of
investigated compounds. In agreement with the data from the PpIX fluorescence experiments,
the ALAꢀHPO conjugates that exhibited a marked enhancement of PpIX fluorescence also
exhibited high phototoxicity. In general, compounds 7c, 7d, 7e, 7f and 23 were found to be the
most phototoxic compounds at low concentrations.
2
The LD values (at 2.5 J/cm ), calculated from Figure 5 are presented in Table 1. The
5
0
values for 7d, 7e and 7f are between 2.3 ꢁM and 12.9 ꢁM for all investigated cell lines, in
contrast to the much higher value of 100 ꢁM for ALA. For 23, the LD values were somewhat
5
0
higher, ranging between 11.3 and 19.6 ꢁM. The LD value of the hexyl ester of ALA (24) was
5
0
found to be similar to that of 7e. Again, ALAꢀHPO conjugates in the present investigation were
more phototoxic upon exposure to light than our previously reported compounds. For instance,
after the incubation of KB cells with 1d, 1e and 1f (10ꢁM), followed by illumination, the
survival percentages of KB cells were under 6%; while the survival percentages of KB cells
3
7
incubated with the previously reported best ALAꢀHPO compound (200 ꢁM) was 7.8%. The
results of phototoxicity of ALAꢀHPO conjugates were in accordance with those of relative PpIX
generation.
Phototoxicity was both drug concentration and irradiation dose dependent. Our results for
the ALA hexyl ester are in accord with previous studies. For example, the hexyl ester was much
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more phototoxic than ALA against nasopharyngeal carcinoma (NPC) cells. To achieve an 80%
2
death rate (LD ), 0.75 mM ALA and 6 J/cm light dose were required, while only 0.03 mM
80
2
ALA hexyl ester and 4 J/cm light dose were needed. In terms of overall photodynamic dose, the
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ALA hexyl ester is about 40 times more potent than ALA in cell inactivation. Although, iron
can affect the generation of ROS through redox processes, the high production of PpIX through
the use of HPOs and improved PDT potency may predominate over any secondary factors
related to the ability of iron to catalyze ROS generation/cascades following PpIXꢀinduced PDT.
Moreover despite the reduction of iron availability, thereby increasing PpIX accumulation, other
transition metals and freshly released labile iron may preserve the ROS cascades via Fenton
4
6
reactions.
2.2.4 Cell viability and dark toxicity
The “dark” toxicity (namely, the cytotoxicity in the absence of irradiation) of the ALAꢀHPOs
conjugates was also evaluated in the KB cells (Figure 6). The data indicated that there was no
appreciable toxicity found for ALA or all the investigated compounds at 200 ꢁM after 4h or 24 h
incubation.
The structural conjugates, where ALA is at one end and HPO moieties on the other end of the
spacers, do not appear to induce significantly higher dark toxicity than ALA itself. In contrast,
ALA ester derivatives, i.e., hexylꢀALA (24) and octylꢀALA (25) showed significant dark toxicity
(
9% and 14% cell survival, respectively at the lower concentration of 100 ꢁM after 4h of
incubation. The incubation of ALAꢀHPO conjugates for 24h did not affect the cell viability more
than a 4h incubation. However, almost no cells survived after 24h incubation with hexylꢀALA
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and octylꢀALA. It also appears with this assay that HPOs with the spacers n = 6 or more elicit no
chemical toxicity to the cells after cleavage and releasing ALA. The much lower dark toxicity of
the HPO conjugate, albeit in a tumour cell line is a significant finding in respect to the prodrug
metabolism, particularly, if it is to be considered for systemic administration, however further in
vivo studies will be required to validate lower dark toxicities.
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2.2.5 Intracellular Prodrug Quantitative Determination
The cellular uptake of the prodrugs and their intracellular conversion to ALA was assessed
by quantitative determination of ALA using a HPLCꢀbased fluorescence method. We adopted a
1
7,45,47,48
previously reported derivatization method.
after 2h incubation at 100 µM particularly in KB and MCFꢀ7R cells using the conjugates (Figure
A). For example, six and eightꢀfold enhancements with 7d and 7e was observed with respect to
A higher accumulation of ALA was observed
7
ALA itself. These results were largely reflected in the intracellular PpIX levels measured at 24h
following 2h incubation by fresh medium without ALA or prodrugs (Figure 7B).
The fluorescence intensity of PpIX in MCFꢀ7R, which is a chemotherapy resistant breast
cancer tumour cell line, elicited higher porphyrin levels than in the MCFꢀ7 nonꢀresistant
counterpart, for the majority of ALAꢀHPO compounds. Malik and coꢀworkers have also
demonstrated that MCFꢀ7R, doxorubicin resistant tumour cells, are capable of producing higher
levels of porphyrins than sensitive MCFꢀ7 cells due to the low expression of the enzyme
4
0
ferrochelatase in combination with high mitochondrial activity. As found in the present study,
higher PpIX levels than the doxorubicin sensitive MCFꢀ7 cells were also observed.
3. Conclusions
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ALAꢀPDT provides a promising therapy for many types of cancer, but it is important to
improve its efficiency when ALA is administered systemically and intravenously in order to
overcome inefficient cell uptake and conversion to PpIX. Iron chelating agents have been
demonstrated to be effective at improving ALAꢀPDT by increasing the cellular PpIX
accumulation. In the present study, a series of ALAꢀHPO conjugates were synthesized and tested
in three cells lines. All ALAꢀHPO conjugates showed significantly elevated cellular PpIX
accumulation in KB, MCFꢀ7 and MCFꢀ7R cell lines when compared to ALA and a combination
of ALA with the corresponding HPO. Overall, compound 7e, with a linker of (CH ) , was
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10
found to be the most effective in both enhancement of cellular PpIX levels and tumour cell
phototoxicity, with comparable efficacy to the ALA hexyl ester.
