Spacer optimization of new conjugates for a melanoma-selective delivery approach

Article abstract of DOI:10.1039/c3ob41428k

In the search for more selective anticancer drugs, we designed and synthesized seven conjugates varying the structure of the linker connecting the 5-iodo-2′-deoxyuridine (IUdR) to the ICF 01012 melanoma-carrier for potential intratumoural specific drug re

Full text of DOI:10.1039/c3ob41428k

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Spacer optimization of new conjugates for a
melanoma-selective delivery approach†
Cite this: Org. Biomol. Chem., 2013, 11,
6372
Mathieu André,a,b Sébastien Tarrit,a,b Marie-Joelle Couret,a,b
Marie-Josèphe Galmier,a,b Eric Débiton,a,b Jean-Michel Chezala,b and
Emmanuelle Mounetou*a,b
In the search for more selective anticancer drugs, we designed and synthesized seven conjugates varying
the structure of the linker connecting the 5-iodo-2’-deoxyuridine (IUdR) to the ICF 01012 melanoma-
carrier for potential intratumoural specific drug release. Chemical and in vitro metabolic stability evalu-
ations showed that, except for the ester conjugate (1), the ketal (2b), acetal (2a), carbonate (4) and car-
bamate (3) conjugates were compatible with our approach. The acetal (2a) and its PEGylated derivative
(2c) were of particular interest for further in vivo development owing to their respective pH-dependent
stability and limited metabolic degradation in order to exploit the acidic subcellular environment of
malignant melanocytes to trigger the release of therapeutics upon internalization in cells.
Received 11th July 2013,
Accepted 30th July 2013
DOI: 10.1039/c3ob41428k
www.rsc.org/obc
Introduction
A major challenge in modern cancer chemotherapy is achiev-
ing tumour-selective treatments. Standard therapies are still
mostly limited by high toxicities against normal tissues indu-
cing severe side-effects (bone marrow, gastrointestinal tract
and hair follicles) for the patients. The administered doses are
therefore reduced, and often given at suboptimal levels, result-
ing in failure of therapy.1
To overcome this lack of selectivity, maximal drug delivery
at the cancer cells is required to spare normal cells, minimize
toxicities and thereby improve anticancer treatment efficacy.
Chemical conjugation of the drug to a targeting moiety is a
typical strategy developed to increase the selectivity of cancer
chemotherapeutic agents.2,3
After our laboratory discovered melanoma tracers such
as ICF 01012 (Fig. 1) with high long-lasting affinity for the
tumour,4 we sought to design melanoma-specific structures
for enhanced drug delivery applications. Melanoma is an extre-
mely aggressive malignancy of melanocytes with high meta-
static potential combined with high therapy resistance and
very poor prognosis.5,6 This ever more prevalent cancer rep-
Fig. 1 Structure of the melanoma tracer ICF 01012.
therapeutic approaches to this disease are therefore urgently
needed.7
We previously demonstrated that our tracers derived from
N,N-diethylaminoethyleneheteroarylamide could be used as
carriers to the pigmented melanoma cells.8,9 Accordingly, we
demonstrated that these tracers, forming reversible complexes
with melanin via both ionic and hydrophobic interactions,10
were highly concentrated in the melanoma tumour in vivo, and
particularly localise into the melanosomes, acidic organelles
of melanocytes.11,12 Although the direct coupling of drugs to
melanoma targeting moieties with non-cleavable linkers is of
major interest, it may also be limited by the scavenger effect
due to melanosomal sequestration of anticancer agents in
melanoma treatments.13
resents
a considerable clinical problem worldwide. New
To overcome these limits, we have developed a strategy for
tumour-selective delivery based on ICF 01012 drug conjugates
with a spacer that incorporates a pre-determined breaking
point allowing the drug to be released at the cellular target
site. As the first challenge is the optimization of the physico-
chemical properties, a model was designed for primary vali-
dation of this strategy: 5-iodo-2′-deoxyuridine (IUdR), an anti-
metabolite, was conjugated to the ICF 01012 carrier via various
cleavable linkers (ester, carbamate, and carbonate) (Fig. 2). As
aINSERM – Université d’Auvergne UMR 990, IMTV, BP 184, F-63005 Clermont-
Ferrand Cedex, France. E-mail: emmanuelle.mounetou@inserm.fr;
Fax: +(33) 4 73 15 08 01; Tel: +(33) 4 73 15 08 00
bClermont Université, Université d’Auvergne, IMTV, BP 10448, F-63005 Clermont-
Ferrand Cedex, France
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
c3ob41428k
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Fig. 2 Structure of the seven synthesized conjugates.
the targeted melanosomes are acidic compartments,14–16 a pH- (Scheme 1). Ester (1) was prepared by a Steglich esterification
dependent cleavage was also investigated with acid-sensitive of IUdR (6) with acrylic acid in 46% yield (Scheme 1). Aza-
spacers containing ketal/acetal functions to exploit the acidic Michael addition of the secondary amine (5) on acrylate (7)
subcellular environment to trigger the release of therapeutics catalyzed by 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) gave the
upon internalization in malignant melanocytes. In addition, conjugate (8) in 18% yield. Ester (1) was then obtained in excel-
variation of the degree of linker methylation and introduction lent yield (95%) by final deprotection of the silyl group under
of poly(ethyleneglycol) PEG polymer linkers were investigated classical conditions, i.e. tetrabutylammonium fluoride (TBAF)
to modulate the metabolic stability. Here we describe the syn- in tetrahydrofuran (THF).
thesis, through three different multistep pathways, of a series
A similar pathway was successfully used for the synthesis of
of conjugates (1, 2a–d, 3, 4, Fig. 2), followed by a preliminary the conjugates (Scheme 2): the ketal or acetal function was
in vitro stability evaluation in various buffers and under physio- generated by condensation of the vinyl ethers (11a–d)19 on the
logical conditions. Additional stability studies in hepatocyte protected IUdR (6) catalyzed by camphorsulfonic acid (CSA).
microsomes allowed us to select conjugates with spacers The yields ranged from 40% (12b) to nearly quantitative (12a).
showing optimal metabolic stability and profile for follow up In the case of the acetal (12b), an additional asymmetric
in vivo testing.
carbon was created leading to a racemic mixture of the two dia-
stereoisomers as confirmed by 1H NMR analysis showing
doubled signals with the same integration. Protecting esters
on the spacer were removed by lithium hydroxide action in
methanol to give the corresponding primary alcohols (13a–d)
efficiently (79–99%), and were converted into methanesulfo-
nates (Ms) (14a–d). Associated yields were similar (87–89%)
except for compound (14c) which was obtained in 65% yield.
The targeting moiety was then introduced by nucleophilic sub-
stitution of the mesylates (14a–d) by the secondary amine (5)
in anhydrous acetonitrile (MeCN). The preparation of the con-
jugates (14d) and (15d) required higher temperatures because
of steric hindrance by the PEGylated chains. Final deprotec-
tion afforded the conjugates (2a–d) in good yields (81–90%)
except for (2d) (50%) synthesized at a smaller scale.
Results and discussion
Synthesis
Synthetic pathways were designed to be mostly convergent
through the introduction of the targeting unit from its second-
ary amine precursor (5) (Fig. 3) prepared according to a litera-
ture procedure.17 Each synthesis started with regioselective
monoprotection of (+)-5-iodo-2′-deoxyuridine (IUdR) with tri-
isopropylsilyl chloride (TIPSCl) by a slightly modified litera-
ture procedure18 to give protected IUdR (6) in 83% yield
Carbamate (3) and carbonate (4) were prepared by conver-
gent routes from the tertiary amine precursors (18) and (19)
(Scheme 3). For the preparation of (19), the secondary amine
(5) was easily alkylated by bromoethanol in dry acetonitrile in
62% yield. By contrast, a three-step sequence was necessary for
the synthesis of (18): protection of the bromoethylamine
Fig. 3 Structure of the precursor (secondary amine) 5.
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Scheme 1 Synthesis of the ester conjugate (1). (i) TIPSCl, imidazole, DMAP (cat.), DMF, rt, 18 h, 83%; (ii) acrylic acid, DCC, DMAP, THF, rt, 1 h, 46%; (iii) amine (5),
DBU, MeCN, rt, 96 h, 18%; (iv) TBAF, THF, rt, 5 h, 95%.
Scheme 2 Synthesis of the conjugates (2a–d). (i) (a) (11a), CSA (cat.), THF, rt, 5 h, 99%; (b) (11b), CSA (cat.), THF, rt, 4 h, 40%; (c) (11c), CSA (cat.), THF, rt, 18 h,
92%; (d) (11d), CSA (cat.), THF, rt, 20 h, 55%; (ii) LiOH, EtOH, rt, 4 h, (13a), 93%, (13b), 87%, (13c), 99%, (13d), 79%; (iii) (a) MsCl, DIPEA, DCM, rt, 1 h, (14a), 89%,
(14b), 88%, (14c), 65%, (14d), 87%; (iv) amine (5), MeCN, DIPEA (a) 55 °C, 88 h, 15%, (b) 50 °C, 96 h, 15%; (c) 65 °C, 90 h, 30%; (d) 70 °C, 70 h, 12%; (v) TBAF, THF,
rt, 4 h, (2a), 81%, (2b), 81%, (2c), 90%, (2d), 50%.
bromohydrate via the tert-butoxycarbonyl (Boc) derivative (16) deprotection carried out with trifluoroacetic acid (TFA) in
followed by nucleophilic substitution by the secondary amine almost quantitative yield (96%). The protected IUdR (6) was
(5) to give the tertiary amine (17) in 53% yield, and final then preactivated at the 3′ position via the formation of the
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Scheme 3 Synthesis of the precursors (18–19). (i) Boc2O, DIPEA, DCM, rt, 15 h, 45%; (ii) amine (5), MeCN, rt, 96 h, 53%; (iii) TFA, DCM, rt, 1 h 30, 96%; (iv) DIPEA,
MeCN, reflux, 48 h, 62%.
