Melanoma-targeted delivery system (part 1): Design, synthesis and evaluation of releasable disulfide drug by glutathione

Article abstract of DOI:10.1016/j.ejmech.2015.06.055

Abstract Here we describe the design and synthesis of a prodrug developed for pigmented melanoma therapy, consisting of a Melanin-Targeting Probe (MTP) conjugated to 5-iodo-2′-deoxyuridine (IUdR) with a reduction-sensitive pre-determined breaking point. C

Full text of DOI:10.1016/j.ejmech.2015.06.055

European Journal of Medicinal Chemistry 101 (2015) 668e680
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Melanoma-targeted delivery system (part 1): Design, synthesis and
evaluation of releasable disulfide drug by glutathione
Radhia El Aissi a, b, Jean-Michel Chezal b, S eꢀ bastien Tarrit a b, Olivier Chavignon a b,
Emmanuel Moreau
a
a
,
,
,
a, b, *
INSERM e Universit ꢀe d'Auvergne, UMR 990, IMTV, BP 184, F-63005 Clermont-Ferrand Cedex, France
Clermont Universit ꢀe , Universit ꢀe d'Auvergne, Imagerie Mol ꢀe culaire et Th ꢀe rapie Vectoris ꢀe e, BP 10448, F-63005 Clermont-Ferrand Cedex, France
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 18 February 2015
Received in revised form
Here we describe the design and synthesis of a prodrug developed for pigmented melanoma therapy,
consisting of a Melanin-Targeting Probe (MTP) conjugated to 5-iodo-2 -deoxyuridine (IUdR) with a
0
reduction-sensitive pre-determined breaking point. Compared with the non-cleavable conjugate (17b),
prodrug (17a) bearing a self-immolative disulfide linker achieved complete release of IUdR within 20 min
in the presence of reducing agents such as DTT or glutathione. Analytical results also showed that
prodrug (17a) was more sensitive than parent non-cleavable conjugate (17b) for a concentration range of
glutathione similar to that found in the intracellular compartment of tumours.
21 June 2015
Accepted 23 June 2015
Available online 13 July 2015
Keywords:
Melanoma-targeted therapy
Prodrug
©
2015 Elsevier Masson SAS. All rights reserved.
Glutathione
Disulfide linker
IUdR
1
. Introduction
that prevent cell division. To date, several drugs have been
approved by the Food and Drug Administration for malignant
metastatic melanoma, e.g. dacarbazine (DNA alkylating agent) [5],
interleukin-2 [6], ipilimumab(CTLA-4 blockade) [7], vemurafenib
and dabrafenib (anti-BRAF) [8]. Compared with chemotherapy, the
potent specific BRAF inhibitors vemurafenib and dabrafenib have
significantly improved response rates, along with progression-free
and overall survival, in patients with metastatic melanoma with BR
AFV600E mutations. However, acquired resistance to BRAF in-
hibitors frequently develops through reactivation of the mitogen-
activated protein kinase (MAPK) pathway, resulting in a median
progression-free survival of 6e8 months [9,10]. Although the use of
combination therapy with BRAF and MEK inhibitors has provided
good responses, only 40% of patients who harbour a BRAFV600
mutation benefit from such an approach [11e13]: dabrafenib plus
trametinib, compared with vemurafenib monotherapy, or nivolu-
mab compared with dacarbazine, were associated with significant
improvements in overall survival and progression-free survival,
among previously untreated patients who had metastatic mela-
noma without a BRAF mutation. Analysis of these recent data
suggests that the treatment of malignant metastatic melanoma
should involve simultaneous blockade of more than one cell-
signalling pathway [14,15]. Taking into account the difficulties
encountered so far and the remaining needs, continued efforts are
According to WHO reports for 2012, cancer now causes more
deaths than all coronary heart diseases, and more than all strokes. It
is estimated that 14.1 million new cancer cases and 8.2 million
cancer deaths occurred in 2012 worldwide compared with 12.7
million and 7.6 million, respectively in 2008. Of all cancers com-
bined, malignant melanoma of the skin accounted for 232,000 new
cancers (1.6% of new cancers) and 55,000 deaths worldwide (0.7%
of cancer deaths) for the year 2012 [1]. Early diagnosis followed by
surgical excision of the nevus before metastasis occurs frequently
offers an encouraging prognosis [2,3]. However, for a minority of
patients, whose melanoma is not diagnosed in the initial stage of
the disease, the development and progression of this cancer
throughout the body, in the brain, liver, or bones, are very fast.
Advanced-stage melanoma (stage IV) is often associated with an
overall median survival period of 6e12 months, with only 5% of
patients surviving beyond 5 years [4]. Melanoma shows resistance
to traditional chemotherapies such as cytotoxic anticancer drugs
*
Corresponding author. INSERM e Universit eꢀ d'Auvergne, UMR 990, IMTV, BP
84, F-63005 Clermont-Ferrand Cedex, France.
1
E-mail address: emmanuel.moreau@udamail.fr (E. Moreau).
http://dx.doi.org/10.1016/j.ejmech.2015.06.055
0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

R. El Aissi et al. / European Journal of Medicinal Chemistry 101 (2015) 668e680
669
required (i) to explore the entire new range of treatment possibil-
ities and (ii) to develop new entities that, in combination with
specific melanoma-targeted drugs, enable effective control of this
disease. Among all the different molecular targets investigated,
melanins are relevant, as they are detected in more than 90% of
primary melanoma cases and in 30e50% of metastatic lesions [16].
The capacity of these amorphous, irregular polymers to bind to
many drugs, especially those with coplanar fused aromatic rings
and/or polyamine side chains, make it a promising target for a
pigmented melanoma-targeting strategy [11,17]. Based on these
findings, in the last two decades our research group has developed
arylcarboxamide families that have potential applications for PET
imaging [18e20] and/or targeting radionuclide therapy [21,22] of
melanoma due to their rapid, specific and long-lasting tumour
uptake. We are now exploring the use of such Melanin-Targeting
Probes (MTPs) [23] to carry an anticancer drug into the mela-
noma tumour site [24,25]. The MTP-anticancer drug conjugate was
to be designed to release the drug after it had reached its intra-
cellular target so as to overcome melanosomal sequestration. This
phenomenon was previously observed for cisplatin, which was
trapped in these subcellular organelles, inhibiting efficient alkyl-
ation of DNA [26].
Accordingly, we planned to use this scavenger effect as a “Trojan
horse” to produce a conjugate that would release the known
anticancer drug only after reaching the tumour environment and
allowing a better and specific tumoral accumulation of the anti-
cancer drug. Thus the use of an appropriate linker between the
drug and MTP is crucial for the efficacy of melanoma-targeted
therapy. The linker has to be stable during handling, storage and
under physiological conditions, while easily cleavable in the
tumour intracellular compartment to release the free parent drug
-2-carboxamide ICF01012 (called MTP) [43] conjugated with a self-
immolative disulfide linker to the antimetabolite IUdR, chosen as a
model anticancer agent.
2. Results and discussion
We recently modified the ICF01012 scaffold in order to deliver
anticancer agents to pigmented melanoma. Given its favourable
clearance and high tumour-melanoma uptake (5.60% ID/g) at 3 h
post-injection in melanoma-bearing mice, compound (1d*) repre-
sents a good building block for the design of new melanoma se-
lective drug delivery systems (Fig. 1) [43]. The synthesis of the
disulfide IUdReSSeMTP-conjugate required first modifying both
the IUdR and MTP moieties in order to form the disulfide linkage.
Before synthesising the IUdReSSeMTP conjugate (17a), carrying
a disulfide cleavable linker and its sulfide homolog IUdR-S-MTP
(17b), which served as a non-cleavable reference, we investigated
the linkage and cleavage of IUdR-(di)thiophenyl conjugate (Fig. 1).