4. Experimental Section
4.1 General
1
13
H and C NMR were recorded on a Jeol EX270 MHz spectrometer (Jeol Ltd, Toxyo, Japan), or
on Bruker Avance 400 or Bruker Avance 500 spectrometer. Chemical shifts are quoted in ppm
measured downfield relative to TMS. Ultravioletꢀvisible spectra were recorded on a Unicam
UV2 spectrometer (PerkinꢀElmer, Beaconsfield, UK) in dichloromethane solution. Infrared
spectra were recorded on a Nicolet FTIR (Fourier Transform Infrared Spectroscopy)
spectrometer (ThermoꢀElectro Corporation, Wisconsin, USA) as thin films in dichloromethane.
Mass spectra were obtained using a EI/CI (Electroionization/Chemical Ionization). Analytical
thin layer chromatography was carried out using Adsorbentien, aluminiumꢀbacked, silicaꢀcoated
plates (VWR, Germany) which were visualized using ultraviolet light (254nm). Column
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chromatography was carried out using Alfa Aester silica gel (220ꢀ440 mesh flash grade). High
Performance Liquid Chromatography (HPLC) was performed on a Si (55w) 250 2.1 mm column
attached to a Agilent instrument (Agilent, Life Sciences & Chemical Analysis Group, UK). All
the chemicals were purchased from Aldrich, Fisher, Alfa Aester and Aladdin, and used without
further purification unless otherwise stated. Analytical analysis was carried out by Electrospray
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(ES) instrument (Quattro Premier XE, Micromass Technologies) which coupled to high
performance liquid chromatograpy (HPLC, Waters/AcQuity Ultrapreformance LC, Waters,
Manchester, UK). Electrospray ionization (ESI) mass spectra were obtained by infusing samples
into an LCQ Deca XP ion trap instrument. High resolution mass spectra (HRMS) were
determined on Waters QTOF micro. The purity of ALAꢀHPO conjugates was determined by
analytical HPLC (Agilent system 1100) coupled with UV−vis/DAD (diode array detector) using
C18 reverseꢀphase column [HYPERSIL (5 ꢁm, 2.1 mm × 150 mm)]. The total run was
monitored with UV detector at two wavelengths 268 and 282 nm. The purities of all the AlAꢀ
HPO conjugates were over 95%.
4.2 Synthetic Procedures
4.2.1 NꢀBenzyloxycarbonylꢀ5ꢀaminolevulinic acid (NꢀCbz ALA)
5
ꢀAminolaevulinic acid (0.22 g, 1.30 mmol) was dissolved in water (3mL) and the solution
was adjusted to pH 8ꢀ10 with sodium carbonate solution. NꢀBenzyloxycarbonyl chloride (0.30
mL, 1.40 mmol) was dissolved in dioxane (3mL) and added to the mixture. After 18 h at room
temperature the solvents were removed in vacuum and 0.1M of sodium hydroxide (20mL) was
added. The mixture was washed with diethyl ether three times. The aqueous layer was
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neutralized to pH 6 and the product was extracted with dichloromethane (5×20mL). The organic
layers were combined, dried with Na SO and evaporated to yield the product as thick oil (0.18
2
4
1
g, 52%). H NMR (270 MHz, CDCl ) δ 2.73 (m, 4H, CH ), 4.10 (s, 2H, CH ), 5.12 (s, 2H, CH ),
3
2
2
2
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5
.45 (br, 1H, NH), 7.31 (m, 5H, Ph); C NMR (67.5 MHz, CDCl ) δ 27.02, 34.20, 46.61, 65.54,
3
ꢀ
71.33, 127.66, 127.86, 128.94, 138.31, 155.00, 171.82. ESIꢀMS: m/z 264 ([MꢀH] ).
4
.2.2 3ꢀ(Benzyloxy)ꢀ2ꢀethylꢀ4Hꢀpyranꢀ4ꢀone (2a)
To a solution of ethyl maltol (84g, 0.6mol) in methanol (300 mL) was added NaOH solution
(
26.4g in 80 mL H O). The mixture was heated to reflux and benzyl bromide (85.6 mL, 0.72mol)
2
o
was added dropwise over a 2h period. The solution was maintained at 60ꢀ65 C overnight. NaBr
was removed by filtration and the filtrate was concentrated. Dichloromethane (400mL) was
added to the residue, and washed with 5% NaOH solution (3×250mL), H O (2×300mL) and
2
dried over anhydrous Na SO . After removal of the solvent, diethyl ether (100mL) was added to
2
4
o
the residue and allowed to stand to 4 C. The product was obtained as white crystals (91.2g, 66
1
%). H NMR (CDCl , 400MHz) δ 0.98 (t, J = 7.6Hz, 3H, CH ), 2.50 (q, J = 7.6Hz, 2H, CH ),
3
3
2
5
.17 (s, 2H, CH ), 6.37 (d, J = 5.6Hz, 1H, C5ꢀH in pyridinone), 7.30ꢀ7.41 (m, 5H, Ph), 7.63 (d, J
2
+
=
5.6Hz, 1H, C6ꢀH in pyridinone). ESIꢀMS: m/z 231 ([M+H] ).
4
.2.3 General synthetic procedure for amino alcohols (3)
8
ꢀAminooctanꢀ1ꢀol (3d): To a mixture of 1,8ꢀoctanediol (13.3g, 91mmol), phthalimide (8.92g,
6
0.6mmol), triphenylphosphine (15.89g, 60.6 mmol) in tetrahydrofuran (THF) (150mL), cooled
on an iceꢀbath, was added dropwise diisopropyl azodicarboxylate (DIPAD) (12.25g, 60.0mmol)
over a period of 1 h. The mixture was stirred at room temperature overnight. After removal of
the solvent, the residue was purified by column chromatography using ethyl acetate/cyclohexane
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(
1:2) as an eluent to obtain 2ꢀ(8ꢀhydroxyoctyl)isoindolineꢀ1,3ꢀdione (3d’) as a white solid
1
(13.2g, 79%). H NMR (CDCl , 400MHz) δ1.32 (m, 8H, CH ), 1.53 (m, 2H, CH ), 1.65 (m, 2H,
3
2
2
CH ), 3.61 (t, J = 6.5Hz, 2H, CH ), 3.66 (t, J = 7.5Hz, 2H, CH ), 7.69 (m, 2H, Ph), 7.82 (m, 2H,
2
2
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
+
Ph). ESIꢀMS: m/z 276 ([M + H] ), 298 ([M + Na] ).