Scheme 4 Synthesis of the carbamate (3) and carbonate (4) conjugates. (i) p-Nitrophenyl chloroformate, pyridine, DCM, 0 °C to rt, 17 h, 85%; (ii) amine (18), NEt3,
DCM, rt, 18 h, 96%; (iii) alcohol (19), pyridine, DMAP, DCM, rt, 72 h, 86%; (iv) TBAF, THF, rt, 2 h, (3), 86%, (4), 91%.
p-nitrophenyl carbonate (20) in good yield after purification nucleophilic enough to displace the equilibrium and provide
(85%) (Scheme 4). Coupling with (18) or (19) was then carried the carbamate (21) in excellent yield (96%), but the primary
out using different conditions. The primary amine (18) was alcohol (19) required catalysis by N,N-dimethylaminopyridine
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(DMAP) to react efficiently and yield the carbonate (22) (86%).
The half-lives of the ester conjugate (1) were around 2 h in
Final silyl groups deprotection using TBAF yielded the conju- PBS, MEM or serum media as expected for this hydrolysis sen-
gates (3) and (4) in 86% and 91% yields respectively.
sitive function. This process was slowed at pH 5 and stopped
over a 3 days period at even more acid pH 3. This result is con-
sistent with the lower hydrolysis tendency of ester in acidic
medium. Conversely, the carbamate conjugate (3) was stable in
Chemical stability
Since we designed conjugates with cleavable spacers, it was PBS, acidic buffers and MEM for more than 3 days and dis-
played a half-life of almost 2 days in serum as this function is
imperative to assess the stability of the different linkers under
physiologically relevant conditions, i.e. in phosphate-buffered chemically more stable. The stability of the carbonate conju-
saline (pH 7.4; PBS), in cell culture minimum essential gate (4) was intermediate with a half-life of ∼6–7 h in PBS and
MEM that decreased to 4 h in serum. Surprisingly, the acetal
medium (MEM) and in human serum. Their pH-dependence
was also tested in acidic buffers. Compounds (1), (2a–b), (3), conjugate (2a) was stable over the experimental time frame
and (4) were incubated at a concentration of 500 μM in PBS, in even at pH 3. Additional tests demonstrated that it could be
hydrolyzed only at pH below 2. However, the ketal conjugate
10 mM acidic buffers (pH from 3 to 6), in the minimum essen-
tial medium (MEM) and in human serum at 37 °C for 3 days. (2b) showed half-lives of around 4–5 days in the neutral PBS
Aliquots were taken at different time intervals and each conju- and MEM that decreased according to the pH from 30 h
at pH 6 to 44 min at pH 3. The ketal conjugate (2b) was
gate was assayed by reversed-phase high performance liquid
chromatography (RP-HPLC) with an internal standard. In the thus more easily hydrolyzed than the acetal conjugate (2a)
case of incubation in human serum, non-specific plasma under both neutral and acidic conditions consistent with the
literature.20,21
protein binding represented less than 5% at any time-point
and therefore did not interfere with the results obtained. Half-
life was calculated for each conjugate from the first-order kine- where only chemical instability can occur, or in MEM (contain-
ing nutriments such as vitamins, amino acids, and glucose)
Finally, the conjugates were generally more stable in PBS,
tics of disappearance plotting the conjugate relative concen-
tration against time. Representative curves are presented for than in human serum containing proteins favouring enzymatic
(1) and (4) in Fig. 4 and 5 respectively. The half-lives calculated degradation. The trend observed in human serum was consist-
ent with the chemical stability following the increasing order
for all conjugates are reported in Table 1.
Fig. 4 Stability of the ester conjugate (1) in various media at 37 °C. (A) Phosphate-buffered saline (pH 7.4; PBS), (B) human serum, (C) cell culture minimum essen-
tial medium (MEM), and (D) acidic buffer (pH 5.0). Relative concentration against time: expressed as the ratio of the peak area at the considered time to the peak
area at time zero.
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Fig. 5 Stability of the carbonate conjugate (4) in various media at 37 °C. (A) Phosphate-buffered saline (pH 7.4; PBS), (B) human serum, (C) cell culture minimum
essential medium (MEM), and (D) acidic buffer (pH 5.0). Relative concentration against time: expressed as the ratio of the peak area at the considered time to the
peak area at time zero.
Table 1 Half-lives of conjugates (1), (2a–b), (3) and (4) under various
using a variety of subcellular fractions from human and
animal liver as the drug metabolising enzyme source. Micro-
conditions
somes (endoplasmic reticulum) isolated from murine hepato-
Human
cytes contain a complete metabolic system including the
cytochrome P450 superfamily and are a widely accepted overall
model for the investigation of drug metabolism. The S9 frac-
tion (post-mitochondrial supernatant fraction), a mixture of
microsomes and cytosol containing a wide variety of phase
I and II enzymes, can also be used. To rationalize the choice of
the system adopted, experiments were both carried out with
pure microsomes and liver S9 fraction. The metabolic stability
was evaluated for the ketal conjugate (2b) and the acetal (2a)
as a reference. It was extended to the two PEG derivatives (2c)
and (2d) as we expect the PEG spacer to modify the metabolic
profile. The conjugates were incubated at a concentration of
50 μM with the microsomes, active or not (thermal inacti-
vation), at 37 °C for various periods of time from 15 min to
4 h. The different samples were analysed by RP-HPLC coupled
to electrospray tandem mass spectrometry (MS/MS) for meta-
bolite identification.
Conjugate PBS
pH = 6 pH = 5 pH = 4 pH = 3 MEM
serum
a
(1)
1 h 49 ND
—
30 h
—
ND
—
—
—
1 h 44
—
1 h 47
42 h
a
a
a
a
a
a
(2a)
(2b)
(3)
—
5 d 6 h 31 h
25 h
—
4 h 46 0 h 44 4 d 21 h 28 h
a
a
a
a
a
a
a
a
—
—
—
—
—
—
—
6 h 42
44 h
4 h 16
(4)
7 h 35 5 h 30 47 h
a Stable over 3 days.
of stability: ester (1) < ketal (2b) ∼ carbonate (4) < acetal (2a) <
carbamate (3). A pH-dependent stability was observed for the
ketal conjugate (2b) which was more rapidly hydrolyzed under
acidic conditions. This result was of great interest as it offers
the opportunity to achieve specific cleavage of the conjugate in
acidic melanosomes. The following evaluation thus focused
especially on the study and modulation of this ketal conjugate
(2b).
Each HPLC chromatograph revealed at least six peaks: one
attributed to the uncleaved parent conjugate and the others to
Metabolic stability
In our approach, it was important to further evaluate the stabi- the metabolites. For all the conjugates (2a–d), the rate of
lity in the presence of intensive drug metabolising enzymatic metabolism was similar, ∼55% of the intact parent compound
systems in order to select conjugates with spacers showing being detected after 4 h of incubation. As the metabolic pro-
optimal metabolic stability and profile for selective drug files were similar for experiments carried out whether in
release. Metabolic stability determinations can be performed microsomes or in liver S9 fraction (Table S1, ESI†), the pure
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Scheme 5 Metabolites identified after microsomal incubation of conjugates (2a–d).
microsomes system was adopted for metabolite identification. carrier and could therefore hamper our targeting strategy. The
Five common metabolites (A–E) were identified for conjugates kinetics of formation of these two metabolites (D and E) were
(2a–d) at all times (Scheme 5). As no degradation was observed further investigated and compared for the conjugates (2a), (2c)
using inactive microsomes, the detected metabolites derived and (2d) (Fig. 6). In all cases, metabolite D was formed more
only from enzymatic reactions. Metabolites A and B (<5% at rapidly than metabolite E. However, whereas the production of
4 h) resulted, respectively, from IUdR and quinoxaline de-ioda- metabolite D constantly increased over the time frame (4 h),
tion. Metabolites C (<1% at 4 h), D (∼15% at 4 h) and E (∼20%
at 4 h) were generated by various N-monodealkylations of the
tertiary amine. Accordingly, N-dealkylation is a metabolic reac-
tion commonly observed for drugs containing secondary and
tertiary amines. Metabolite G (<10% at 4 h) was formed by the
cleavage of the ketal function. For conjugates (2c) and (2d),
metabolites F (<5% at 4 h) resulting from the cleavage at
various sites of the PEG chain were also identified. Interestingly,
the same cleavage pattern and metabolic profile were found for
the ester (1), carbamate (3) and carbonate (4) (data not shown).