To this end, compounds (11a, b) were synthesized, and their sta-
bility in the presence of (2S,3S)-dithiothreitol (DTT), known to
reduce the disulfide bond, were evaluated. For compounds (11a, b),
we planned to develop a synthesis between IUdR and primary al-
cohols (2a, b) obtained from pyridyl disulfide (1) (Fig. 2).
As depicted in Scheme 1A, the asymmetrical disulfide (1) was
easily obtained using a literature procedure by oxidation of the
thiol function of 2-mercaptopyridine with sulfuryl chloride in the
presence of 2-mercaptoethanol [44]. Using a similar method,
compound (2a) was obtained in 71% yield from 4-
methoxybenzenethiol and 2-mercaptoethanol. S-alkylation of 4-
methoxybenzenethiol by 2-chloroethanol gave compound (2b)
(75%) using the protocol of Fujihara and co-workers, slightly
modified [45].
[27]. Besides the possibility of conjugating drugs to the carrier
through acid-cleavable bonds, which has been previously explored
in our laboratory with some success [28], another interesting
strategy could involve the use of (i) reductive systems induced by
hypoxia [29] (quinines [30], nitro(hetero)aryls [31] and N-oxides
Our synthesis then started by the regioselective monoprotection
of IUdR with tert-butyldimethylsilyl chloride to give 5 -protected
0
IUdR (6) according to the procedure of Moharram and co-workers
(79%) (Scheme 2) [46]. The monoprotected IUdR (6) was then
0
[32]) or disulfide-bearing linkers by a thiol exchange reaction [33],
converted in the presence of 4-nitrophenylchloroformate into 3 -
or (ii) self-immolative linkers activated by tumour-associated en-
zymes (such as proteases, glucoridinases, carboxylesterases,
cathepsin, for example) [34,35]. The concept of specific release of
cytotoxic agents in melanocytes from tyrosinase-activated pro-
drugs, known as Melanocyte-Directed Enzyme Prodrug Therapy
activated carbonate ester (7) with good yield (72%) after silica gel
purification. Compound (8) was obtained by treatment of IUdR
derivative (7) with primary alcohol (1). We note that compound (8)
can be alternatively synthesized from (6) with excellent yield (90%)
using a one-pot procedure avoiding the carbonate isolation step (7).
The condensation of 4-methoxybenzenethiol with derivative (8)
gave disulfide compound (10a) in moderate yield (58%). More
conveniently, disulfide (10a) can be obtained using a one-pot
method from (6) and (2a) in 74% yield. Final deprotection of the
(
MDEPT), is also attractive [36]. However, for this last strategy, and
despite significant efforts, the release of the active substance was
unlikely when carbamate, urea or hydrazine linkers were used
[37e39]. Among all the above-mentioned linker units, we opted to
use the promising disulfide bond function, which is stable in the
blood circulation, but efficiently cleaved by intracellular reducing
systems present in the tumour cells [33]. Thiols are scarce in the
blood circulation, while glutathione (GSH) levels are 1000 times
higher in tumour cells than in the blood plasma [39]. This linker is
increasingly used to form small molecule drug conjugates. Recently,
Santra et al. described a tumour-targeted prodrug (folate-doxoru-
bicin conjugate) that became activated within the cell by GSH-
mediated dissociation [40]. Janssen et al. reported a disulfide pro-
drug conjugate SN38, an active metabolite of irinotecan, linked to
the nontoxic B-subunit of the Shiga toxin, a natural ligand of
pancreatic cells. The cytotoxic effect of the SN38 conjugate was
increased more than 100-fold compared with irinotecan [41]. We
can also cite the promising folate-targeted prodrug EC145 bearing a
disulfide linker, which is currently being tested in a global phase 3
study in women with platinum-resistant ovarian cancer [42].
Here, we describe the design, synthesis and preliminary evalu-
ation of a melanoma-targeted prodrug based on a melanin-
targeting derivative of N-(2-diethylaminoethyl)-6-iodoquinoxaline
3
silyl group in the presence of InCl in refluxing acetonitrile/water
afforded prodrug (11a) in 83% yield (34% overall yield from IUdR
and (1)). Prodrug (11a) can also be obtained from (8) in 55% overall
yield by soft deprotection of the silyl group, yielding compound (9),
followed by treatment with 4-methoxybenzenethiol. Obviously, in
our case, use of the disulfide precursor (1) to generate the disulfide
(11a) was not advantageous. The best procedure was the straight-
forward formation of the disulfide bond with (2a), followed by
reaction with activated IUdR (7) prepared in situ (49% overall yield
from IUdR and (1)).
A similar three-step procedure was successfully used for the
synthesis of analogue (11b) from (6) and (2b) after a final soft
deprotection of compound (10b). Finally, conjugates (11a, b) were
synthesized from IUdR and (2a, b) in four steps, with overall yields
of 49 and 39%, respectively.
2.1. Evaluation of IUdR release from (11a, b) conjugates
Compounds (11a, b) in 0.9 mM PBS (pH ¼ 7.4)/acetonitrile (98/2,

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R. El Aissi et al. / European Journal of Medicinal Chemistry 101 (2015) 668e680
Fig. 1. Rational for the design of releasable disulfide prodrug IUdReSSeMTP (17a) and its non-cleavable derivative IUdR-S-MTP (17b).
ꢀ
v/v) solution were treated with one equivalent of DTT at 37 C, and
the advancement of the reaction was monitored by analytical RP-
HPLC (see details in Experimental Section).
has no disulfide bond, no degradation or cleavage was observed
over 60 min (Fig. 3B, retention time: 14.65 min). These results also
highlight the excellent stability of the carbonate function in PBS/
acetonitrile solution with or without DTT. We therefore assume
that the IUdR release observed for (11b) was mainly due to the
reduction of the disulfide bond in the presence of DTT, followed by
self-immolative elimination of the thioethyl carbonate residue.
As depicted in Fig. 3A, free IUdR (retention time: 2.98 min) was
released after 30 min of treatment, but the reaction was incom-
plete. Over the next 30 min, compound (11a) was completely
converted into free IUdR. By contrast, compound (11a) was found to
remain stable in the PBS/acetonitrile mixture for more than 24 h in
the absence of DTT (Fig. 3A, retention time: 17.98 min).
When the same method was applied to compound (11b), which
Scheme 1. Synthesis of alcohols (1, 2a, b) and their protected derivatives (4a, b).
a
Reagents and conditions: i) DCM, sulfuryl chloride, rt; ii) 2-mercaptoethanol; DCM, rt;
Fig. 2. Rational for the synthesis of releasable disulfide prodrug IUdReSSeMTP (11a,
Z ¼ SS) and its non-cleavable derivative (11b).
iii) NCS; DCM, rt; iv) 2-chloroethanol, Na
DMF, rt; vi) 4-thiophenol, DCM, rt; vii) 2-bromoethanol, Na
2
CO
3
, DMF, rt; v) TBDMSCl, imidazole, DMAP,
CO , DMF, rt.
2
3

R. El Aissi et al. / European Journal of Medicinal Chemistry 101 (2015) 668e680
671
Scheme 2. Synthesis of IUdReSSeMTP (11a) and IUdR-S-MTP (11b). aReagents and conditions: i) TBDMSCl, imidazole, DMAP, DMF, rt; ii) 4-nitrophenylchloroformate, DMAP, THF,
rt; iii) (1), DMAP, THF, rt; iv) a) 4-nitrophenylchloroformate, DMAP, THF, rt; b) (1), DMAP, THF, rt; v) 4-methoxybenzenethiol, THF; rt; vi) a) 4-nitrophenylchloroformate, DMAP, THF,
rt; b) (2a), DMAP, THF; rt; vii) InCl , CH CN/H O (1/1, v/v), reflux; viii) a) 4-nitrophenylchloroformate, DMAP, THF, rt; b) (2b), DMAP, THF, rt.