To a solution of 3d’ (12.5g, 45.5mmol) in 95% ethanol (150mL) was added 50% hydrazine
4.35g in 15mL H O, 68.2mmol). The mixture was refluxed for 3 h, and then 10M HCl (15mL)
(
2
was added. Reflux was continued for a further 15min, cooled with an iceꢀbath and then filtered.
The filtrate was concentrated, and 15 mL of water was added, washed with diethyl ether. The
aqueous layer was adjusted to pH 12 with 10 M NaOH, and then extracted with dichloromethane
(5×120mL). The combined organic layer was dried over anhydrous Na SO . After removal of the
2 4
1
solvent, 8ꢀaminooctanꢀ1ꢀol (3d) was obtained as a white solid (5.41g, 82%). H NMR (CDCl ,
3
5
00MHz) δ1.32 (m, 8H, CH ), 1.45 (m, 2H, CH ), 1.56 (m, 2H, CH ), 2.58 (t, J = 7.0Hz, 2H,
2 2 2
+
CH ), 2.68 (t, J = 7.0Hz, 2H, CH ), 3.63 (t, J = 6.5Hz, 2H, CH ). ESIꢀMS: m/z 146 ([M + H] ).
2
2
2
1
2ꢀ(10ꢀhydroxydecyl)isoindolineꢀ1,3ꢀdione (3e’): H NMR (CDCl , 500MHz) δ1.25 (m, 10H,
3
CH ), 1.31 (m, 2H, CH ), 1.55 (m, 2H, CH ), 1.66 (m, 2H, CH ), 3.63 (t, J = 6.5Hz, 2H, CH ),
2
2
2
2
2
+
3
.67 (t, J = 7.5Hz, 2H, CH ), 7.70 (m, 2H, Ph), 7.83 (m, 2H, Ph). ESIꢀMS: m/z 304 ([M + H] ),
2
+
326 ([M + Na] ).
1
1
0ꢀAminodecanꢀ1ꢀol (3e): H NMR (CDCl , 500MHz) δ1.28 (m, 10H, CH ), 1.42 (m, 4H, CH ),
3 2 2
1
.55 (m, 2H, CH ), 2.67 (t, J = 7.0Hz, 2H, CH ), 3.62 (t, J = 6.5Hz, 2H, CH ). ESIꢀMS: m/z 174
2
2
2
+
([M + H] ).
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ꢀ(12ꢀhydroxydodecyl)isoindolineꢀ1,3ꢀdione (3f’): H NMR (CDCl , 500MHz) δ1.26 (m, 16H,
3
CH ), 1.32 (m, 2H, CH ), 1.66 (m, 2H, CH ), 3.63 (t, J = 6.5Hz, 2H, CH ), 3.67 (t, J = 7.5Hz,
2
2
2
2
+
+
2
H, CH ), 7.70 (m, 2H, Ph), 7.84 (m, 2H, Ph). ESIꢀMS: m/z 332 ([M + H] ), 354 ([M + Na] ).
2
10
11
12
13
14
15
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49
50
51
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53
54
55
56
57
58
59
60
1
1
2ꢀaminododecanꢀ1ꢀol (3f): H NMR (CDCl , 500MHz) δ1.27 (m, 16H, CH ), 1.43 (m, 2H,
3
2
CH ), 1.55 (m, 2H, CH ), 2.67 (t, J = 7.0Hz, 2H, CH ), 3.62 (t, J = 6.5Hz, 2H, CH ). ESIꢀMS:
2
2
2
2
+
m/z 202 ([M + H] ).
4
.2.4 General synthetic procedure for hydroxypyridinones (4)
3
ꢀ(Benzyloxy)ꢀ2ꢀethylꢀ1ꢀ(2ꢀhydroxyethyl)pyridinꢀ4(1H)ꢀone (4a): Compound 2a (4.6g, 20 mmol)
was dissolved in ethanol (30mL)/H O(30mL), followed by the addition of ethanolamine (3.05g,
2
5
0mmol) and 2M NaOH solution (2mL). The mixture was refluxed for 18h. After cooling to
room temperature, the solution was adjusted to pH 1 with concentrated hydrochloric acid, and
then concentrated to half volume. 50 mL of H O was added. The solution was washed with
2
diethyl ether (2×50mL), adjusted to pH 9 with 10 M NaOH, extracted with DCM (3×50mL). The
combined organic layers were dried over anhydrous Na SO . After removal of the solvent, 4a
2
4
1
was obtained by crystallization from ethyl acetate as yellow crystals (4.06g, 74%). H NMR
(
CDCl , 400MHz) δ 0.96 (t, J = 7.6Hz, 3H, CH ), 2.68 (q, J = 7.6Hz, 2H, CH ), 3.86 (m, 2H,
3
3
2
CH ), 3.90 (m, 2H, CH ), 5.06 (s, 2H, CH ), 6.15 (d, J = 7.4Hz, 1H, C5ꢀH in pyridinone), 7.30ꢀ
2
2
2
+
7
.40 (m, 4H, Ph and buried C6ꢀH in pyridinone), 7.42 (m, 2H, Ph). ESIꢀMS: m/z 274 ([M + H] ),
+
296 ([M + Na] ).
1
3
4
7
ꢀ(Benzyloxy)ꢀ2ꢀethylꢀ1ꢀ(4ꢀhydroxybutyl)pyridinꢀ4(1H)ꢀone (4b): 69% yield. H NMR (CDCl ,
3
00MHz) δ 1.02 (t, J = 7.6Hz, 3H, CH ), 1.54 (m, 2H, CH ), 1.78 (m, 2H, CH ), 2.57 (q, J =
3
2
2
.6Hz, 2H, CH ), 3.66 (t, J = 6.0Hz, 2H, CH ), 3.82 (t, J = 7.6Hz, 2H, CH ), 5.22 (s, 2H, CH ),
2
2
2
2
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.36 (d, J = 7.6Hz, 1H, C5ꢀH in pyridinone), 7.25 (d, J = 7.6Hz, 1H, C6ꢀH in pyridinone), 7.28ꢀ
+
+
7
.35 (m, 3H, Ph), 7.41 (m, 2H, Ph); ESIꢀMS: m/z 302 ([M + H] ), 324 ([M + Na] ).