In our study, the N-dealkylated products D and E were
always the two main metabolites. Metabolite E was formed by
N-deethylation of the parent conjugate. We have previously
demonstrated that melanoma tracers of the N-ethylaminoethy-
leneamido series maintain the tumour affinity observed for
their N,N-diethylaminoethyleneamido counterparts.4 Even
Fig. 6 Kinetics of formation of dealkylated metabolites D and E by microsomal
■
◆
▲
incubation of conjugates (2a) ( ), (2c) ( ) and (2d) ( ). Dotted lines, metabolite
E; solid lines, metabolite D. Relative concentration against time: expressed as the
ratio of the peak area of the metabolite to total area of all metabolite peaks at
a certain time interval.
after N-deethylation, metabolite E could therefore reach the
tumour cells. Conversely, the N-dealkylation pathway generat-
ing metabolite D was associated with the loss of the ICF 01012
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for the acetal conjugate (2a), the metabolic reaction kinetics further development of melanoma-targeted drug delivery
was slowed for the PEGylated congeners (2c) and (2d) and systems.
reached a plateau after 1 h. In addition, the extent and rate of
N-dealkylation/N-deethylation were reduced for (2d) and
especially for (2c). This result was anticipated as PEGylation
Experimental
has been shown to improve metabolic stability.22,23 The
General
advantage of PEG for the design of targeted conjugates is the
Proton nuclear magnetic resonance (NMR) spectra were
recorded using a Bruker AM-400 spectrometer. Chemical shifts
(δ) are reported in parts per million relative to the internal tetra-
methylsilane standard. Melting points (mp), uncorrected,
were determined using an electrothermal melting point appar-
atus. Elemental analyses were performed by the CNRS Service
Central d’Analyses (Vernaison, France). Chemicals were sup-
plied by Sigma-Aldrich Chimie SARL. Analytical thin layer
chromatography (TLC) was conducted on precoated silica gel
plates (60 F254, 0.25 mm thick) with both detection by ultra-
violet light at 254 nm and visualization by iodine. The HPLC
system was a Hewlett-Packard HP 1050 liquid chromatograph.
Electrospray ionization mass spectrometry (ESI-MS) was per-
formed using a Brüker ESQUIRE-LC for the structural determi-
nation of the conjugates and using a Thermo Finnigan TSQ
7000 mass spectrometer for the stability and metabolism
studies.
possibility of linking a targeting residue (carrier) on one
side and the active molecule on the other side. The increased
steric hindrance generally reduces enzyme degradation and
metabolic effects. Finally, the length of the glycol spacer
also seemed to affect the rate of N-dealkylation and the
shorter PEG chain conjugate (2c) presented a better metabolic
profile.
Conclusion
We prepared a series of seven conjugates of IUdR and the mela-
noma carrier ICF 01012 containing various spacer units: ester
(1), acetal (2a), ketal (2b), PEGylated acetal (2c and 2d), carba-
mate (3) and carbonate (4) for melanoma-selective delivery.
Preliminary evaluation consisting of stability tests under
various physiological conditions indicated that the ester conju-
gate (1) was too unstable for further development. Conversely,
carbamate (3), carbonate (4), acetal (2a) and ketal (2b) dis-
played relevant half-lives of several hours considering the
maximal tumoural concentration of the carrier ICF 01012 at
6 h post-injection.4 Importantly, the ketal conjugate (2b) stabi-
The tertiary amine precursors (18–19) and the vinyl ether
precursors (11a–d) were prepared as described in the ESI.†
Synthesis
5-Iodo-5′-O-triisopropylsilyl-2′-deoxyuridine (6). To a stirred,
lity was pH-dependent and decreased as the acidity increased. cooled at 0 °C solution of IUdR (5 g, 14.12 mmol), imidazole
Hydrolysis occurred 5 to 25 times faster in mildly acidic solu- (2.115 g, 31.07 mmol) and DMAP (172 mg, 1.41 mmol) in
tions (pH 5 and 4, respectively) compared to solutions at physio- anhydrous DMF (70 mL) was added a solution of triisopropyl
logical pH. We demonstrated that this class of molecules silyl chloride (2.995 g, 15.53 mmol) in anhydrous DMF
undergoes accelerated hydrolysis under mildly acidic con- (30 mL). The mixture was stirred for 1 h at 0 °C and for 18 h at
ditions. Moreover, the subcellular targets of the ICF 01012 room temperature, poured into sat. aq. NH4Cl (500 mL) and
melanoma-carrier are highly specialised organelles of the extracted with EtOAc (3 × 150 mL). The organic extracts were
melanocytes, i.e. the melanosomes.14 Melanosomes, early or combined, washed with sat. aq. NH4Cl (250 mL) and brine
late, are acidic compartments (pH 4–6).15,16 This property is (250 mL), dried over MgSO4 and concentrated in vacuo to give
particularly interesting and would allow rapid hydrolysis of the a clear oil. After column chromatography on silica gel (EtOAc–
ketal conjugate (2b) in melanoma cells specifically. Conjugates DCM, 40 : 60, v/v) the pure product (6) was obtained as white
such as (2b) that decompose in mildly acidic environments crystals (5.96 g, 83%). Mp 185–187 °C; Rf 0.28 (EtOAc–DCM,
1
i
may therefore be useful for the development of melanoma 40 : 60, v/v); H NMR: δ 1.07–1.29 (21H, m, 3 × Pr), 2.04–2.19
drug delivery systems where an acid-sensitive spacer could and 2.40–2.51 (1H each, two m, H-2′), 2.60 (1H, br s, OH),
exploit the lower subcellular pH of melanosomes to trigger the 3.88–3.97 (2H, m, H-5′), 4.06–4.17 (1H, m, H-4′), 4.52–4.58 (1H,
release of covalently linked therapeutics. Moreover, the ketal m, H-3′), 6.29 (dd, 1H, J 8.1, 5.6, H-1′), 8.06 (1H, s, H-6), 9.05
conjugate (2b) was also relatively metabolically stable as at (1H, br s, NH); 13C NMR: δ 12.1 (Si[CH(CH3)2]3), 18.3 (Si[CH-
least 50% of the molecules were intact after a 4 h microsomal (CH3)2]3), 41.8 (C-2′), 63.7 (C-5′), 68.7 (C-5), 72.5 (C-3′), 85.9
incubation and the metabolic stability was even enhanced for (C-1′), 87.8 (C-4′), 144.4 (C-6), 150.1 (C-2 CvO), 160.1 (C-4
their PEGylated congeners (2d) and (2c).
CvO); m/z (ESI) 508.83 [M − H]−.
Taken together, these results support that the ketal linker
3′-O-Acryloyl-5-iodo-5′-O-triisopropylsilyl-2′-deoxyuridine (7).
may be useful for constructing specific delivery systems that To a stirred, cooled at 0 °C solution of DCC (606 mg,
would be metabolically stable enough to reach the targeted 2.94 mmol) in anhydrous DCM (8 mL) was slowly added a
melanoma cell, precisely the melanosome, mostly in its intact solution of acrylic acid (202 μL, 2.94 mmol) in anhydrous DCM
form and then allow rapid drug release by hydrolysis under (8 mL). The mixture was stirred for 1 h at 0 °C and for 1 h at rt.
the acidic environment of the melanosomal compartment. A solution of (6) (1 g, 1.96 mmol) and DMAP (36 mg,
These data were essential to validate our approach before 0.29 mmol) in anhydrous DCM (24 mL) was then added and
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the mixture was stirred at reflux for 18 h, cooled at 0 °C, fil- silica gel (EtOAc–EtOH–NH4OH, 90 : 10 : 0.5, v/v/v) the pure
tered and the filtrate was poured into water (50 mL). The product (1) was obtained as a yellow foam (114 mg, 95%). Rf
1
aqueous layer was extracted with DCM (2 × 30 mL) and the 0.26 (EtOAc–EtOH–NH4OH, 90 : 10 : 0.5, v/v/v); H NMR: δ 1.06
organic layers were combined, dried over MgSO4 and concen- (2H, t, J 6.8, NCH2CH3), 2.31–2.42 (2H, m, H-2′), 2.56–2.92 (8H,
trated in vacuo to give a white solid. After column chromato- m, 3 × NCH2, OCOCH2), 3.56 (2H, q, J 7.1, NCH2CH2NH),
graphy on silica gel (EtOAc–cyclohexane, 65 : 35, v/v) the pure 3.75–3.82 (2H, m, H-5′), 4.10–4.14 (1H, m, H-4′), 5.32–5.37 (1H,
product (7) was obtained as white crystals (510 mg, 46%). Mp m, H-3′), 6.23 (1H, dd, J 8.2, 6.1, H-1′), 7.93 (1H, d, J 8.8, ArH
1
167–169 °C; Rf 0.30 (EtOAc–cyclohexane, 65 : 35, v/v); H NMR: ‘m’ to I), 8.21 (1H, dd, J 8.8, 1.8, ArH ‘o’ to I), 8.44 (1H, s, H-6),
δ 0.98–1.39 (21H, m, 3 × iPr), 2.10–2.21 (1H, m, H-2′), 2.53 (1H, 8.53 (1H, br s, ArCONH), 8.60 (1H, d, J 1.8, ArH ‘o’ to I), 9.52
dd, J 13.8, 4.9 Hz, H-2′), 3.98–4.04 (2H, m, H-5′), 4.11–4.15 (1H, (1H, s, ArH ‘o’ to CONH); 13C NMR: δ 11.9 (NCH2CH3), 32.4
m, H-4′), 5.41 (d, 1H, m, J 6.0, H-3′), 5.88 (1H, dd, J 9.7, 1.2, (OCOCH2), 37.1 (C-2′), 47.0 (NCH2CH3), 48.8 (OCOCH2CH2),
OCOCHCH2 cis), 6.12 (1H, dd, J 16.9, 9.7, OCOCHCH2), 6.37 51.6 (NCH2CH2NH), 61.1 (C-5′), 69.9 (C-5), 74.7 (C-3′), 84.4
(1H, dd, J 9.0, 4.9, H-1′), 6.45 (1H, dd, J 16.9, 1.2, OCOCHCH2 (C-1′), 85.0 (C-4′), 99.2 (ArCI), 130.7 (ArCH ‘m’ to I), 137.6
trans), 8.07 (1H, s, H-6), 9.83 (1H, br s, NH); 13C NMR: δ 11.9 (ArCH ‘β’ to N), 139.0 (ArC ‘p’ to I), 139.8 (ArCH ‘o’ to I), 143.6
(Si[CH(CH3)2]3), 18.2 (Si[CH(CH3)2]3), 38.7 (C-2′), 63.7 (C-5′), (ArC ‘m’ to I), 144.3 (ArCH ‘o’ to CONH), 144.5 (ArCCONH),
69.2 (C-5), 75.3 (C-3′), 85.4 (C-1′), 86.1 (C-4′), 127.7 144.7 (C-6), 150.1 (C-2 CvO), 160.4 (C-4 CvO), 162.6
(OCOCHCH2), 132.3 OCOCHCH2, 143.8 (C-6), 150.3 (C-2 (ArCONH), 171.8 (OCO); m/z (ESI) 779.26 [M + H]+; Found C,
CvO), 160.2 (C-4 CvO), 165.7 (OCOCHCH2); m/z (ESI) 563.12 38.26; H, 3.76; N, 10.58. C25H28I2N6O7 requires C, 38.58; H,
[M − H]−.