3 3 2
We then determined the effective half-life time of prodrug (11a)
in the presence of DTT. We studied the kinetics of IUdR release from
prodrug (11a) using compound (11b) as an internal standard. At
different times (1, 2, 6, 10, 15, 20, 25, 30 and 60 min), aliquots were
withdrawn and analyzed by RP-HPLC and the amount of released
IUdR was determined from a standard curve (see Experimental
Section for details). Using DTT, the half-life of compound (11a) was
conditions, yielding compound (5) (78%), followed by a regiose-
lective TBDMS protection of the aliphatic alcohol to give phenol
derivative (4b) (88%) (Scheme 1B).
Scheme 3 depicts the synthesis in five steps of IUdR-MTP con-
jugates (17a, b). The key intermediate (13) was obtained by N-
acylation of compound (12) [41] in the presence of 2-bromoacetyl
chloride in DMF. Compound (13) was then converted into benzyl
ethers (14a, b) by condensation of the corresponding phenol de-
rivatives (4a, b). After deprotection of the silyl groups under mild
conditions, alcohols (15a, b) were treated with IUdR carbonate (7)
to afford (16a, b) with 78 and 76% yields, respectively. Finally,
2
(
.3 min and the IUdR was completely released within 20 min
Fig. 3C).
Taken together, these preliminary results are evidence that di-
sulfide IUdR prodrugs may enable drug release in the reductive
conditions found in tumour cells, and encouraged us to develop
melanoma-targeted drug (17a, b) (Fig. 1).
3
TBDMS deprotection in the presence of InCl gave IUdR-MTP con-
jugates (17a, b) in 68 and 43% yields, respectively.
2.3. Evaluation of IUdR release from conjugates (17a, b)
2.2. Synthesis of conjugates (17a, b)
Synthesis of compounds (17a, b) was based on the same pro-
Using the internal standard method, compounds (17a, b) were
cedure used for derivatives (11a, b), i.e a condensation of IUdR
carbonate (7) with the alcohols (15a, b), obtained from 6-
aminoquinoxaline (12) and (di)sulfides (4a, b) (Fig. 4) [47].
Asymmetrical disulfide (4a) was obtained by silylation of (1)
followed by a thiol-disulfide exchange using 4-thiophenol (overall
yield 88%) (Scheme 1B). Compound (4b) was prepared by S-alkyl-
ation of 4-mercaptophenol with 2-bromoethanol, under basic
treated by DTT to evaluate both their half-life and their ability to
release IUdR under reductive conditions at 37 C. After DTT treat-
ꢀ
ment, fast degradation of compound (17a) associated with a strong
release of IUdR were observed (Fig. 5A), while compound (17b)
remained stable over 24 h (Fig. 5B). Also, compounds (17a, b) were
found to remain stable for at least 24 h in the PBS/acetonitrile so-
lution. In the presence of DDT, the half-life of compound (17a) was

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R. El Aissi et al. / European Journal of Medicinal Chemistry 101 (2015) 668e680
Fig. 4. Rational for the synthesis of releasable disulfide prodrug IUDReSSeMTP (17a)
and its non-cleavable derivative IUdR-S-MTP (17b).
3.1 min and the IUdR was completely released after 12 min (Fig. 5C).
From these results, we decided to evaluate the release of IUdR
ꢀ
from compound (17a) in the presence of glutathione (GSH) at 37 C.
The cytoplasmic concentration of this thiol-containing tripeptide is
in the millimolar range (1e10 mM), whereas it is present in the
micromolar range in the blood (1e10 mM) [48]. In order to assess
the influence of intracellular and extracellular glutathione con-
centrations on the parent drug release, prodrug (17a) was incu-
bated with two different concentrations of GSH (5 mM and 5 mM,
Fig. 3. Evaluation of IUdR release after treatment of disulfide conjugates
IUdReSSeMTP (11a) (A) and IUdR-S-MTP (11b) (B) by DTT. A) orange: IUdR (retention
time 2.98 min); purple: (11a) in PBS/acetonitrile (98/2, v/v) (retention time 17.98 min);
blue: (11a) treated with one equivalent of DTT during 30 min; pink: (11a) treated with
one equivalent of DTT during 60 min; B) orange: IUdR (retention time 2.98 min);
purple: (11b) in PBS/acetonitrile (98/2, v/v) (retention time 14.65 min); blue: (11b)
treated with one equivalent of DTT during 30 min; pink: (11b) treated with one
equivalent of DTT during 60 min; C) Kinetic rate of cleavage for compound (11a) with
DTT (1 eq.) and determination of its half-life; red: released IUdR; purple: (11a). (For
interpretation of the references to colour in this figure legend, the reader is referred to
the web version of this article.)
Scheme 3. Synthesis of IUdReSSeMTP (11a) and IUdR-S-MTP (11b). aReagents and
conditions: i) 2-bromoacetyl chloride, DMF, rt; ii) (4a) or (4b), NaH, THF, rt; iii) InCl
CH CN/H O (1/1, v/v), reflux; iv) (7), DMAP, THF, rt.
3
,
3
2

R. El Aissi et al. / European Journal of Medicinal Chemistry 101 (2015) 668e680
673
Fig. 6. Evaluation of IUdR release after treatment of disulfide prodrug IUdReSSeMTP
17a) by GSH. A) orange: (17a) treated with GSH (5 mM) during 60 min; blue: (17a)
treated with GSH (5 M) during 60 min; B) Kinetic of cleavage of (17a) with GSH
5 mM) and determination of its half-life; red: released IUdR; purple: (17a). (For
(
m
(
interpretation of the references to colour in this figure legend, the reader is referred to
the web version of this article.)
compound (17a) was treated with a 5 mM GSH solution. On the basis
of these data and similar protocol described by Santra et al., these
results further indicate the stability of the prodrug in the blood-
stream and its activation only in the presence of intracellular GSH
[
40].
We went on to study the kinetics of IUdR release from prodrug
17a) treated with a GSH intracellular concentration of 5 mM using
(
compound (11b) as an internal standard. At different times (1, 2, 6,
0, 15, 20, 25, 30 and 60 min), aliquots were withdrawn and
1
analyzed by RP-HPLC, and the amount of IUdR released was
determined from the standard curve. The half-life of compound
(17a) was 2.1 min, while the IUdR release reached a plateau within
20 min (Fig. 6B). We assume that after cleavage of disulfide linker
Fig. 5. Evaluation of IUdR release after treatment of disulfide prodrug IUdReSSeMTP
17a) (A) and IUdR-S-MTP (17b) (B) by DTT. A) orange: IUdR (retention time 3.02 min);
by GSH, the resultant thiol would then undergo cyclization leading
to the release: i) of alkylcarbamate IUdR derivative, then free IUdR
(
purple: (17a) in PBS/acetonitrile (retention time 11.63 min); blue: (17a) treated with
one equivalent of DTT during 30 min; pink: (17a) treated with one equivalent of DTT
during 60 min; B) orange: IUdR (retention time 3.02 min); purple: (17b) in PBS/
acetonitrile (retention time 10.68 min); blue: (17b) treated with one equivalent of DTT
during 30 min; pink: (17b) treated with one equivalent of DTT during 60 min; C) Ki-
netic of cleavage of (17a) with DTT (1 eq.) and determination of its half-life; red:
released IUdR; purple: (17a). (For interpretation of the references to colour in this
figure legend, the reader is referred to the web version of this article.)
(Fig. S1, Path A) or ii) free IUdR by attack of the resulting thiol on
carbonyl function of carbamate group as described in the literature
(Fig. S1, Path B) [49].