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3ꢀ(Benzyloxy)ꢀ2ꢀethylꢀ1ꢀ(6ꢀhydroxyhexyl)pyridinꢀ4(1H)ꢀone (4c): 79% yield. H NMR (CDCl ,
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
00MHz) δ 1.03 (t, J = 7.6Hz, 3H, CH ), 1.30ꢀ1.44 (m, 4H, CH ), 1.56 (m, 2H, CH ), 1.67 (m,
3 2 2
2H, CH ), 2.56 (q, J = 7.6Hz, 2H, CH ), 3.64 (t, J = 6.4Hz, 2H, CH ), 3.75 (t, J = 7.6Hz, 2H,
2
2
2
CH ), 5.26 (s, 2H, CH ), 6.41 (d, J = 7.6Hz, 1H, C5ꢀH in pyridinone), 7.18 (d, J = 7.6Hz, 1H,
2
2
+
C6ꢀH in pyridinone), 7.27ꢀ7.35 (m, 3H, Ph), 7.42 (m, 2H, Ph); ESIꢀMS: m/z 330 ([M + H] ), 352
+
([M + Na] ).
1
3ꢀ(Benzyloxy)ꢀ2ꢀethylꢀ1ꢀ(8ꢀhydroxyoctyl)pyridinꢀ4(1H)ꢀone (4d): 67% yield. H NMR (CDCl ,
3
4
00MHz) δ 1.03 (t, J = 7.6Hz, 3H, CH ), 1.32 (m, 8H, CH ), 1.56 (m, 2H, CH ), 1.66 (m, 2H,
3 2 2
CH ), 2.57 (q, J = 7.6Hz, 2H, CH ), 3.64 (t, J = 6.6Hz, 2H, CH ), 3.74 (t, J = 7.6Hz, 2H, CH ),
2
2
2
2
5.28 (s, 2H, CH ), 6.43 (d, J = 7.6Hz, 1H, C5ꢀH in pyridinone), 7.17 (d, J = 7.6Hz, 1H, C6ꢀH in
2
+
pyridinone), 7.29ꢀ7.35 (m, 3H, Ph), 7.43 (m, 2H, Ph). ESIꢀMS: m/z 358 ([M + H] ), 380 ([M +
+
Na] ).
1
3ꢀ(Benzyloxy)ꢀ2ꢀethylꢀ1ꢀ(10ꢀhydroxydecyl)pyridinꢀ4(1H)ꢀone (4e): 63% yield. H NMR (CDCl ,
3
5
00MHz) δ 1.04 (t, J = 7.5Hz, 3H, CH ), 1.29 (m, 10H, CH ), 1.55 (m, 2H, CH ), 1.66 (m, 4H,
3
2
2
CH ), 2.57 (q, J = 7.5Hz, 2H, CH ), 3.64 (t, J = 6.5Hz, 2H, CH ), 3.74 (t, J = 7.5Hz, 2H, CH ),
2
2
2
2
5.29 (s, 2H, CH ), 6.42 (d, J = 7.5Hz, 1H, C5ꢀH in pyridinone), 7.17 (d, J = 7.5Hz, 1H, C6ꢀH in
2
+
pyridinone), 7.29ꢀ7.35 (m, 3H, Ph), 7.44 (m, 2H, Ph). ESIꢀMS: m/z 386 ([M + H] ), 408 ([M +
+
Na] ).
1
3ꢀ(Benzyloxy)ꢀ2ꢀethylꢀ1ꢀ(12ꢀhydroxydodecyl)pyridinꢀ4(1H)ꢀone (4f): 62% yield. H NMR
(CDCl , 500MHz) δ 1.04 (t, J = 7.5Hz, 3H, CH ), 1.28 (m, 16H, CH ), 1.55 (m, 2H, CH ), 1.66
3
3
2
2
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(
m, 2H, CH ), 2.57 (q, J = 7.5Hz, 2H, CH ), 3.64 (t, J = 6.5Hz, 2H, CH ), 3.74 (t, J = 7.5Hz, 2H,
2
2
2
CH ), 5.28 (s, 2H, CH ), 6.42 (d, J = 7.5Hz, 1H, C5ꢀH in pyridinone), 7.17 (d, J = 7.5Hz, 1H,
2
2
+
C6ꢀH in pyridinone), 7.29ꢀ7.35 (m, 3H, Ph), 7.43 (m, 2H, Ph). ESIꢀMS: m/z 414 ([M + H] ), 436
10
11
12
13
14
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16
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49
50
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52
53
54
55
56
57
58
59
60
+
(
[M + Na] ).
4
.2.5 General procedure for ALA coupling to pyridinones
NꢀBenzyloxycarbonylꢀ5ꢀaminolevulinic acid (2.2 mmol) was dissolved in DCM (20 mL)
and DMF (5mL) under argon, DCC (2 mmol) and DMAP (0.2 mmol). The mixture was left to
stir for 50 min at room temperature. Compound 4 (2 mmol) was dissolved in DCM (10mL) and
added dropwise over 30min. The mixture was stirring overnight at room temperature. The heavy
precipitate was filtered off and the solvents were evaporated in vacuo. The residue was dissolved
in DCM and washed with water then saturated sodium hydrogen carbonate. The solvent was
removed and the residue was purified by flash chromatography on silica gel using a gradient of
petroleum ether/ethyl acetate (1:4), followed by 100% ethyl acetate and then methanol/ethyl
acetate (1:9). The products 6 were isolated as yellow pastes with yields of 72ꢀ80%.