3.63; N, 10.80%.
3′-O-{3-[Ethyl-2-({[(6-iodoquinoxalin-2-yl)carbonyl]amino}-
ethyl)amino]propanoyl}-5-iodo-5′-O-triisopropylsilyl-2′-deoxyur-
idine (8). To a stirred suspension of (7) (510 mg, 0.90 mmol)
in anhydrous MeCN (7.5 mL) were added the secondary amine
(5) (334 mg, 0.90 mmol) and DBU (67 μL, 0.45 mmol). The
mixture was stirred for 96 h at rt, poured into water (50 mL)
and extracted with DCM (3 × 20 mL). The organic layers were
combined, dried over MgSO4 and concentrated in vacuo to give
a brown oil. After column chromatography on silica gel
(EtOAc–EtOH–NH4OH, 97 : 3 : 0.5, v/v/v) the pure product (8)
was obtained as a yellow foam (151 mg, 18%). Rf 0.25 (EtOAc–
EtOH–NH4OH, 97 : 3 : 0.5, v/v/v); 1H NMR: δ 1.02–1.17 (21H,
General procedure for the preparation of compounds (12a–d)
To a solution of (6) and camphorsulfonic acid (0.04 equivalent)
in anhydrous THF (10 mL mmol−1) was added a solution of
the corresponding vinyl ether (9a–d) in anhydrous THF (5 mL
mmol−1). The mixture was stirred for different times at rt and
then poured into a mixture of water (30 mL mmol−1 (6)) and
sat. aq. NaHCO3 (15 mL mmol−1 (6)) which was extracted with
DCM (3 × 15 mL mmol−1). The organic layers were combined,
dried over MgSO4 and concentrated in vacuo.
3′-O-{1-[2-(Acetyloxy)ethoxy]ethyl}-5-iodo-5′-O-triisopropylsi-
lyl-2′-deoxyuridine (12a). Prepared from (6) (1.5 g, 2.94 mmol)
and vinyl ether (11a) (1.147 g, 8.82 mmol). Reaction time: 5 h.
After column chromatography on silica gel (EtOAc–cyclo-
hexane, 35 : 65, v/v) the pure product (12a) was obtained as a
white solid (1.859 g, 99%). Mp 96–98 °C; Rf 0.24 (EtOAc–cyclo-
i
m, 3 × Pr), 1.25 (2H, t, J 6.8, NCH2CH3), 1.98–2.13 (1H, m,
H-2′), 2.40–2.65 (5H, m, H-2′, OCOCH2, NCH2CH3), 2.73 (2H, t,
J 6.2, OCOCH2CH2), 2.89 (2H, t, J 7.1, NCH2CH2NH), 3.58 (2H,
q, J 7.1, NCH2CH2NH), 3.76–3.92 (2H, m, H-5′), 4.06–4.13 (1H,
m, H-4′), 5.30 (1H, d, J = 5.7, H-3′), 6.22 (1H, dd, J 9.0, 4.9,
H-1′), 7.80 (1H, d, J 8.8, ArH ‘m’ to I), 8.00 (1H, s, H-6), 8.06
(1H, dd, J 8.8, 1.8, ArH ‘o’ to I), 8.26 (1H, br s, ArCONH), 8.60
(1H, d, J 1.8, ArH ‘o’ to I), 9.64 (1H, s, ArH ‘o’ to CONH); 13C
NMR: δ 11.9 (Si[CH(CH3)2]3), 12.0 (NCH2CH3), 18.1 (Si[CH-
(CH3)2]3), 32.8 (OCOCH2), 37.4 (NCH2CH2NH), 38.5 (C-2′), 47.4
(NCH2CH3), 48.9 (OCOCH2CH2), 52.3 (NCH2CH2NH), 63.6
(C-5′), 69.3 (C-5), 75.4 (C-3′), 85.3 (C-1′), 86.0 (C-4′), 98.1 (ArCI),
130.8 (ArCH ‘m’ to I), 138.6 (ArCH ‘β’ to N), 139.5 (ArC ‘p’ to I),
139.7 (ArCH ‘o’ to I), 143.6 (C-6), 144.09, 144.3 (ArCCONH, ArC
‘m’ to I), 144.7 (ArCH ‘o’ to CONH), 150.2 (C-2 CvO), 160.1
(C-4 CvO), 163.0 (ArCONH), 172.3 (OCO); m/z (ESI) 935.48
[M + H]+.
i
hexane, 35 : 65, v/v); 1H NMR: δ 1.09–1.29 (21H, m, 3 × Pr),
1.35 (3H, d, J 5.0, O[CHCH3]O), 2.03–2.13 (4H, m, H-2′,
OCOCH3), 2.43–2.54 (1H, m, H-2′), 3.61–3.92 (4H, m, H-5′,
OCH2CH2OCO), 3.96–4.25 (3H, m, H-4′, OCH2CH2OCO),
4.44–4.51 (1H, m, H-3′), 4.85 (1H, q, J = 5.0, O[CHCH3]O),
6.18–6.25 (1H, m, H-1′), 8.04 (1H, s, H-6), 8.36 (1H, br s, NH);
13C NMR: δ 12.0 (Si[CH(CH3)2]3), 18.2 (Si[CH(CH3)2]3), 20.0
(O[CHCH3]O), 41.0 (C-2′), 62.2, 63.5, 63.7 (C-5′, OCH2CH2OCO),
68.7 (C-5), 74.9 (C-3′), 85.7 (C-1′), 86.2 (C4′), 99.2 (O[CHCH3]O),
144.1 (C-6), 150.1 (C-2 CvO), 160.1 (C-4 CvO), 171.0
(OCOCH3); m/z (ESI) 639.05 [M − H]−.
The intermediates (12b–d) were prepared by using a similar
procedure and fully characterised (see the ESI†).
3′-O-{3-[Ethyl-2-({[(6-iodoquinoxalin-2-yl)carbonyl]amino}-
ethyl)amino]propanoyl}-5-iodo-2′-deoxyuridine (1). To a solu-
tion of (8) (143 mg, 0.15 mmol) in anhydrous THF (4.5 mL)
General procedure for the preparation of compounds (13a–d)
was added Bu4NF (1 M in THF, 230 μL) and the mixture was To a solution of (12a–d) in anhydrous EtOH (20 mL mmol−1
)
stirred for 5 h at rt and poured into sat. aq. NaHCO3 (30 mL). was added LiOH (1.5 equivalents). The mixture was stirred for
The mixture was extracted with DCM (3 × 10 mL), the organic different times at rt and then poured into a mixture of 5% aq.
layers were combined, dried over MgSO4 and concentrated Na2CO3 (80 mL mmol−1) and sat. aq. NaCl (30 mL mmol−1
)
in vacuo to give a brown oil. After column chromatography on which was extracted with DCM (4 × 30 mL mmol−1). The
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organic layers were combined, dried over MgSO4 and concen- mmol−1). The organic layers were combined, dried over MgSO4
trated in vacuo.
and concentrated in vacuo.
3′-O-[1-(2-Hydroxyethoxy)ethyl]-5-iodo-5′-O-triisopropylsilyl-
3′-O-(1-{2-[Ethyl(2-{[(6-iodoquinoxalin-2-yl)carbonyl]amino}-
2′-deoxyuridine (13a). Prepared from (12a) (1.959 g, ethyl)amino]ethoxy}ethyl)-5-iodo-5′-O-triisopropylsilyl-2′-deoxy-
2.94 mmol). Reaction time: 1 h. After column chromatography uridine (15a). Prepared from (14a) (1.244 g, 1.84 mmol).