3. Conclusion
Our approach to improving the efficiency of melanoma-targeted
therapies was based on the use of a cytotoxic drug connected to an
MTP via a self-immolative linker. In this study, we successively
synthesized four IUdR conjugates (11a, b) and (17a, b). We
demonstrate that only compounds (11a) and (17a), bearing a self-
immolative disulfide linker, were able to release IUdR efficiently
under disulfide reductive conditions. Also, GSH treatment of (17a)
respectively). This study was monitored by analytical RP-HPLC to
follow the IUdR release. Compound (11b) served as an internal
standard. As shown in Fig. 6A, the release of the parent drug from
prodrug (17a) was only observed for the GSH concentration of
5
mM (Fig. 6A). Conversely, no degradation was detected when
suggests
a potential activation in the intracellular tumour

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R. El Aissi et al. / European Journal of Medicinal Chemistry 101 (2015) 668e680
compartment. We consider that the resulting anticancer agent-
MTP prodrug holds the promise of tumour-specific delivery of the
cytotoxic drug (targeted therapy), thus avoiding the dose-limiting
toxicity of standard chemotherapies. Our next objective will be to
study our activatable IUdReSSeMTP prodrug in melanoma-bearing
mice.
methoxythiophenol (1.0 g, 7.13 mmol, 1 eq.) dissolved in anhy-
drous dichloromethane (4 mL) was added. After stirring at room
temperature for 45 min, a solution of 2-mercaptoethanol (602 mL,
8.55 mmol, 1.2 eq.) dissolved in anhydrous dichloromethane (3 mL)
was added dropwise. The reaction mixture was stirred at room
temperature for 48 h, then washed with brine (2 ꢂ 25 mL), dried
over magnesium sulfate, filtered and evaporated under reduced
pressure. The crude product was purified on silica gel eluted with a
gradient ethyl acetate/cyclohexane (20/80 to 30/70, v/v) to afford
4
. Experimental section
ꢁ
1
4.1. Materials for chemical syntheses
compound (2a) (1.1 g, 5.09 mmol). Yield: 71%; IR (KBr)
n
(cm
)
1
3
373, 2937, 2835, 1590, 1571, 1491, 1461, 1288, 1247, 1172; H NMR
3
Unless otherwise mentioned, all manipulations were performed
(200 MHz, CDCl
3
)
d
ppm 7.45 (d, J ¼ 8.9 Hz, 2H, H-4), 6.83 (d,
3
3
under argon; all reagents were purchased from the following
commercial suppliers: SigmaeAldrich, Acros Organics, Carlo Erba,
TCI Europa. Anhydrous DMF and anhydrous triethylamine were
purchased from Acros Organics. THF was distilled over benzophe-
none and sodium. Dichloromethane was distilled over calcium
hydride. Nuclear magnetic resonance (NMR) spectra were acquired
J ¼ 8.9 Hz, 2H, H-5), 3.81 (t, J ¼ 6.0 Hz, 2H, H-1), 3.75 (s, 3H, H-7),
3
1
3
2.84 (t, J ¼ 6.0 Hz, 2H, H-2), 2.69 (brs, 1H, OH); C NMR (50 MHz,
CDCl
3
)
d
ppm 159.9 (C-6), 132.1 (C-4), 127.9 (C-3), 114.9 (C-5), 60.2 (C-
þ þ
1), 55.6 (C-7), 40.9 (C-2); HRMS m/z for [M þ H ] : calc: 217.0357;
found: 217.0351.
1
on a Bruker AC-200 operating at 200 and 50 MHz for H NMR and
4.2.3. 2-((4-Methoxyphenyl)thio)ethanol (2b)
13
1
C NMR, respectively. All H NMR spectra are reported in
d
units,
To a solution of 4-methoxythiophenol (1.0 g, 7.13 mmol, 1 eq.)
ꢀ
parts per million (ppm) and the coupling constants are indicated in
hertz (Hz). The following abbreviations are used for spin multi-
plicity: s singlet, d doublet, t triplet, q quadruplet, m multiplet, and
br broad. Electrospray ionization mass spectra (ESI-MS) were
recorded on an Esquire (Bruker Daltonics, Wissenbourg, France)
spectrometer. High resolution mass spectra (HRMS) were recorded
on an Alliance 2695 (Waters) liquid chromatography coupled with
a Q-ToF micro (Waters/Micromass) spectrometer. Tyrosyl tyrosine,
an internal mass standard, is used to correct mass values. TLC was
performed on aluminum backed silica gel sheets (60F254 plates)
and visualized under UV light (254 nm). Column chromatography
was performed using silica gel normal phase (Merck or SDS)
dissolved in anhydrous DMF (5 mL) cooled to 0 C, sodium car-
bonate (3.1 g, 22.38 mmol, 3.1 eq.) and 2-chloroethanol (472 mL,
7.05 mmol, 1.05 eq.) were added, successively. The resulting
ꢀ
mixture was stirred at 0 C for 15 min, then at room temperature for
1 h 15 min. Water (25 mL) was added and the reaction mixture was
extracted with ethyl acetate (2 ꢂ 30 mL). The combined organic
layers were washed with brine (2 ꢂ 25 mL), dried over magnesium
sulfate, filtered and evaporated under reduced pressure. The crude
product was purified on silica gel eluted with a gradient ethyl ac-
etate/cyclohexane (25/75 to 30/70, v/v) to afford compound (2b)
ꢁ
1
(980 mg, 5.32 mmol). Yield: 75%; IR (KBr)
n
(cm ) 3296, 2953,
1
2833, 1632, 1596, 1495, 1285, 1247, 1178; H NMR (200 MHz, CDCl
3
)
3
3
(
35e70
mm). Uncorrected melting points (Mp) were recorded on a
d
ppm 7.37 (d, J ¼ 8.8 Hz, 2H, H-4), 6.83 (d, J ¼ 8.8 Hz, 2H, H-5), 3.79
3
3
Wagner & Munz Heizbank-Koffler apparatus or an Electrothermal
IA9300. Infrared spectra (IR) were recorded on a Bruker FT Vector
(s, 3H, H-7), 3.66 (t, J ¼ 5.9 Hz, 2H, H-1), 2.98 (t, J ¼ 5.9 Hz, 2H, H-
13
3
2), 2.03 (brs, 1H, OH); C NMR (50 MHz, CDCl ) dppm 159.4 (C-6),
2
4
4
2.
134.1 (C-4),124.6 (C-3),114.7 (C-5), 60.0 (C-1), 55.3 (C-7), 39.3 (C-2).
HRMS m/z for [M þ H ] : calc:185.0636; found: 185.0639.