2
ꢀ(3ꢀ(Benzyloxy)ꢀ2ꢀethylꢀ4ꢀoxopyridinꢀ1(4H)ꢀyl)ethylꢀ5ꢀ(((benzyloxy)carbonyl)amino)ꢀ4ꢀ
1
oxopentanoate (6a): Yield 77%. H NMR (CDCl , 270MHz) δ 0.95 (t, J = 7.6Hz, 3H CH ), 2.47
3
3
(m, 2H, CH ), 2.55 (m, 2H, CH ), 2.59 (q, J = 7.6Hz, 2H, CH ), 3.95 (m, 4H, CH ), 4.12 (s, 2H,
2
2
2
2
CH ), 5.01 (s, 2H, CH ), 5.14 (s, 2H, CH ), 6.30 (s, 1H, NH), 6.32 (d, J= 7.4, 1H, C5ꢀH in
2
2
2
1
3
pyridinone), 7.2ꢀ7.36 (m, 11H, 10H from 2Ph and 1H from C6ꢀH in pyridinone); C NMR: δ
13.2, 17.7, 27.7, 34.5, 49.9, 50.6, 62.6, 66.8, 71.7, 117.2, 123.3, 127.9, 128.1, 128.3, 128.6,
1
28.7, 136.1, 138.5, 142.0, 142.4, 146.2, 156.2, 173.1, 173.5, 206.7. ESIꢀMS: m/z 521 ([M +
+
+
H] ), 543 ([M + Na] ).
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ꢀ(3ꢀ(Benzyloxy)ꢀ2ꢀethylꢀ4ꢀoxopyridinꢀ1(4H)ꢀyl)butylꢀ5ꢀ(((benzyloxy)carbonyl)amino)ꢀ4ꢀ
1
oxopentanoate (6b): Yield 79%. H NMR (CDCl , 270MHz): 0.95 (t, J = 7.6Hz, 3H CH ), 1.52
3
3
(m, 2H, CH ), 1.67 (m, 2H, CH ), 2.47 (m, 2H, CH ), 2.49 (m, 2H, CH ), 2.52 (q, J = 7.6Hz, 2H,
2 2 2 2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
CH ), 3.63 (m, 2H, CH ), 3.93 (m, 4H, CH ), 4.98 (s, 2H, CH ), 5.11 (s, 2H, CH ), 6.01 (s, 1H,
2
2
2
2
2
NH), 6.31 (d, J = 7.4, 1H, C5ꢀH in pyridinone), 7.19ꢀ7.22 (m, 11H, 10H from 2Ph and 1H from
1
3
C6ꢀH in pyridinone). C NMR: δ13.3, 25.7, 26.3, 27.9, 28.5, 29.1, 31.5, 50.7, 53.3, 64.8, 66.9,
2.9, 76.8, 117.4, 127.9, 128.1, 128.2, 128.3, 128.6, 128.7, 136.2, 138.5, 145.2, 156.3, 172.5,
7
+
+
173.4, 204.0. ESIꢀMS: m/z 549 ([M+H] ). 571 ([M+Na] ).
6ꢀ(3ꢀ(Benzyloxy)ꢀ2ꢀethylꢀ4ꢀoxopyridinꢀ1(4H)ꢀyl)hexylꢀ5ꢀ(((benzyloxy)carbonyl)amino)ꢀ4ꢀ
1
oxopentanoate (6c): Yield 80%. H NMR (CDCl , 270MHz): δ 0.98 (t, J = 7.6Hz, 3H, CH ),
3
3
1.21ꢀ1.23 (m, 4H, CH ), 1.52ꢀ1.55(m, 4H, CH ), 2.44–2.47 (m, 4H, CH ), 3.63 (q, J = 7.4Hz,
2
2
2
2
H, CH ), 3.84ꢀ3.98 (m, 6H, CH ), 5.01 (s, 2H, CH ), 5.11 (s, 2H, CH ), 6.02( br, 1H, NH), 6.31
2 2 2 2
(d, J = 7.4, 1H, C5ꢀH in pyridinone), 7.18 (d, J = 7.4Hz, C6ꢀH in pyridinone), 7.21 (m, 10H, Ph);
1
3
C NMR: δ 13.9, 19.5, 25.7, 26.7, 27.5, 28.9, 29.2, 29.3, 29.4, 31.7, 34.5, 49.9, 50.7, 52.6, 65.0,
6
7.0, 71.2, 117.5 127.9, 128.2, 128.8 136.4, 138.1, 138.5, 142.0, 156.2, 172.5, 173.1, 206.7. ESIꢀ
+
+
MS: m/z 577 ([M+H] ), 599 ([M+Na] ).
8ꢀ(3ꢀ(Benzyloxy)ꢀ2ꢀethylꢀ4ꢀoxopyridinꢀ1(4H)ꢀyl)octyl
5ꢀ(((benzyloxy)carbonyl)amino)ꢀ4ꢀ
1
oxopentanoate (6d): Yield 75%: H NMR (CDCl , 270MHz): δ 1.02 (t, J = 7.6Hz, 3H, CH ),
3
3
1.29 (m, 8H, CH ), 1.63(m, 4H, CH ), 2.55–2.70 (m, 6H, CH ), 3.75 (m 2H, CH ), 4.03–4.13(m,
2
2
2
2
4H, CH ), 5.09 (s, 2H, CH ), 5.25 (s, 2H, CH ), 5.57( br, 1H, NH), 6.49 (d, 1H, J= 7.4Hz, 1H,
2 2 2
1
3
C5ꢀH in pyridinone), 7.28–7.64(m, 11H, 2Ph and C6ꢀH in pyridinone); C NMR: δ13.4, 20.2,
2
5.9, 26.5, 27.9, 28.2, 28.6, 29.2, 29.3, 29.4, 31.5, 50.7, 53.3, 64.8, 67.0, 72.9, 117.5, 127.9,
28.1, 128.3, 128.5, 128.6, 128.8, 134.2, 137.9, 138.1, 146.8, 156.3, 172.6, 173.6, 204.1. ESIꢀ
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+
+
MS: m/z, 605 ([M+H] ), 627 ([M+Na] ).