on silica gel (EtOAc–cyclohexane, 80 : 20, v/v) the pure product Time of reaction: 88 h, temperature: 55 °C. After column
(11a) was obtained as a colourless caramel (1.630 g, 93%). Rf chromatography on silica gel (EtOAc–EOH–NH4OH, 95 : 5 : 0.5,
0.29 (EtOAc–cyclohexane, 80 : 20, v/v); 1H NMR: δ 1.06–1.19 v/v/v) the pure product (15a) was obtained as a light brown
i
(21H, m, 3 × Pr), 1.37 (3H, d, J 5.0, O[CHCH3]O), 1.93–2.13 foam (0.265 g, 15%). Rf 0.29 (EtOAc–EOH–NH4OH, 95 : 5 : 0.5,
1
i
(2H, H-2′, OH), 2.43–2.63 (1H, m, H-2′), 3.52–3.76 (2H, m, v/v/v); H NMR: δ 1.08–1.16 (24H, m, 3 × Pr, NCH2CH3), 1.30
OCH2CH2OCO), 3.71–3.75 (2H, m, OCH2CH2OCO), 3.82–3.98 (3H, d, J 5.0, O[CHCH3]O), 1.96–2.04 (1H, m, H-2′), 2.39–2.44
(2H, m, H-5′), 3.95–4.15 (1H, m, H-4′), 4.43–4.55 (1H, m, H-3′), (1H, m, H-2′), 2.65–2.82 (6H, m, 3 × NCH2), 3.51–3.85 (4H, m,
4.87 (1H, q, J = 5.0, O[CHCH3]O), 6.18–6.25 (1H, m, H-1′), 8.04 OCH2CH2N, NCH2CH2NH), 3.87–3.98 (2H, m, H-5′), 4.04–4.10
(1H, s, H-6), 8.63 (1H, br s, NH); 13C NMR: δ 12.0 (Si[CH- (1H, m, H-4′), 4.38–4.44 (1H, m, H-3′), 4.78 (1H, q, J = 5.0, O
(CH3)2]3), 18.2 (Si[CH(CH3)2]3), 20.1 (O[CHCH3]O), 39.6 (C-2′), [CHCH3]O), 6.17 (1H, q, J = 7.8, 5.2, H-1′), 7.80 (1H, d, J 8.8,
61.9 (OCH2CH2OCO), 63.5 (C-5′), 66.0 (OCH2CH2OCO), 68.7 ArH ‘m’ to I), 7.96–8.09 (2H, m, H-6, ArH ‘o’ to I), 8.38 (1H, br
(C-5), 75.3 (C-3′), 86.1 (C-1′), 86.5 (C-4′), 99.4 (O[CHCH3]O), s, ArCONH), 8.60 (d, 1H, J 1.8, ArH ‘o’ to I), 9.63 (1H, s, ArH ‘o’
144.2 (C-6), 150.1 (C-2 CvO), 160.0 (C-4 CvO); m/z (ESI) to CONH); 13C NMR: δ 12.0 (Si[CH(CH3)2]3, NCH2CH3), 18.3 (Si-
596.98 [M − H]−.
The intermediates (13b–d) were prepared by using a similar (C-2′), 48.7 (NCH2CH3), 52.9, 53.2 (OCH2CH2N, NCH2CH2NH),
[CH(CH3)2]3), 20.2 (O[CHCH3]O), 37.5 (NCH2CH2NH), 39.7
procedure and fully characterised (see the ESI†).
63.1, 63.5 (C-5′, OCH2CH2N), 68.7 (C-5), 75.0 (C-3′), 85.8 (C-1′),
86.4 (C-4′), 98.2 (ArCI), 99.5 (O[CHCH3]O), 130.8 (ArCH ‘m’ to
I), 138.7 (ArCH ‘β’ to N), 139.6 (ArC ‘p’ to I), 139.9 (ArCH ‘o’ to
General procedure for the preparation of compounds (14a–d)
To a solution of (13a–d) and DIPEA (2.5 equivalents) in anhy- I), 144.1 (C-6), 144.2, 144.5 (ArCCONH, ArC ‘m’ to I), 144.8
drous DCM (12 mL mmol−1) was added MsCl (1.2 equivalents). (ArCH ‘o’ to CONH), 149.9 (C-2 CvO), 160.0 (C-4 CvO), 163.2
The mixture was stirred for 1 h at rt and then poured into a (ArCONH).
mixture of water (110 mL mmol−1) and sat. aq. NaCl (15 mL
The intermediates (15b–d) were prepared using a similar
mmol−1) which was extracted with DCM (3 × 40 mL mmol−1). procedure and fully characterised (see the ESI†).
The organic layers were combined, dried over MgSO4 and con-
centrated in vacuo.
General procedure for the preparation of target compounds
(2a–d)
5-Iodo-3′-O-{1-[2-(methylsulfonyloxy)ethoxy]ethyl}-5′-O-triiso-
propylsilyl-2′-deoxyuridine (14a). Prepared from (13a) (1.630 g, To a solution of (15a–d) in anhydrous THF (30 mL mmol−1
)
2.72 mmol). After column chromatography on silica gel was added Bu4NF (1 M in THF, 2 equivalents) and the mixture
(EtOAc–cyclohexane, 65 : 35, v/v) the pure product (14a) was was stirred for 4 h at rt and poured into sat. aq. NaHCO3
obtained as a white foam (1.639 g, 89%). Rf 0.29 (EtOAc–cyclo- (100 mL mmol−1). The mixture was extracted with DCM (3 ×
i
hexane, 65 : 35, v/v); 1H NMR: δ 1.09–1.26 (21H, m, 3 × Pr), 30 mL mmol−1), the organic layers were combined, dried over
1.36 (3H, d, J 5.0, O[CHCH3]O), 2.02–2.14 (1H, m, H-2′), MgSO4 and concentrated in vacuo.
2.39–2.48 (1H, m, H-2′), 3.04 (3H, s, OSO2CH3), 3.67–4.15 (5H,
3′-O-(1-{2-[Ethyl(2-{[(6-iodoquinoxalin-2-yl)carbonyl]amino}-
m, H-4′, H-5′, OCH2CH2OSO2), 4.32–4.38 (2H, m, OCH2- ethyl)amino]ethoxy}ethyl)-5-iodo-2′-deoxyuridine (2a). Pre-
CH2OSO2), 4.43–4.50 (1H, m, H-3′), 4.86 (1H, q, J = 5.0, O pared from (15a) (265 mg, 0.28 mmol). After column chrom-
[CHCH3]O), 6.20 (1H, dd, J = 7.9, 5.2, H-1′), 8.03 (1H, s, H-6), atography on silica gel (EtOAc–EtOH–NH4OH, 90 : 10 : 0.5,
8.36 (1H, br s, NH); 13C NMR: δ 12.0 (Si[CH(CH3)2]3), 18.2 (Si- v/v/v) the pure product (2a) was obtained as a yellow foam
[CH(CH3)2]3), 20.0 (O[CHCH3]O), 37.7 (OSO2CH3), 39.3, 39.8 (180 mg, 81%). Rf 0.28 (EtOAc–EtOH–NH4OH, 90 : 10 : 0.5,
1
(C-2′), 62.3 (C-5′), 63.4 (OCH2CH2OSO2), 68.9, 69.0 (C-5, OCH2- v/v/v); H NMR: δ 1.10 (3H, t, J 6.8, NCH2CH3), 1.22–1.31 (3H,
CH2OSO2), 75.3 (C-3′), 85.7 (C-1′), 86.2 (C-4′), 99.4 (O[CHCH3]- m, O[CHCH3]O), 2.33–2.45 (2H, m, H-2′), 2.65–2.93 (6H, m, 3 ×
O), 144.1 (C-6), 150.2 (C-2 CvO), 160.2 (C-4 CvO); m/z (ESI) NCH2), 3.53–3.71 (4H, m, OCH2CH2N, NCH2CH2NH),
675.02 [M − H]−.
3.86–4.02 (3H, m, H-4′, H-5′), 4.49–4.82 (2H, m, H-3′,
The intermediates (14b–d) were prepared by using a similar O[CHCH3]O), 6.04–6.14 (1H, m, H-1′), 7.82 (1H, d, J 8.8, ArH ‘m’
procedure and fully characterised (see the ESI†).
to I), 8.08 (1H, dd, J 8.8, 1.8, ArH ‘o’ to I), 8.30–8.39 (1H, br s,
ArCONH), 8.50 (1H, s, H-6), 8.61 (d, 1H, J 1.8, ArH ‘o’ to I), 9.62
(1H, s, ArH ‘o’ to CONH); 13C NMR: δ 11.2 (NCH2CH3), 20.1
(O[CHCH3]O), 37.4 (NCH2CH2NH), 39.5 (C-2′), 48.4 (NCH2CH3),
General procedure for the preparation of compounds (15a–d)
To a solution of (14a–d) in anhydrous MeCN (20 mL mmol−1
)
was added secondary amine (5) (1.5 equivalents). The mixture 52.9, 53.0 (OCH2CH2N, NCH2CH2NH), 60.6 (C-5′), 63.4
was stirred for different times at different temperatures (for (OCH2CH2N), 68.2 (C-5), 72.0 (C-3′), 85.9 (C-1′), 86.3 (C-4′), 98.3
details see below) and then poured into sat. aq. NaHCO3 (ArCI), 99.4 (O[CHCH3]O), 130.8 (ArCH ‘m’ to I), 138.5 (ArCH
(160 mL mmol−1) and extracted with DCM (3 × 50 mL ‘β’ to N), 139.5 (ArC ‘p’ to I), 139.9 (ArCH ‘o’ to I), 143.9, 144.3
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(ArCCONH, ArC ‘m’ to I), 144.5 (ArCH ‘o’ to CONH), 145.6 product (2d) was obtained as a brown caramel (0.009 g, 50%).
(C-6), 150.3 (C-2 CvO), 160.7 (C-4 CvO), 163.3 (ArCONH); m/z Rf 0.27 (EtOAC–DCM–EtOH–NH4OH, 20 : 65 : 15 : 0.5, v/v/v/v);
(ESI) 795.23 [M + H]+; Found C, 38.62; H, 4.15; N, 10.18. 1H NMR: δ 1.09 (3H, t, J 6.9 NCH2CH3), 1.31 (3H, d, J 5.0,
C26H32I2N6O7 requires C, 39.31; H, 4.06; N, 10.58%.