þ þ
.2. Access to compounds (1), (2a, b) (3), (4a, b) and (5)
4.2.4. 2-((2-((tert-Butyldimethylsilyl)oxy)ethyl)disulfanyl)pyridine
.2.1. 2-(Pyridin-2-yldisulfanyl)ethanol (1)
(3)
To a solution of 2-mercaptopyridine (2.0 g, 17.22 mmol, 1 eq.) in
To a solution of (1) (3.2 g, 17.24 mmol, 1 eq.) dissolved in
ꢀ
ꢀ
anhydrous dichloromethane (50 mL), cooled at ꢁ5 C, sulfuryl
chloride (1.8 mL, 22.23 mmol, 1.3 eq.) was added dropwise. The
reaction mixture was stirred at room temperature for 2 h, then
concentrated under reduced pressure. To this crude product taken
anhydrous DMF (30 mL), cooled at ꢁ5 C, imidazole (56.89 mmol,
3.3 eq.) and DMAP (2.24 mmol, 0.13 eq.) were added, followed by a
solution of tert-butyldimethylsilane chloride (1.1 eq.) dissolved in
ꢀ
anhydrous DMF (15 mL). The reaction mixture was stirred at ꢁ5 C
ꢀ
up in anhydrous dichloromethane (40 mL) and cooled at ꢁ5 C, was
for 30 min and then at room temperature for 2 h 30 min. An
aqueous saturated ammonium chloride solution (60 mL) was added
and the reaction mixture was extracted with ethyl acetate
(3 ꢂ 30 mL). The combined organic layers were washed with water
(40 mL), brine (2 ꢂ 30 mL), dried over sulfate magnesium, filtered
and evaporated under reduced pressure. The crude product was
purified on silica gel eluted by ethyl acetate/cyclohexane (10/90, v/
added a solution of 2-mercaptoethanol (1.51 mL, 22.23 mmol,
1
.3 eq.) dissolved in anhydrous dichloromethane (2 mL). The
resulting mixture was stirred at room temperature for 6 h. After
ꢀ
cooling to ꢁ5 C, the obtained precipitate was filtered, taken up in
dichloromethane (20 mL) and DMAP (1.89 g, 15.47 mmol, 0.9 eq.)
was added. After stirring at room temperature for 10 min, the re-
action solvent was concentrated under reduced pressure. The crude
product was purified on silica gel eluted with dichloromethane/
methanol (95/5, v/v) to afford (1) (2.17 g,11.58 mmol). Yield: 67%; IR
v) to afford compound (3) (4.5 g, 14.92 mmol). Yield: 87%; IR (KBr)
n
ꢁ
1
1
(cm ) 2954, 2856, 1574, 1446, 1417, 1255, 1112; H NMR (200 MHz,
CDCl ppm 8.46 (m, 1H, H-6), 7.78 (m, 1H, H-3), 7.65 (m, 1H, H-4),
7.09 (m, 1H, H-5), 3.87 (t, J ¼ 6.5 Hz, 2H, H-8), 2.93 (t, J ¼ 6.5 Hz,
3
) d
ꢁ
1
3
3
(
(
1
2
d
KBr)
200 MHz, CDCl
n
(cm ) 3344, 2866, 1576, 1561, 1448, 1417, 1117; 1H NMR
13
3
)
d
ppm 8.26 (m, 1H, H-5), 7.42 (m, 1H, H-7), 7.32 (m,
2H, H-7), 0.90 (s, 9H, H-11), 0.06 (s, 6H, H-9); C NMR (50 MHz,
CDCl ppm 160.5 (C-2), 149.5 (C-6), 137.0 (C-4), 120.5 (C-5), 119.6
3
H, H-8), 6.95 (m, 1H, H-6), 5.46 (brs, 1H, OH), 3.64 (t, J ¼ 5.3 Hz,
3
) d
3
13
H, H-1), 2.77 (t, J ¼ 5.3 Hz, 2H, H-2); C NMR (50 MHz, CDCl
3
)
(C-3), 61.3 (C-8), 41.5 (C-7), 25.9 (C-11),17.9 (C-10), -5.3 (C-9). HRMS
þ
15Si þ H]þ calc: 188.0204; found: 188.0204.
ppm 159.2 (C-3), 149.5 (C-5), 137.0 (C-7), 121.3 (C-6, C-8), 58.6 (C-1),
m/z for [MH -C
6
H
þ þ
4
2.3 (C-2); ESI-MS: m/z 187.94 [M þ H ] .
4
.2.5. 4-((2-((tert-Butyldimethylsilyl)oxy)ethyl)disulfanyl)phenol
4.2.2. 2-((4-Methoxyphenyl)disulfanyl)ethanol (2a)
(4a)
To a suspension of N-chlorosuccinimide (952.5 mg, 7.13 mmol, 1
To a solution of (3) (3.0 g, 9.94 mmol, 1 eq.) dissolved in
eq.) in anhydrous dichloromethane (40 mL), a solution of 4-
dichloromethane (60 mL), a solution of 4-mercaptophenol (1.5 g,

R. El Aissi et al. / European Journal of Medicinal Chemistry 101 (2015) 668e680
675
0
0
1
1.92 mmol, 1.2 eq.) dissolved in dichloromethane (5 mL) was
added. The resulting mixture was stirred at room temperature for
6 h. The reaction solvent was evaporated under reduced pressure
4.3.2. 3 -O-((4-Nitrophenyloxy)carbonyl)-5-iodo-5 -O-tert-
0
butyldimethylsilyl-2 -deoxyuridine (7)
1
To a solution of (6) (1.1 g, 2.42 mmol, 1.05 eq.) and 4-nitrophenyl
chloroformate (465.1 mg, 2.31 mmol, 1 eq.) dissolved in anhydrous
THF (15 mL), a solution of DMAP (422.8 mg, 3.46 mmol, 1.5 eq.)
dissolved in anhydrous THF (5 mL) was added dropwise. The re-
action mixture was stirred at room temperature for 3 h 30 min. The
reaction mixture was quenched by an aqueous saturated ammo-
nium chloride solution (50 mL) and extracted with ethyl acetate
(3 ꢂ 30 mL). The combined organic layers were washed with water
(40 mL) and brine (40 mL), dried over sulfate magnesium, filtered
and evaporated under reduced pressure. The crude product was
purified on silica gel eluted with a gradient of ethyl acetate/cyclo-
hexane (20/80 to 40/60, v/v) to afford product (7) (1.05 g,
and the crude product was purified on silica gel eluted with ethyl
acetate/cyclohexane (10/90, v/v) to afford compound (4a) (3.1 g,
ꢁ
1
9
.79 mmol). Yield: 98%; IR (KBr)
n
(cm ) 3328, 2929, 2857, 1598,
583, 1493, 1471, 1257, 1089; H NMR (200 MHz, CDCl ppm 7.35 (d,
J ¼ 8.6 Hz, 2H, H-3), 6.71 (d, J ¼ 8.6 Hz, 2H, H-2), 5.99 (brs, 1H,
1
1
3
) d
3
3
3
3
OH), 3.83 (t, J ¼ 7.0 Hz, 2H, H-6), 2.81 (t, J ¼ 7.0 Hz, 2H, H-5), 0.85
13
(
(
(
3
s, 9H, H-9), 0.03 (s, 6H, H-7); C NMR (50 MHz, CDCl ) dppm 155.9
C-1), 132.1 (C-3), 128.3 (C-4), 116.2 (C-2), 61.9 (C-6), 40.8 (C-5), 30.0
þ þ
C-9), 18.4 (C-8), -5.2 (C-7); HRMS m/z for [M þ Na ] : calc:
3
39.0885; found: 339.0887.
ꢀ
ꢁ1
1
1
.66 mmol). Yield: 72%; Mp: 95 ± 1 C; IR (KBr) n (cm ) 2929, 2856,
765 (CO), 1689 (CO), 1524, 1251, 1191, 1082; H NMR (200 MHz,
4.2.6. 4-((2-Hydroxyethyl)thio)phenol (5)
1
To a solution of 4-mercaptophenol (1.6 g, 12.52 mmol, 1 eq.)