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0ꢀ(3ꢀ(Benzyloxy)ꢀ2ꢀethylꢀ4ꢀoxopyridinꢀ1(4H)ꢀyl)decyl
5ꢀ(((benzyloxy)carbonyl)amino)ꢀ4ꢀ
1
oxopentanoate (6e): Yield 72%. H NMR (CDCl , 270MHz): δ 1.02 (t, J = 7.6Hz, 3H, CH ),
3
3
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
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45
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47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
.24 (m, 12H, CH ), 1.56 (m, 4H, CH ), 2.51ꢀ2.66 (m, 6H, CH ), 3.7 (m, 2H, CH ), 4.01ꢀ4.09
2 2 2 2
(m, 4H, CH ), 4.95 (s, 2H, CH ), 5.23 (s, 2H, CH ), 5.69 (br, 1H, NH), 6.36 (d, J= 7.4, 1H, C5ꢀH
2
2
2
1
3
in pyridinone), 7.12 (d, J= 7.4, 1H, C6ꢀH in pyridinone), 7.26ꢀ7.31 (m, 10H, Ph); CNMR: 13.4,
0.3, 25.9, 26.5, 27.3, 27.9, 28.2, 28.6, 28.9, 29.2, 29.3, 29.4, 31.5, 50.7, 53.3, 64.8, 67.0, 72.9,
117.5, 127.9, 128.1, 128.3, 128.5, 128.6, 128.8, 134.2, 137.9, 138.1, 146.2, 156.3, 172.6, 173.6,
2
+
+
204.1. ESIꢀMS: m/z 633 ([M+H] ), 655 ([M+Na] ).
12ꢀ(3ꢀ(Benzyloxy)ꢀ2ꢀethylꢀ4ꢀoxopyridinꢀ1(4H)ꢀyl)dodecyl
5ꢀ(((benzyloxy)carbonyl)amino)ꢀ4ꢀ
1
oxopentanoate (6f): Yield 73%. H NMR (CDCl , 270MHz) 0.98 (t, J = 7.6Hz, 3H, CH ), 1.03
3
3
(m, 16H, CH ), 1.57ꢀ1.60 (m, 4H, CH ), 2.53–2.64 (m, 6H, CH ), 3.71 (q, J=7.4Hz, 2H, CH ),
2 2 2 2
4
6
7
.02 (m, 2H, CH ), 4,11 (s, 2H, CH ), 5.06 (s, 2H, CH ), 5.24 (s, 2H, CH ), 5.59 (br, 1H, NH),
2 2 2 2
.40 (d, J = 7.4Hz, 1H, C5ꢀH in pyridinone), 7.28 (d, J = 7.4Hz, 1H, C6ꢀH in pyridinone), 7.30–
13
.39 (m, 10H, Ph); C NMR: 13.3, 20.1, 25.9, 26.4, 27.6, 28.2, 28.6, 28.9, 29.2, 29.4, 29.5, 29.7,
31.6, 50.7, 53.3, 60.5, 65.0, 67.0, 72.9, 117.5, 128.8, 127.9, 128.1, 128.2, 128.6, 128.8, 136.5,
+
+
137.9, 138.7, 145.6, 156.3, 172.5, 173.6, 204.1. ESIꢀMS: m/z 661 ([M+H] ), 683 ([M+Na] ).
4.2.6 General procedure for the deprotection of amino and hydroxyl groups of ALA-HPO
conjugates
Compounds 6a–6f, 11, 17 and 22 (1 equiv.) were dissolved in methanol/ethyl acetate (50mL,
1:4). Catalytic amount of palladium on carbon (5% Pd/C, 10% weight of protected ALAꢀHPO
conjugates) and benzyl chloride (2.2 equiv.) were added. The hydrogenation was carried out
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under 40 psi H at room temperature for 10 min. Pd/C was filtered off and the filtrate was
2
evaporated to dryness. The residues were purified by semiꢀpreparative Agilent HPLC using
reversꢀphase Waters column (4.6mm X 250 mm) eluted with acetonitrile/methanol (30:70, V/V)
with flow rate 1mL/min. AlAꢀHPO conjugates 7, 12, 18 and 23 were isolated as thick oils. The
purity of all compounds was determined by analytical HPLC (Agilent system 1100) coupled with
UV−vis/DAD using C18 reverseꢀphase column [HYPERSIL (5 ꢁm, 2.1 mm × 150 mm)]. The
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mobile phase was A (0.1% TFA in MeOH:H O, 2:98), 100% isocratic for 5 min, B (0.1% TFA
2
in MeOH:H O, 98:2), gradient from 0ꢀ50% between 6ꢀ10 min, 50% B isocratic from 11ꢀ15 min,
2
then equilibrate with 100% A for 2 min. The total elution time was 18 min and flow rate was
0.25 mL/min. The total run was monitored with UV detector at two wavelengths 268 and 282 nm.
(2ꢀ(2ꢀEthylꢀ3ꢀhydroxyꢀ4ꢀoxopyridinꢀ1(4H)ꢀyl)ethoxy)ꢀ2,5ꢀdioxopentanꢀ1ꢀaminium chloride (7a):
1
The product was isolated as thick oil, yield 35%, HPLCꢀDAD (R : 2.3 min; purity 95.6%) H
t
NMR (DMSO, 270MHz): 1.23 (t, J = 7.5Hz, 3H, CH ), 2.64 (m, 2H, CH ), 2.85 (m, 2H, CH ),
3
2
2
3.06 (m, 2H, CH ), 3.59 (m, 2H, CH ), 4.02 (s, 2H, CH ), 4.38 (m, 2H, CH ), 7.13 (d, J=6.7Hz,
2
2
2
2
+
13
1
H, C5ꢀH in pyridinone), 7.66 (m, 3H, H N ), 8.10 (d, J = 6.9Hz, 1H, C6ꢀH in pyridinone); C
3
NMR: δ12.1, 15.3, 27.7, 36.2, 44.3, 53.7, 55.5, 113.3 127.9, 134.0, 143.0, 164.9, 172.1, 206.2.
+
+
ESIꢀMS: m/z 297 ([M+H] ); HRMS: HRMS: Calcd. for C H N O : 297.1450 ([M+H] ), found:
14
21
2
5 ,
297.1441.