O[CHCH3]O), 2.15–2.51 (1H, m, H-2′), 2.66–2.86 (6H, m, 3 ×
3′-O-(1-(2-(Ethyl(2-(((6-iodoquinoxalin-2-yl)carbonyl)amino)- NCH2), 3.59–3.65 (24H, m, 9 × OCH2CH2O, OCH2CH2N,
ethyl)amino)ethoxy)-1-methylethyl)-5-iodo-2′-deoxyuridine (2b). NCH2CH2NH), 3.86–3.97 (2H, m, H-5′), 4.04–4.10 (1H, m, H-4′),
Prepared from (15b) (0.134 g, 0.14 mmol). After column 4.54–4.67 (1H, m, H-3′), 4.77–4.87 (1H, m, O[CHCH3]O),
chromatography on silica gel (EtOAc–EtOH–NH4OH, 6.12–6.20 (1H, m, H-1′), 7.82 (1H, d, J 8.9, ArH ‘m’ to I), 8.08
90 : 10 : 0.5, v/v/v) the pure product (2b) was obtained as a (1H, dd, J 8.9, 1.8, ArH ‘o’ to I), 8.48 (1H, br s, ArCONH), 8.51
yellow foam (0.091 g, 81%). Rf 0.28 (EtOAc–EtOH–NH4OH, (1H, s, H-6), 8.60 (d, 1H, J 1.8, ArH ‘o’ to I), 9.63 (1H, s, ArH ‘o’
90 : 10 : 0.5, v/v/v); 1H NMR: δ 1.12 (3H, t, J 6.8, NCH2CH3), 1.35 to CONH); 13C NMR: δ 11.9 (NCH2CH3), 20.0 (O[CHCH3]O),
(6H, s, O[C(CH3)2]O), 2.37–2.53 (2H, m, H-2′), 2.65–3.03 (6H, 37.4 (NCH2CH2NH), 39.7 (C-2′), 48.7 (NCH2CH3), 52.8
m, 3 × NCH2), 3.52–4.04 (7H, m, OCH2CH2N, NCH2CH2NH, (NCH2CH2NH), 53.0 (OCH2CH2N), 61.3 (C-5′), 63.7 (O[CHCH3]
H-4′, H-5′), 4.62–4.66 (1H, m, H-3′), 6.08 (1H, q, J = 6.2, 4.2, OCH2CH2), 67.9 (C-5), 69.8–70.8 (OCH2CH2N, OCH2CH2N, 8 ×
H-1′), 7.82 (1H, d, J 8.8, ArH ‘m’ to I), 8.08 (1H, dd, J 8.8, 1.9, OCH2CH2O), 72.4 (C-3′), 86.0 (C-4′), 86.4 (C-1′), 98.1 (ArCI),
ArH ‘o’ to I), 8.40 (1H, br s, ArCONH), 8.61 (d, 1H, J 1.9, ArH ‘o’ 98.6 (O[CHCH3]O), 131.0 (ArCH ‘m’ to I), 138.6 (ArCH ‘β’ to N),
to I), 8.74 (1H, s, H-6), 9.59 (1H, s, ArH ‘o’ to CONH); 13C NMR: 139.7 (ArC ‘p’ to I), 139.8 (ArCH ‘o’ to I), 144.2, 144.4
δ
10.9 (NCH2CH3), 25.1, 26.4 (O[C(CH3)2]O), 37.5 (ArCCONH, ArC ‘m’ to I), 144.8 (ArCH ‘o’ to CONH),), 145.7
(NCH2CH2NH), 41.1 (C-2′), 49.3 (NCH2CH3), 52.9, 53.0 (C-6), 150.0 (C-2 CvO), 160.1 (C-4 CvO), 163.2 (ArCONH); m/z
(OCH2CH2N, NCH2CH2NH), 59.7 (C-5′), 61.4 (OCH2CH2N), (ESI) 1191.33 [M + H]+.
66.8 (C-3′), 67.6 (C-5), 86.3 (C-1′), 86.5 (C-4′), 98.3 (ArCI), 100.9
5-Iodo-3′-O-[(4-nitrophenony)carbonyl]-5′-O-triisopropylsilyl-
(O[C(CH3)2]O), 130.8 (ArCH ‘m’ to I), 138.6 (ArCH ‘β’ to N), 2′-deoxyuridine (20). To a cooled at 0 °C solution of (6)
139.6 (ArC ‘p’ to I), 139.9 (ArCH ‘o’ to I), 143.9, 144.4 (750 mg, 1.47 mmol) in anhydrous pyridine (5.3 mL) was
(ArCCONH, ArC ‘m’ to I), 144.6 (ArCH ‘o’ to CONH), 145.8 added a solution of p-nitrophenyl chloroformate (370 mg,
(C-6), 150.3 (C-2 CvO), 160.6 (C-4 CvO), 163.4 (ArCONH); m/z 1.84 mmol) in anhydrous DCM (5.3 mL). The mixture was
(ESI) 809.11 [M + H]+; Found C, 40.11; H, 4.55; N, 10.51. stirred for 2 h at 0 °C and for 15 h at rt and then concentrated
C27H34I2N6O7 requires C, 40.11; H, 4.24; N, 10.40%.
in vacuo (co-evaporation with 2 × 30 mL toluene) to give a
3′-O-(20-Ethyl-24-(6-iodoquinoxalin-2-yl)-1-methyl-24-oxo- yellow solid. After column chromatography on silica gel
2,5,8,11,14,17-hexaoxa-20,23-diazatetracos-1-yl)-5-iodo-2′-deoxy- (EtOAc–cyclohexane, 30 : 70, v/v) the pure product (18) was
uridine (2c). Prepared from (15c) (0.170 g, 0.15 mmol). After obtained as a white solid (845 mg, 85%). Mp 175–177 °C; Rf
column chromatography on silica gel (DCM–EtOH–NH4OH, 0.22 (EtOAc–cyclohexane, 30 : 70, v/v); 1H NMR: δ 1.08–1.29
i
92 : 8 : 1, v/v/v) the pure product (2c) was obtained as a yellow (21H, m, 3 × Pr), 2.12–2.31 (1H, m, H-2′), 2.69 (1H, dd, J 13.7,
foam (0.132 g, 90%). Rf 0.29 (DCM–EtOH–NH4OH, 92 : 8 : 1, v/ 5.2, H-2′), 4.04–4.05 (2H, m, H-5′), 4.33 (1H, m, H-4′), 5.41 (1H,
1
v/v); H NMR: δ 1.07 (3H, t, J 6.9 NCH2CH3), 1.30 (3H, d, J 5.0, d, J 6.1, H-3′), 6.36 (1H, dd, J 8.8, 5.2, H-1′), 7.40 (2H, d, J 8.8, 2
O[CHCH3]O), 2.14–2.51 (1H, m, H-2′), 2.64–2.84 (6H, m, 3 × × ArH ‘m’ to NO2), 8.04 (1H, s, H-6), 8.30 (2H, J 8.8, 2 × ArH ‘o’
NCH2), 3.58–3.65 (24H, m, 5 × OCH2CH2O, OCH2CH2N, to NO2), 8.42 (1H, br s, H-3); 13C NMR: δ 12.0 (Si[CH(CH3)2]3),
NCH2CH2NH), 3.80–3.97 (2H, m, H-5′), 4.03–4.10 (1H, m, H-4′), 18.2 (Si[CH(CH3)2]3), 38.7 (C-2′), 63.8 (C-5′), 69.2 (C-5), 80.1
4.55–4.66 (1H, m, H-3′), 4.77–4.86 (1H, m, O[CHCH3]O), (C-3′), 85.4 (C-1′), 85.6 (C-4′), 121.8 (2 × ArCH ‘m’ to NO2), 125.5
6.10–6.18 (1H, m, H-1′), 7.82 (1H, d, J 8.9, ArH ‘m’ to I), 8.08 (2 × ArCH ‘o’ to NO2), 143.7 (C-6), 145.6 (ArCNO2), 150.0 (C-2
(1H, dd, J 8.9, 1.8, ArH ‘o’ to I), 8.44 (1H, br s, ArCONH), 8.49 CvO), 152.0 (OCOO), 155.1 (ArC ‘p’ to NO2), 160.3 (C-4 CvO).
(1H, s, H-6), 8.60 (d, 1H, J 1.8, ArH ‘o’ to I), 9.63 (1H, s, ArH ‘o’
to CONH); 13C NMR: δ 11.9 (NCH2CH3), 20.1 (O[CHCH3]O), ethyl)amino]ethyl}amino)carbonyl]-5-iodo-5′-O-triisopropylsi-
37.5 (NCH2CH2NH), 39.6 (C-2′), 48.6 (NCH2CH3), 52.7 lyl-2′-deoxyuridine (21). To solution of (20) (311 mg,
3′-O-[({2-[Ethyl-(2-{[(6-iodoquinoxalin-2-yl)carbonyl]amino}-
a
(NCH2CH2NH), 53.0 (OCH2CH2N), 61.3 (C-5′), 63.6 (O[CHCH3] 0.46 mmol) in anhydrous DCM (4.2 mL) was added a solution
OCH2CH2), 68.0 (C-5), 69.9–70.7 (OCH2CH2N, OCH2CH2N, 4 × of (18) (285 mg, 0.69 mmol) and NEt3 (97 μL, 0.69 mmol) in
OCH2CH2O), 72.5 (C-3′), 85.9 (C-4′), 86.4 (C-1′), 98.1 (ArCI), anhydrous DCM (4.2 mL). The mixture was stirred for 18 h at
98.7 (O[CHCH3]O), 130.9 (ArCH ‘m’ to I), 138.6 (ArCH ‘β’ to N), rt and then poured into 5% aq. Na2CO3 (75 mL). The layers
139.7 (ArC ‘p’ to I), 139.8 (ArCH ‘o’ to I), 144.3, 144.4 were separated and the aqueous layer was extracted with DCM
(ArCCONH, ArC ‘m’ to I), 144.8 (ArCH ‘o’ to CONH), 145.7 (3 × 25 mL). The organic layers were combined, washed with
(C-6), 150.1 (C-2 CvO), 160.3 (C-4 CvO), 163.1 (ArCONH); m/z 5% aq. Na2CO3 (30 mL), dried over MgSO4 and concentrated in
(ESI) 1015.25 [M + H]+.