3
CDCl
3
)
d
ppm 8.29 (d, J ¼ 9.2 Hz, 2H, H-13), 8.12 (s, 1H, H-6), 7.40 (d,
ꢀ
dissolved in anhydrous DMF(10 mL), cooled to ꢁ5 C, potassium
3
3
3
0
J ¼ 9.2 Hz, 2H, H-12), 6.37 (dd, J ¼ 9.1 Hz, J ¼ 5.2 Hz, 1H, H-1 ),
carbonate (1.9 g, 13.74 mmol, 1.1 eq.) and 2-bromoethanol (932
mL,
0
0
0
5
.29 (m, 1H, H-3 ), 4.36 (m, 1H, H-4 ), 3.96 (m, 2H, H-5 ), 2.70 (dd,
13.14 mmol, 1.05 eq.) were added, successively. The resulting
2
3
0
0
J ¼ 14.1 Hz, J 5.2 Hz, 1H, H-2 ), 2.20 (m, 1H, H-2 ), 0.94 (s, 9H, H-9),
ꢀ
mixture was stirred at ꢁ5 C for 30 min, then at room temperature
0
0
13
0
.18 (s, 3H, H-7 or H-7 ), 0.16 (s, 3H, H-7 or H-7 ). C NMR (50 MHz,
for 1 h 30 min. Ethyl acetate (25 mL) was added and the organic
layer was washed with water (2 ꢂ 20 mL), dried over magnesium
sulfate, filtered and evaporated under reduced pressure. The crude
product was purified on silica gel eluted with a gradient cyclo-
hexane/ethyl acetate (100/0 to 60/40, v/v) to afford compound (5)
CDCl ppm 160.6 (C-4), 155.8 (C-10), 152.8 (C-2), 150.8 (C-11), 146.3
3
) d
0 0
C-14), 144.5 (C-6), 126.1 (C-13), 122.4 (C-12), 86.1 (C-1 , C-4 ), 81.2
(
(
0
0
0
C-3 ), 69.8 (C-5), 64.3 (C-5 ), 39.4 (C-2 ), 26.9 (C-9), 19.1 (C-8), ꢁ4.4
0
þ ꢁ
and ꢁ4.6 (C-7, C-7 ). ESI-MS: m/z 631.97 [M-H ] .
ꢁ1
(
2
1.66 g, 9.75 mmol). Yield: 78%; IR (KBr)
n
(cm ) 3328, 2931, 2877,
487, 1598, 1584, 1494, 1262; H NMR (200 MHz, MeOD) ppm 7.30
d, J ¼ 8.7 Hz, 2H, H-3), 6.76 (d, J ¼ 8.7 Hz, 2H, H-2), 3.61 (t,
4
.3.3. 2-(((tert-Butyldimethylsilyl)oxy)methyl)-5-(5-iodo-2,4-
1
d
dioxo-3,4-dihydropyrimidin-1(2H)-yl)tetrahydrofuran-3-yl (2-
pyridin-2-yldisulfanyl)ethyl) carbonate (8)
.3.3.1. Method A. To a solution of (7) (540 mg, 0.85 mmol, 1 eq.)
dissolved in anhydrous THF (10 mL), DMAP (270.1 mg, 2.22 mmol,
.6 eq.) and a solution of (1) (286.4 mg,1.28 mmol,1.5 eq.) dissolved
3
3
(
(
4
3
3
13
J ¼ 6.9 Hz, 2H, H-6), 2.89 (t, J ¼ 6.9 Hz, 2H, H-5); C NMR
(
50 MHz, MeOD) ppm 158.3 (C-1), 135.1 (C-3), 125.4 (C-4), 117.0 (C-
d
2
), 61.7 (C-6), 39.2 (C-5).
2
in anhydrous THF (10 mL) were added, successively. After stirring at
room temperature for 16 h, the reaction solvent was evaporated
under reduced pressure and the crude product was dissolved in
ethyl acetate (15 mL). The organic layer was washed with water
4
.2.7. 4-((2-((tert-Butyldimethylsilyl)oxy)ethyl)thio)phenol (4b)
Compound (4b) was synthesized from (5) (684.3 mg,
.02 mmol) according to the previously method described for the
4
(
2 ꢂ 30 mL) and brine (2 ꢂ 20 mL), dried over magnesium sulfate,
synthesis of compound (3). The crude product was purified on silica
gel eluted with ethyl acetate/cyclohexane (15/85, v/v) to afford
compound (4b) (1.0 g, 3.52 mmol). Yield: 88%; IR (KBr)
3
filtered and evaporated under reduced pressure. The crude product
was purified on silica gel eluted by ethyl acetate/cyclohexane (40/
60, v/v) to afford compound (8) (390 mg, 0.57 mmol). Yield: 67%.
ꢁ1
n
(cm
)
1
329, 2955, 2857, 1600, 1583, 1495, 1471, 1258; H NMR (200 MHz,
3
3
CDCl
2
3
)
d
ppm 7.27 (d, J ¼ 8.7 Hz, 2H, H-3), 6.74 (d, J ¼ 8.7 Hz, 2H, H-
3
4.3.3.2. Method B. To a solution of (6) (1.0 g, 2.13 mmol, 1 eq.) and
4-nitrophenyl chloroformate (430.3 mg, 2.13 mmol, 1 eq.) dissolved
in anhydrous THF (25 mL), a solution of DMAP (416.4 mg,
), 6.67 (brs, 1H, OH), 3.78 (t, J ¼ 7.6 Hz, 2H, H-6), 2.96 (t,
3
13
J ¼ 7.6 Hz, 2H, H-5), 0.90 (s, 9H, H-9), 0.07 (s, 6H, H-7); C NMR
50 MHz, CDCl ppm 155.2 (C-1), 133.5 (C-3), 125.8 (C-4), 116.1 (C-
), 62.6 (C-6), 37.7 (C-5), 25.9 (C-9), 18.4 (C-8), -5.2 (C-7). HRMS m/z
(
2
3
) d
3
.41 mmol, 1.6 eq.) dissolved in anhydrous THF (6 mL) was added
dropwise. After stirring at room temperature for 3 h 30 min, DMAP
169.1 mg, 1.38 mmol, 0.65 eq.) then a solution of (1) (478.7 mg,
.56 mmol, 1.2 eq.) dissolved in anhydrous THF (5 mL) were added.
þ þ
[M þ Na ] : calc: 307.1164; found: 307.1163.
(
2
4
4
.3. Access to compounds (6), (7), (8), (9), (10a, b) and (11a, b)
After stirring at room temperature for 16 h, the solvent reaction was
evaporated under reduced pressure. The crude product was taken
up in dichloromethane (40 mL) then washed with water (30 mL)
and brine (2 ꢂ 30 mL). The organic layer was dried over magnesium
sulfate, filtered and evaporated under reduced pressure. The crude
product was purified on silica gel eluted with a gradient ethyl ac-
etate/cyclohexane (30/70 to 60/40, v/v) to afford compound (8)
0
0
.3.1. 5-Iodo-5 -O-tert-butyldimethylsilyl-2 -deoxyuridine (6)
0
Compound (6) was synthesized from 5-iodo-2 -deoxyuridine
(
4.5 g, 12.70 mmol) according to the previous described protocol to
synthesize compound (3). The crude product was purified on silica
gel eluted with a gradient of dichloromethane/ethyl acetate (60/40
to 50/50, v/v) to afford product (6) (4.68 g, 9.99 mmol). Yield: 79%;
ꢁ1
(1.31 g, 1.92 mmol). Yield: 90%. IR (KBr)
(CO), 1689 (CO), 1447, 1418, 1259, 1197, 1124; H NMR (200 MHz,
CDCl ppm 9.89 (brs, 1H, NH), 8.45 (m, 1H, H-15), 8.06 (s, 1H, H-6),
n (cm ) 2953, 2856, 1746
ꢀ
ꢁ1
1
1
Mp: 223 ± 1 C; IR (KBr)
n
(cm ) 3568, 3173, 2953, 2852,1733 (CO),
1676 (CO), 1608, 1261, 1119; H NMR (200 MHz, acetone-d
6
)
d
ppm
3
) d
3
3
3
1
1
2
3
0.37 (brs,1H, NH), 8.13 (s,1H, H-6), 6.23 (dd, J ¼ 9.1 Hz, J ¼ 5.2 Hz,
7.63 (m, 2H, H-17, H-18), 7.08 (m, 1H, H-16), 6.25 (dd, J ¼ 9.1 Hz,
0
0
0
0
3
3
3
0
3
0
H, H-1 ), 4.43 (m, 1H, H-3 ), 4.01 (m, 1H, H-4 ), 3.90 (m, 2H, H-5 ),
J ¼ 5.2 Hz, 1H, H-1 ), 5.08 (d, J ¼ 5.6 Hz, 1H, H-3 ), 4.37 (t,
0
0
0
0
.27 (m, 2H, H-2 ), 0.95 (s, 9H, H-9), 0.17 (s, 3H, H-7 or H-7 ), 0.16 (s,
H, H-7 or H-7 ). C NMR (50 MHz, acetone-d
J ¼ 6.5 Hz, 2H, H-11), 4.18 (m, 1H, H-4 ), 3.87 (m, 2H, H-5 ), 3.07 (t,
2 3
0
13
0
6
)
d
ppm 161.4 (C
4
O),
J ¼ 6.5 Hz, 2H, H-12), 2.54 (dd, J ¼ 13.9 Hz, J ¼ 5.2 Hz, 1H, H-2 ),
0
0
0
0
0
151.5 (C
2
O), 145.9 (C-6), 89.4 (C-4 ), 87.0 (C-1 ), 73.1 (C-3 ), 69.4 (C-
2.06 (m, 1H, H-2 ), 0.89 (s, 9H, H-9), 0.13 (s, 3H, H-7 ou H-7 ), 0.11 (s,
0
0
0
13
5
), 65.1 (C-5 ), 42.5 (C-2 ), 27.2 (C-9), 19.7 (C-8), -4.3 and ꢁ4.4 (C-7,
3H, H-7 ou H-7); C NMR (50 MHz, CDCl
3
) dppm 161.7 (C-4), 160.8
0
C-7 ).