5ꢀ(4ꢀ(2ꢀEthylꢀ3ꢀhydroxyꢀ4ꢀoxopyridinꢀ1(4H)ꢀyl)butoxy)ꢀ2,5ꢀdioxopentanꢀ1ꢀaminium
chloride
1
(7b): Yield 42%, HPLCꢀDAD (R : 3.2 min; purity 96.3%). H NMR (CD OH, 270MHz): δ 1.26
t
3
(
t, J = 7.1Hz, 3H, CH ), 1.82 (m, 2H, CH ) 1.98 (m, 2H,CH ), 2.64 (m, 2H, CH ), 2.83 (m, 2H,
3 2 2 2
CH ), 3.06 (q, J = 5.4Hz, 2H, CH ), 4.09 (m, 4H, CH ), 4.43 (m, 2H, CH ), 7.12 (d, J = 6.4Hz,
2
2
2
2
1
3
1
H, C5ꢀH in pyridinone), 8.32 (d, J = 6.7Hz, 1H, C6ꢀH in pyridinone); C NMR: δ 11.1, 15.3,
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9.4, 26.8, 27.1, 36.2, 50.3, 52.6, 64.4, 116.8, 126.2, 137.4, 140.5, 156.2, 172.2, 201.2. ESIꢀMS:
+
+
+
m/z 325 ([M+H] ), 347 ([M+Na] ); HRMS: Calcd for C H N O : 325.1763 ([M+H] ), found:
16 25
2
5 ,
3
25.1743.
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5
ꢀ((6ꢀ(2ꢀEthylꢀ3ꢀhydroxyꢀ4ꢀoxopyridinꢀ1(4H)ꢀyl)hexyl)oxy)ꢀ2,5ꢀdioxopentanꢀ1ꢀaminium chloride
1
(7c): Yield 42%, HPLCꢀDAD (R : 3.7 min; purity 96.6%). H NMR (CD OH, 270MHz): δ 1.29
t
3
(t, J = 7.1Hz, 3H, CH ), 1.45 (m, 4H, CH ) 1.89 (m, 4H, CH ), 2.67 (m, 4H, CH ), 2.82 (m, 2H,
3
2
2
2
CH ), 3.29 (m, 2H, CH ), 3.55 (m, 2H, CH ), 4.06 (m, 2H, CH ), 7.15 (d, J = 6.4Hz, 1H, C5ꢀH in
2
2
2
2
1
3
pyridinone), 8.20 (d, J = 6.7Hz, 1H, C6ꢀH in pyridinone); C NMR: δ 11.1, 15.2, 19.4, 25.4,
6.8, 29.1, 34.2, 36.2, 50.3, 52.6, 64.4, 111.9 136.4, 138.5, 156.2, 172.2, 173.1, 201.2. ESIꢀMS:
2
+
+
+
m/z 353.3 ([M+H] ), 375.3 ([M+Na] ); HRMS: Calcd for C H N O : 353.2076 ([M+H] ),
1
8
29
2
5 ,
found: 353.2065.
5ꢀ((8ꢀ(2ꢀEthylꢀ3ꢀhydroxyꢀ4ꢀoxopyridinꢀ1(4H)ꢀyl)octyl)oxy)ꢀ2,5ꢀdioxopentanꢀ1ꢀaminium)
1
dichloride (7d): Yield 48%, HPLCꢀDAD (R : 4.3 min; purity 97.7%). H NMR (CD OH,
t
3
270MHz): δ 1.17 (t, 3H, J = 7.1Hz, CH ), 1.26 (m, 12H, CH ), 2.66 (m, 2H, CH ), 2.85 (m, 2H,
3
2
2
CH ), 3.49 (m, 2H, CH ), 3.61, (m, 2H, CH ), 4.05ꢀ4.11 (m, 4H, CH ), 7.33 (d, J = 6.4Hz, 1H,
2
2
2
2
1
3
C5ꢀH in pyridinone) 8.22 (d, J = 6.7Hz, 1H, C6ꢀH in pyridinone); C NMR: δ 11.3, 19.9, 25.7,
26.4, 29.0, 29.1, 31.3, 34.2, 36.2, 50.3, 52.6, 64.4, 110.8 138.4, 143.4, 146.4, 158.4, 172.9, 201.7.
+
+
ESIꢀMS: m/z 382.1 ([M+H] ), 404.2 ([M+Na] ). HRMS: Calcd. for C H N O : 381.2389
20
33
2
5 ,
+
([M+H] ), found: 381.2383.
5ꢀ((10ꢀ(2ꢀEthylꢀ3ꢀhydroxyꢀ4ꢀoxopyridinꢀ1(4H)ꢀyl)decyl)oxy)ꢀ2,5ꢀdioxopentanꢀ1ꢀaminium
1
chloride (7e): Yield 45%, HPLCꢀDAD (R : 5.4 min; purity 97.4%). H NMR (CD OH,
t
3
270MHz): δ 1.17 (t, J = 7.1Hz, 3H, CH ), 1.26 (m, 16H, CH ), 2.66 (m, 2H, CH ), 2.85 (m, 2H,
3 2 2
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CH ), 3.15 (m, 2H, CH ), 3.91 (m, 2H, CH ), 4.05 (s, 2H, CH ), 4.60 (m, 2H, CH ), 7.15 (d, J =
2
2
2
2
2
1
3
6
1
1
.4Hz, 1H, C5ꢀH in pyridinone), 8.20 (d, J = 6.7Hz, 1H, C6ꢀH in pyridinone); C NMR: δ 11.3,
9.8, 25.0, 26.4, 29.0, 29.1, 29.3, 31.2, 34.2, 50.3, 52.6, 56.4, 61.7, 64.7, 110.8, 138.4, 143.5,
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+
46.0, 158.2, 172.9, 201.2. ESꢀMS: 409.2 ([M+H] ); HRMS: Calcd for C H N O : 409.2702
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37
2
5 ,
+
([M+H] ), found: 409.2700.