vacuo to give an orange oil. After column chromatography on
3′-O-(32-Ethyl-36-(6-iodoquinoxalin-2-yl)-1-methyl-36-oxo- silica gel (EtOAc–EtOH–NH4OH, 90 : 10 : 0.5, v/v/v) the pure
2,5,8,11,14,17,20,23,26,29-decaoxa-32,35-diazahexatriacont-1- product (19) was obtained as a light yellow foam (420 mg,
yl)-5-iodo-2′-deoxyuridine (2d). Prepared from (15d) (0.022 g, 96%). Rf 0.31 (EtOAc–EtOH–NH4OH, 90 : 10 : 0.5, v/v/v); 1H
i
0.02 mmol). After column chromatography on silica gel NMR: δ 1.03–1.25 (24H, m, 3 × Pr, NCH2CH3), 1.96–2.10 (1H,
(EtOAC–DCM–EtOH–NH4OH, 20 : 65 : 15 : 0.5, v/v/v/v) the pure m, H-2′), 2.28–2.38 (1H, m, H-2′), 2.60–2.78 (6H, m, 3 × NCH2),
6382 | Org. Biomol. Chem., 2013, 11, 6372–6384
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3.27 (2H, q, 5.0, OCONHCH2), 3.60 (2H, q,
Paper
J
J
4.8, 3 × NCH2), 3.08 (2H, q, J 5.7, OCONHCH2), 3.32–3.40 (2H, m,
ArCONHCH2), 3.94–3.98 (3H, m, H-4′, H-5′), 5.21 (1H, d, J 5.7, ArCONHCH2), 3.56–3.58 (2H, m, H-5′), 3.89–3.91 (1H, m, H-4′),
H-3′), 5.57 (1H, t, J 5.0, OCONH), 6.05 (1H, dd, J 8.8, 5.1 Hz, 5.04–5.06 (1H, m, H-3′), 5.25 (1H, t, J 4.8, OH), 6.05 (1H, t, J
H-1′), 7.81 (1H, d, J 8.9, ArCH ‘m’ to I), 8.04 (1H, s, H-6), 8.08 7.2 Hz, H-1′), 7.20 (1H, t, J 5.7, OCONH), 7.89 (1H, d, J 8.7,
(1H, dd, J = 8.9, 1.8, ArCH ‘o’ to I), 8.23 (1H, t, J 4.8, ArCONH), ArCH ‘m’ to I), 8.20 (1H, dd, J = 8.7, 1.7, ArCH ‘o’ to I), 8.33
8.60 (1H, d, J 1.8, ArCH ‘o’ to I), 9.65 (1H, s, ArCH ‘o’ to (1H, s, H-6), 8.58 (1H, d, J 1.7, ArCH ‘o’ to I), 8.93 (1H, t, J 4.8,
CONH); 13C NMR: δ 11.5 (NCH2CH3), 11.8 (Si[CH(CH3)2]3), ArCONH), 9.42 (1H, s, ArCH ‘o’ to CONH), 11.66 (1H, br s, NH);
18.1 (Si[CH(CH3)2]3), 37.4 (ArCONHCH2), 38.4 (C-2′), 38.9 13C NMR: δ 11.9 (NCH2CH3), 37.3 (ArCONHCH2), 37.5 (C-2′),
(OCONHCH2), 47.6 (NCH2CH3), 52.6 (2 × NHCH2CH2N), 63.6 38.3 (OCONHCH2), 47.4 (NCH2CH3), 52.1, 52.6 (2
×
(C-5′), 69.2 (C-5), 75.4 (C-3′), 85.2 (C-1′), 86.0 (C-4′), 98.1 (ArCI), NHCH2CH2N), 61.2 (C-5′), 69.8 (C-5), 74.6 (C-3′), 84.4 (C-1′),
130.6 (ArCH ‘m’ to I), 138.4 (ArCH ‘β’ to N), 139.3 (ArC ‘p’ to I), 85.3 (C-4′), 99.2 (ArCI), 130.8 (ArCH ‘m’ to I), 137.5 (ArCH ‘β’ to
139.7 (ArCH ‘o’ to I), 143.7 (C-6), 143.9, 144.2 (ArC ‘m’ to I, N), 139.0 (ArC ‘p’ to I), 139.8 (ArCH ‘o’ to I), 143.6 (ArC ‘m’ to
ArCCONH), 144.5 (ArCH ‘o’ to CONH), 150.3 (C-2 CvO), 155.6 I), 144.3 (ArCH ‘o’ to CONH), 144.6 (ArCCONH), 144.7 (C-6),
(OCONH), 160.5 (C-4 CvO), 163.0 (ArCONH); m/z (ESI) 950.30 150.1 (C-2 CvO), 155.4 (OCONH), 160.4 (C-4 CvO), 162.7
[M + H]+.
(ArCONH); m/z (ESI) 794.21 [M + H]+; Found C, 36.05; H, 3.73;
3′-O-({2-[Ethyl-(2-{[(6-iodoquinoxalin-2-yl)carbonyl]amino}- N, 11.80. C25H29I2N7O7, 2H2O requires C, 36.20; H, 4.01; N,
ethyl)amino]ethyloxy}carbonyl)-5-iodo-5′-O-triisopropylsilyl-2′- 11.82%.
deoxyuridine (22). To a solution of (19) (400 mg, 0.97 mmol),
3′-O-({2-[Ethyl-(2-{[(6-iodoquinoxalin-2-yl)carbonyl]amino}-
pyridine (78 μL, 0.97 mmol) and DMAP (102 mg, 0.84 mmol) ethyl)amino]ethyloxy}carbonyl)-5-iodo-2′-deoxyuridine (4). To
in anhydrous DCM (6 mL) was added a solution of (20) a solution of (22) (500 mg, 0.53 mmol) in anhydrous THF
(434 mg, 0.64 mmol) in anhydrous DCM (2 mL). The mixture (15 mL) was added Bu4NF (1 M in THF, 789 μL). The mixture
was stirred for 72 h at rt and then poured into EtOAc (50 mL), was stirred for 2 h at rt and then poured into sat. aq. NaHCO3
washed successively with sat. aq. NaHCO3 (2 × 40 mL), water (70 mL), extracted with DCM (3 × 20 mL), dried over MgSO4
(40 mL) and brine (40 mL), dried over MgSO4 and concentrated and concentrated in vacuo to give a brown oil. After column
in vacuo to give an orange oil. After column chromatography chromatography on silica gel (EtOAc–EtOH, 90 : 10, v/v) the
on silica gel (EtOAc–EtOH, 90 : 10, v/v) the pure product (22) pure product (4) was obtained as a yellow foam (380 mg, 91%).
1
was obtained as a light brown foam (527 mg, 86%). Rf 0.28 Rf 0.25 (EtOAc–EtOH, 90 : 10, v/v); H NMR: δ 1.10 (3H, t, J 7.1,
1
(EtOAc–EtOH, 90 : 10, v/v); H NMR: δ 1.08–1.18 (21H, m, 3 × NCH2CH3), 2.32–2.41 (2H, m, H-2′), 2.65–2.89 (6H, m, 3 ×
iPr), 1.26 (3H, t, J 6.9, NCH2CH3), 1.99–2.14 (1H, m, H-2′), 2.47 NCH2), 3.59 (2H, q, J 5.8, ArCONHCH2), 3.80–3.96 (2H, m,
(1H, dd, J 13.8, 5.2, H-2′), 2.70 (2H, q, J 6.9, NCH2CH3), H-5′), 4.10–4.13 (1H, m, H-4′), 4.26 (2H, t, J 5.9, OCOOCH2),
2.77–2.89 (4H, m, 2 × NCH2), 3.59 (2H, q, J 4.8, ArCONHCH2), 5.15–5.20 (1H, m, H-3′), 6.12 (1H, dd, J 8.0, 6.1 Hz, H-1′), 7.82
3.92–3.93 (2H, m, H-5′), 4.07–4.10 (1H, m, H-4′), 4.26 (2H, t, J (1H, d, J 8.9, ArCH ‘m’ to I), 8.08 (1H, dd, J = 8.9, 2.0, ArCH ‘o’
5.9, OCOOCH2), 5.21 (1H, d, J 5.8, H-3′), 6.16 (1H, dd, J 8.7, to I), 8.23 (1H, s, H-6), 8.32 (1H, br s, ArCONH), 8.61 (1H, d, J
5.2 Hz, H-1′), 7.82 (1H, d, J 9.0, ArCH ‘m’ to I), 8.01 (1H, s, H-6), 2.0, ArCH ‘o’ to I), 9.63 (1H, s, ArCH ‘o’ to CONH); 13C NMR: δ
8.08 (1H, dd, J = 9.0, 1.9, ArCH ‘o’ to I), 8.30–8.33 (2H, m, 12.1 (NCH2CH3), 37.7 (ArCONHCH2), 38.1 (C-2′), 48.4
ArCONH, NH), 8.60 (1H, d, J 1.9, ArCH ‘o’ to I), 9.64 (1H, s, (NCH2CH3), 52.1, 52.9 (2
× NCH2), 62.5 (C-5′), 66.8
ArCH ‘o’ to CONH); 13C NMR: δ 12.0 (Si[CH(CH3)2]3), 12.1 (OCOOCH2), 68.8 (C-5), 78.4 (C-3′), 85.3 (C-4′), 86.5 (C-1′), 98.3
(NCH2CH3), 18.2 (Si[CH(CH3)2]3), 37.6 (ArCONHCH2), 38.7 (ArCI), 130.9 (ArCH ‘m’ to I), 138.6 (ArCH ‘β’ to N), 139.6 (ArC
(C-2′), 48.4 (NCH2CH3), 52.0, 52.9 (2 × NCH2), 63.8 (C-5′), 66.8 ‘p’ to I), 139.9 (ArCH ‘o’ to I), 144.1 (ArC ‘m’ to I), 144.4
(OCOOCH2), 69.0 (C-5), 78.6 (C-3′), 85.3 (C-1′), 85.7 (C-4′), 98.2 (ArCCONH), 144.7 (ArCH ‘o’ to CONH), 145.6 (C-6), 150.1 (C-2
(ArCI), 130.9 (ArCH ‘m’ to I), 138.7 (ArCH ‘β’ to N), 139.6 (ArC CvO), 154.6 (OCOO), 160.2 (C-4 CvO), 163.2 (ArCONH); m/z
‘p’ to I), 139.8 (ArCH ‘o’ to I), 143.8 (C-6), 144.1, 144.5 (ArC ‘m’ (ESI) 795.32 [M + H]+; Found C, 38.02; H, 3.75; N, 10.36.
to I, ArCCONH), 144.7 (ArCH ‘o’ to CONH), 149.8 (C-2 CvO), C25H28I2N6O8 requires C, 37.80; H, 3.55; N, 10.58%.