(C-10), 155.7 (C-2), 151.6 (C-13), 151.3 (C-15), 145.4 (C-6), 138.7 (C-

676
R. El Aissi et al. / European Journal of Medicinal Chemistry 101 (2015) 668e680
0
0
0
3
0
3
1
5
(
7), 122.5 (C-16), 121.5 (C-18), 87.0 (C-1 , C-4 ), 80.6 (C-3 ), 70.6 (C-
J ¼ 5.7 Hz, 1H, H-3 ), 4.38 (t, J ¼ 6.7 Hz, 2H, H-11), 4.21 (m, 1H, H-
3
0
0
0
0
), 67.3 (C-11), 65.1 (C-5 ), 40.2 (C-2 ), 38.4 (C-12), 27.7 (C-9), 19.9
C-8), -3.5 and ꢁ3.8 (C-7, C-7 ). ESI-MS: m/z 680.03 [M-H ] .
4 ), 3.90 (m, 2H, H-5 ), 3.78 (s, 3H, H-17), 2.94 (t, J ¼ 6.7 Hz, 2H, H-
0
þ ꢁ
2 3
0
0
12), 2.56 (dd, J ¼ 14.0 Hz, J ¼ 5.3 Hz, 1H, H-2 ), 2.10 (m, 1H, H-2 ),
0
0
0
.93 (s, 9H, H-9), 0.16 (s, 3H, H-7 or H-7 ), 0.15 (s, 3H, H-7 or H-7);
1
3
4
.3.4. 2-(Hydroxymethyl)-5-(5-iodo-2,4-dioxo-3,4-
C NMR (50 MHz, CDCl
3
) dppm 160.1 (C-4), 159.9 (C-16), 154.2 (C-
dihydropyrimidin-1(2H)-yl)tetrahydrofuran-3-yl (2-(pyridin-2-
yldisulfanyl)ethyl) carbonate (9)
10), 150.1 (C-2), 143.9 (C-6), 132.4 (C-14), 127.4 (C-13), 114.8 (C-15),
0
0
0
85.6 (C-1, C-4 ), 79.1 (C-3 ), 68.9 (C-5), 65.8 (C-11), 63.6 (C-5 ), 55.5
0
To a solution of compound (8) (200 mg, 0.29 mmol, 1 eq.) dis-
solved in acetonitrile/eau (40 mL, 1/1, v/v), indium chloride (1 eq.)
was added. The resulting mixture was heated at reflux for 7 h. After
cooling to room temperature, acetonitrile was evaporated under
reduced pressure. The resulting aqueous solution was extracted
with acetate (3 ꢂ 20 mL). The combined organic layers were
washed with brine (2 ꢂ 15 mL), dried over magnesium sulfate,
filtered and evaporated under reduced pressure. The crude product
was purified on silica gel eluted with a gradient ethyl acetate/
cyclohexane (40/60 to 60/40, v/v) to afford compound (9) (110 mg,
(C-17), 38.7 (C-2 ), 36.2 (C-12), 26.2 (C-9), 18.5 (C-8), ꢁ5.0 and ꢁ5.3
0
þ þ
(C-7, C-7 ). HRMS m/z [M þ H ] :calc: 711.0727; found: 711.0724.
4.3.6. ((4-Methoxyphenyl)disulfanyl)ethyl) carbonate (11a)
4.3.6.1. Method A. Compound (11a) was synthesized from (10a)
(100 mg, 0.14 mmol) according to the method previously described
for the synthesis of compound (9) (reaction time: 4 h). The crude
product was purified on silica gel eluted with a gradient ethyl ac-
etate/cyclohexane (10/90 to 35/65, v/v) to afford compound (11a)
(72 mg, 0.12 mmol). Yield: 83%.
ꢁ
1
0
(
(
.19 mmol). Yield: 66%; IR (KBr)
n
(cm ) 3450, 1745 (CO), 1685
CO), 1448, 1418, 1263, 1101; H NMR (200 MHz, CDCl ppm 8.51
m,1H, H-12), 8.43 (brs, 1H, NH), 8.31 (s,1H, H-6), 7.69 (m, 2H, H-14,
1
3
)
d
4.3.6.2. Method B. To a solution of (9) (100 mg, 0.18 mmol, 1 eq.)
ꢀ
dissolved in anhydrous THF (5 mL), cooled at 0 C, a solution of 4-
3
3
0
0
0
H-15), 7.14 (m, 1H, H-13), 6.23 (dd, J ¼ 9.2 Hz, J ¼ 5.2 Hz, 1H, H-1 ),
methoxythiophenol (27.2 mg, 0.19 mmol, 1.1 eq.) dissolved in
anhydrous THF (3 mL) was added. The resulting mixture was stirred
0
0
3
5
3
.27 (m, 1H, H-3 ), 4.44 (t, J ¼ 6.4 Hz, 2H, H-8), 4.23 (m, 1H, H-4 ),
3
ꢀ
.98 (m, 2H, H-5 ), 3.09 (t, J ¼ 6.4 Hz, 2H, H-9), 2.50 (m, 2H, H-2 );
at 0 C for 1 h 30 min, then at room temperature for 4 h. The re-
1
3
C NMR (50 MHz, CDCl
3
)
d
ppm 160.9 (C-4), 160.5 (C-7), 155.6 (C-2),
action solvent was evaporated under reduced pressure and the
crude product was purified on silica gel eluted with a gradient ethyl
acetate/cyclohexane (10/90 to 40/60, v/v) to afford compound (11a)
1
1
51.1 (C-12, C-10), 151.3 (C-15), 146.8 (C-6), 138.7 (C-14), 122.5 (C-
0 0 0
3), 121.4 (C-15), 87.8 (C-1 ), 86.4 (C-4 ), 78.6 (C-3 ), 69.7 (C-5), 67.4
0
0
(
C-8), 63.7 (C-5 ), 39.3 (C-2 ), 38.1 (C-9). ESI-MS: m/z 565.95 [M-
(89 mg, 0.15 mmol). Yield: 83%.