5ꢀ((12ꢀ(2ꢀEthylꢀ3ꢀhydroxyꢀ4ꢀoxopyridinꢀ1(4H)ꢀyl)dodecyl)oxy)ꢀ2,5ꢀdioxopentanꢀ1ꢀaminium
1
chloride (7f): Yield 43%, HPLCꢀDAD (R : 6.2 min; purity 96.8%). H NMR (CD OH,
t
3
2
70MHz): δ 1.11 (t, J = 7.1Hz, 3H, CH ), 1.13 (m, 20H, CH ), 2.66 (m, 2H, CH ), 2.90 (m, 2H,
3 2 2
CH ), 3.32 (m, 2H, CH ), 3.91 (m, 2H, CH ), 4.09 (s, 2H, CH ), 4.60 (m, 2H, CH ), 7.33 (d, J =
2
2
2
2
2
13
6
.4Hz, 1H, C5ꢀH in pyridinone), 8.22 (d, J = 6.7Hz, 1H, C6ꢀH in pyridinone); C NMR: δ 11.3,
19.8, 25.7, 26.1, 28.7, 28.9, 29.0, 29.1, 29.3, 31.3, 32.4, 34.2, 50.2, 52.3, 56.4, 61.7, 64.7, 110.7,
+
+
1
38.1, 143.5, 146.4, 158.4, 172.9, 201.2. ESIꢀMS: m/z 437.0 ([M+H] ), 459.1 ([M+Na] );
+
HRMS: Calcd for C H N O : 437.3015 ([M+H] ), found: 437.3025.
2
4
41
2
5 ,
4.2.7 Synthesis of ALA-HPO conjugate 12
3ꢀ(Benzyloxy)ꢀ2ꢀ(1ꢀhydroxyethyl)ꢀ4Hꢀpyranꢀ4ꢀone (8): A mixture of 3ꢀ(benzyloxy)ꢀ2ꢀethylꢀ4Hꢀ
pyranꢀ4ꢀone (2a) (10g, 43.4mmol), SeO (14.5g, 130.4mmol) and phenyl bromide (50mL) was
2
o
heated to 135ꢀ140 C with vigorous stirring for 4h. After filtration, the filtrate was concentrated
and the residue was purified by column chromatography using ethyl acetate/cycle hexane (2:1)
1
as an eluent to obtain product as a brown oil (4.6g, 43%). H NMR (CDCl , 400MHz) δ 1.23 (d,
3
J = 6.7Hz, 3H, CH ), 4.85 (q, J = 6.7Hz, 1H, CH), 5.22 (d, J = 2.0Hz, 2H, CH ), 6.46 (d, J =
3
2
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.6Hz, 1H, C5ꢀH in pyridinone), 7.37 (m, 5H, Ph), 7.71 (d, J = 5.6Hz, 1H, C6ꢀH in pyridinone).
+
ESIꢀMS: m/z 247 ([M + H] ).
3ꢀ(Benzyloxy)ꢀ2ꢀ(1ꢀ(benzyloxy)ethyl)ꢀ4Hꢀpyranꢀ4ꢀone (9): To a solution of 3ꢀ(benzyloxy)ꢀ2ꢀ(1ꢀ
hydroxyethyl)ꢀ4Hꢀpyranꢀ4ꢀone (8) (4.0g, 16.2mmol) in dichloromethane (50mL) was added 50%
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+
ꢀ
KOH (5.45g, 48.6mmol) and Bu N HSO (0.1g). The mixture was stirred at room temperature
4
4
for 30 min before benzyl bromide (4.17g, 24.4mmol) was added dropwise. The reaction was
monitored by TLC until the starting material disappeared. The reaction mixture was washed with
brine, and the organic layer was dried over anhydrous Na SO . After removal of the solvent, the
2
4
residue was purified by column chromatography using ethyl acetate/cycle hexane (1:2) as an
1
eluent to obtain product 9 as a pale yellow oil (3.4g, 62%). H NMR (CDCl , 400MHz) δ 1.14
3
(d, J = 6.6Hz, 3H, CH ), 4.19ꢀ4.28 (m, 2H, CH ), 4.67 (q, J = 6.6Hz, 1H, CH), 5.19 (s, 2H, CH ),
3
2
2
6
.40 (d, J = 5.6Hz, 1H, C5ꢀH in pyridinone), 7.24ꢀ7.33 (m, 10H, Ph), 7.71 (d, J = 5.6Hz, 1H, C6ꢀ
+
H in pyridinone). ESIꢀMS: m/z 337 ([M + H] ).
3ꢀ(Benzyloxy)ꢀ2ꢀ(1ꢀ(benzyloxy)ethyl)ꢀ1ꢀ(6ꢀhydroxyhexyl)pyridinꢀ4(1H)ꢀone (10): The synthetic
1
procedure was similar to that adopted for the preparation of 4. Yield: 66%. H NMR (CDCl ,
3
4
00MHz) δ 1.14ꢀ1.29 (m, 4H, CH ), 1.22 (d, J = 7.0Hz, 3H, CH ), 1.44 (m, 2H, CH ), 1.62 (m,
2 3 2
2H, CH ), 3.56 (t, J = 6.5Hz, 2H, CH ), 3.98 and 4.10 (m, 2H, CH ), 4.16 (s, 2H, CH ), 5.26 (s,
2
2
2
2
2
H, CH ), 6.48 (d, J = 7.5Hz, 1H, C5ꢀH in pyridinone), 7.21 (m, 3H, Ph and buried C6ꢀH in
2
+
+
pyridinone), 7.28ꢀ7.34 (m, 8H, Ph). ESIꢀMS: m/z 436 ([M + H] ), 458 ([M + Na] ).
6ꢀ(3ꢀ(Benzyloxy)ꢀ2ꢀ(1ꢀ(benzyloxy)ethyl)ꢀ4ꢀoxopyridinꢀ1(4H)ꢀyl)hexylꢀ5ꢀ
1
(
(benzyloxycarbonyl)amino)ꢀ4ꢀoxopentanoate (11): H NMR (CDCl , 270MHz) δ 1.22 (m, 7H,
3
2
CH and CH ), 1.44ꢀ1.59 (m, 4H, 2CH ), 2.58ꢀ2.69 (m, 4H, 2CH ), 3.93ꢀ4.14 (m, 7H, 3CH and
2
3
2
2
2
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