154.7 (OCOO), 159.9 (C-4 CvO), 163.1 (ArCONH).
In vitro stability
3′-O-[({2-[Ethyl-(2-{[(6-iodoquinoxalin-2-yl)carbonyl]amino}-
ethyl)amino]ethyl}amino)carbonyl]-5-iodo-2′-deoxyuridine (3). Stock solutions of the conjugates in MeCN (2 mM) were
To a solution of (21) (420 mg, 0.44 mmol) in anhydrous THF diluted (1 : 4, v/v) in the relevant medium (acidic buffer, PBS,
(12.6 mL) was added Bu4NF (1 M in THF, 530 μL). The mixture MEM, human plasma) and incubated at 37 °C. Aliquots
was stirred for 2 h at rt and then poured into sat. aq. NaHCO3 (200 μL) were removed at various times and quenched by
(50 mL), extracted with DCM (3 × 20 mL), dried over MgSO4 addition of 400 μL of MeCN or ammonium formate buffer
and concentrated in vacuo to give a brown oil. After column (10 mM, pH 3) followed by 25 μL of the internal standard
chromatography on silica gel (EtOAc–EtOH–NH4OH, (4-hydroxybenzoic acid, 0.5 mg mL−1 in ethanol) and 375 μL of
85 : 15 : 0.5, v/v/v) the pure product (3) was obtained as a yellow MeCN. After centrifugation, the supernatant was filtered and
solid (303 mg, 86%). Mp 109–111 °C (dec.); Rf 0.29 (EtOAc– 10 μL aliquots were analyzed by RP-HPLC using a reverse
EtOH–NH4OH, 85 : 15 : 0.5, v/v/v); 1H NMR: δ 0.96 (3H, t, phase Agilent Zorbax Eclipse XDB-C18 column (5 μm, 150 ×
J 6.8, NCH2CH3), 2.17–2.20 (2H, m, H-2′), 2.47–2.66 (6H, m, 4.6 mm). Mobile phase A was 10 mM ammonium formate
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buffer, adjusted to pH 3 with formic acid, and mobile phase B
was MeCN. The flow rate was 0.5 mL min−1, and a gradient
mobile phase composition was used: isocratic hold for 3 min
at 5%, 12 min gradient to 40% B, and 3 min isocratic hold at
40% B. All the experiments were carried out in triplicate. The
natural log of the percent remaining was plotted against time,
the slope of the linear regression was determined, and the
half-lives (T1/2) were calculated.
J. C. Teulade, J. C. Madelmont and N. Moins, J. Med.
Chem., 2008, 51, 3133–3144.
5 J. A. Stein and I. Brewnell, J. Drugs Dermatol., 2008, 62,
296–318.
6 A. P. Algazi, C. S. Soon and A. I. Daud, Cancer Manage. Res.,
2012, 2, 197–211.
7 C. Garbe, T. K. Eigentler, U. Keilhotz, A. Hauschild and
J. M. Kirkwood, Oncologist, 2011, 16, 5–24.
8 M. Rapp, J. C. Maurizis, J. Papon, P. Labarre, T. Wu,
A. Croisy, J. L. Guerquin-Kern, J. C. Madelmont and
E. Mounetou, J. Pharmacol. Exp. Ther., 2008, 326, 171–177.
9 M. Vivier, M. Rapp, J. Papon, P. Labarre, M. J. Galmier,
J. Sauzière and J. C. Madelmont, J. Med. Chem., 2008, 51,
1043–1047.
10 P. Labarre, J. Papon, M. F. Moreau, N. Moins, M. Bayle,
A. Veyre and J. C. Madelmont, Melanoma Res., 2002, 12,
115–121.
11 M. Bonnet, F. Mishellany, J. Papon, A. Cayre, F. Penault-
Llorca, J. C. Madelmont, E. Moit-Noirault, J. M. Chezal and
N. Moins, Pigm. Cell Melanoma Res., 2012, 23, 1–11.
12 J. L. Guerquin-Kern, F. Hillion, J. C. Mademont, P. Labarre,
J. Papon and A. Croisy, Biomed. Eng., 2004, 3, 10–15.
13 K. G. Chen, J. C. Valencia, B. Lai, G. Zhang, J. K. Paterson,
F. Rouzaud, W. Berens, S. M. Wincovitche, S. H. Garfield,
R. D. Leapmean, V. J. Hearing and M. Gottesman, Proc.
Natl. Acad. Sci. U. S. A., 2006, 103, 9903–9907.
14 H. Y. Park and M. Yaar, in Fitzpatrick’s Dermatology in
General Medicine, ed. L. A. Goldsmith, S. I. Katz,
B. A. Gilchrest, A. S. Paller, D. J. Leffel and K. Wolff,
McGraw-Hill, New-York, 8th edn, 2012, pp. 765–781.
15 V. Bhatnagar, S. Anjaiah, N. Puri, B. N. Darshanam and
A. Ramaiah, Arch. Biochem. Biophys., 1993, 307, 183–192.
16 J. Ancans, D. J. Tobin, M. J. Hoogduijn, N. P. Smit,
K. Wakamatsu and A. J. Thody, Exp. Cell Res., 2001, 268,
26–35.
Metabolic stability
Balb-C mouse liver microsomes (MIC255028) were purchased
from Biopredic International. Solvents were HPLC grade.
Microsomal incubations were done in triplicate. Incubation
mixtures containing 1 mg mL−1 of microsomal proteins, 1 mM
NADPH, H+, 50 μL THF-dissolved conjugate in 100 mM potass-
ium phosphate buffer (pH 7.4, 1 mL total volume) were incu-
bated at 37 °C for various times (30 min, 1 h, 2 h, 4 h). All
aliquots were quenched by addition of ethanol (1 mL) at
−20 °C, and centrifuged at 11 000g for 5 min at 4 °C to sedi-
ment the protein precipitate. The supernatant was evaporated
to dryness and reconstituted in 500 μL. The same experiments
were performed as the control with inactive microsomes
heated at 100 °C for 5 min. Samples were analyzed using an
LC-MS/MS system. The HPLC analysis was performed as
described above for the stability studies. The mass spectro-
meter was set for full scanning (m/z 50–1300) and was operated
in positive-ion mode. Parameters were as follows: spray
voltage, 4.5 kV; capillary temperature, 350 °C; nitrogen heath
gas, 80 Psi; nitrogen auxiliary gas, 40 AU; argon collision
pressure, 2.5 mTorr. The mass spectrometry data were
acquired and analyzed using Xcalibur® 1.1 software package
(Thermo Finnigan). Each MS spectrum was recorded by aver-
aging 20 spectra. MS analyses were performed using total ion
current (TIC) chromatograms. Specific compounds (m/z corres-
ponding to compounds and/or metabolites) were detected
using extracted ion current (EIC) chromatograms. Relative con-
centrations are expressed as the ratio of the peak area of the
metabolite to the area of all metabolites at each time of
incubation.
17 D. Denoyer, P. Labarre, J. Papon, E. Miot-Noirault,
M. J. Galmier, J. C. Madelmont, J. M. Chezal and N. Moins,
J. Chromatogr., B: Biomed. Appl., 2008, 875, 411–418.
18 S. Moharram, A. Zhou, L. I. Wiebe and E. E. Knaus, J. Med.
Chem., 2004, 47, 1840–1846.
19 M. André, B. Letribot, M. Bayle, E. Mounetou and
J. M. Chezal, Tetrahedron Lett., 2012, 53, 6107–6110.
References
1 A. Kamb, S. Wee and C. Lengauer, Nat. Rev. Drug Discovery, 20 R. Jain, M. Standley and M. J. Fréchet, Macromolecules,
2007, 5, 65–71. 2007, 40, 452–457.
2 K. Tanabe, Z. Zhang, T. Ito, H. Hatta and S. Nishimoto, 21 Y. J. Kwon, M. Standley, P. G. Andrew, E. R. Gillies and
Org. Biomol. Chem., 2007, 5, 3745–3757. M. J. Fréchet, Mol. Pharmaceutics, 2005, 2, 83–91.
3 Y. H. Lu, X. Q. Gao, M. Wu, D. Zhang-Negrerie and Q. Gao, 22 S. R. Ross, X. Y. Kyl and R. M. Williams, Chem. Rev., 1998,
Mini-Rev. Med. Chem., 2011, 11, 611–624. 98, 2723–2731.
4 J. M. Chezal, J. Papon, P. Labarre, C. Lartigue, 23 F. M. Veronese and G. Pasut, Drug Discovery Today, 2005,
M. J. Galmier, C. Decombat, O. Chavignon, J. Maublant,
10, 1451–1458.
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