þ ꢁ
ꢁ1
1
H ] .
IR (KBr)
n
(cm ) 3465, 3055, 1743 (CO), 1685 (CO), 1589, 1491,
1
448, 1247, 1099; H NMR (200 MHz, CDCl
3
)
d
ppm 9.75 (brs, 1H, NH),
3
3
4.3.5. 2-(((tert-Butyldimethylsilyl)oxy)methyl)-5-(5-iodo-2,4-
8.29 (s, 1H, H-6), 7.45 (d, J ¼ 8.8 Hz, 2H, H-11), 6.83 (d, J ¼ 8.8 Hz,
3
0
0
dioxo-3,4-dihydropyrimidin-1(2H)-yl)tetrahydrofuran-3-yl(2-((4-
methoxyphenyl)disulfanyl)ethyl) carbonate (10a)
2H, H-12), 6.20 (t, J ¼ 6.1 Hz, 1H, H-1 ), 5.24 (m, 1H, H-3 ), 4.36 (t,
3
0
0
J ¼ 6.6 Hz, 2H, H-8), 4.17 (m, 1H, H-4 ), 4.03 (m, 2H, H-5 ), 3.77 (s,
3
0
13
4
.3.5.1. Method A. To a solution of (9) (200 mg, 0.29 mmol, 1 eq.)
3H, H-14), 2.92 (t, J ¼ 6.6 Hz, 2H, H-9), 2.46 (m, 2H, H-2 ). C NMR
ꢀ
dissolved in anhydrous THF (4 mL) cooled at 0 C, a solution of 4-
methoxythiophenol (45.1 mg, 0.32 mmol, 1.1 eq.) dissolved in
anhydrous THF (3 mL) was added. The resulting mixture was stirred
(50 MHz, CDCl
3
)
d
ppm 160.4 (C-4), 159.9 (C-13), 154.3 (C-7), 150.1 (C-
0
2), 145.7 (C-6), 132.4 (C-11), 127.5 (C-10), 114.9 (C-12), 86.5 (C-1 ),
85.2 (C-4 ), 78.4 (C-3 ), 68.7 (C-5), 65.9 (C-8), 62.5 (C-5 ), 55.5 (C-
0
0
0
ꢀ
0
þ þ
for 1 h at 0 C, then 3 h at room temperature. The solvent reaction
14), 38.0 (C-2 ), 36.3 (C-9). HRMS m/z for [M þ H ] : calc:
was evaporated under reduced pressure and the crude product was
dissolved in dichloromethane (20 mL). The resulting solution was
washed with brine (2 ꢂ 20 mL), dried over magnesium sulfate,
filtered and evaporated under reduced pressure. The crude product
was purified on silica gel eluted with a gradient ethyl acetate/
cyclohexane (10/90 to 30/70, v/v) to afford compound (10a)
596.9862; found: 596.9838.
4.3.7. 2-(((tert-Butyldimethylsilyl)oxy)methyl)-5-(5-iodo-2,4-
dioxo-3,4-dihydropyrimidin-1(2H)-yl)tetrahydrofuran-3-yl (2-((4-
methoxyphenyl)thio)ethyl) carbonate (10b)
To a solution of (6) (500 mg, 1.07 mmol, 1 eq.) and 4-nitrophenyl
chloroformate (215.1 mg, 1.07 mmol, 1 eq.) dissolved in anhydrous
THF (15 mL), a solution of DMAP (208.6 mg, 1.70 mmol, 1.6 eq.)
dissolved in anhydrous THF (3 mL) was added. The resulting
mixture was stirred at room temperature for 3 h 30 min. DMAP
(85 mg, 0.70 mmol, 0.65 eq.) was added, followed by a solution of
(2b) (236 mg, 1.28 mmol, 1.2 eq.) dissolved in anhydrous THF
(3 mL). The reaction mixture was stirred at room temperature for
16 h. After evaporation of THF under reduced pressure, the crude
product was dissolved in dichloromethane (20 mL), washed with
water (25 mL) and brine (2 ꢂ 20 mL), dried over magnesium sulfate,
filtered and evaporated under reduced pressure. The crude product
was purified on silica gel eluted with a gradient ethyl acetate/
cyclohexane (10/90 to 35/65, v/v) to afford compound (10b)
(
120 mg, 0.17 mmol). Yield: 58%.
4.3.5.2. Method B. To a solution of (6) (500 mg, 1.07 mmol, 1 eq.)
and 4-nitrophenyl chloroformate (215.1 mg, 1.07 mmol, 1 eq.) dis-
solved in anhydrous THF (15 mL), a solution of DMAP (208.6 mg,
1
.70 mmol, 1.6 eq.) dissolved in anhydrous THF(3 mL) was added
dropwise. After stirring at room temperature for 3 h 30 min, DMAP
85.0 mg, 0.70 mmol, 0.65 eq.) and a solution of (2a) (277.0 mg,
.28 mmol, 1.2 eq.) dissolved in anhydrous THF (3 mL) were added.
(
1
The reaction mixture was stirred at room temperature for 16 h. The
reaction solvent was evaporated under reduced pressure. The crude
product was dissolved in dichloromethane (20 mL) and the
resulting solution was washed with water (25 mL) and brine
ꢁ
1
(
2 ꢂ 20 mL). The organic layer was dried over magnesium sulfate,
(430 mg, 0.63 mmol). Yield: 59%; IR (KBr)
n
(cm ) 1745 (CO), 1713
(CO), 1420, 1362, 1269, 1222; H NMR (200 MHz, CDCl ppm 9.26
(brs, 1H, NH), 8.09 (s, 1H, H-6), 7.37 (d, J ¼ 8.9 Hz, 2H, H-14), 6.83
1
filtered and evaporated under reduced pressure. The crude product
was purified on silica gel eluted with a gradient ethyl acetate/
cyclohexane (10/90 to 30/70, v/v) to afford compound (10a)
3
) d
3
3
3
3
0
(d, J ¼ 8.9 Hz, 2H, H-15), 6.26 (dd, J ¼ 9.1, J ¼ 5.2 Hz, 1H, H-1 ),
3
0
3
(
560 mg, 0.79 mmol). Yield: 74%.
5.10 (d, J ¼ 5.9 Hz, 1H, H-3 ), 4.23 (t, J ¼ 7.0 Hz, 2H, H-11), 4.19 (m,
3
(cmꢁ1) 2927, 2855, 1756 (CO), 1686 (CO), 1590, 1491,
1H, H-4 ), 3.89 (m, 2H, H-5 ), 3.77 (s, 3H, H-17), 3.03 (t, J ¼ 7.0 Hz,
0
0
IR (KBr)
n
1
2 3
0
1
8
2
257, 1196, 1125; H NMR (200 MHz, CDCl
.09 (s, 1H, H-6), 7.46 (d, J ¼ 8.9 Hz, 2H, H-14), 6.85 (d, J ¼ 8.9 Hz,
3
)
d
ppm 9.60 (brs, 1H, NH),
2H, H-12), 2.54 (dd, J ¼ 13.9 Hz, J ¼ 5.2 Hz, 1H, H-2 ), 2.08 (m, 1H,
3
3
0
0
0
H-2 ), 0.92 (s, 9H, H-9), 0.15 (s, 3H, H-7 or H-7 ), 0.14 (s, 3H, H-7 or
H, H-15), 6.27 (dd, 3J ¼ 9.2 Hz, J ¼ 5.3 Hz, 1H, H-1 ), 5.13 (d,
3
0
13
3
H-7); C NMR (50 MHz, CDCl ): dppm 160.1 (C-4),159.5 (C-16),154.3

R. El Aissi et al. / European Journal of Medicinal Chemistry 101 (2015) 668e680
677
3
(
1
5
C-10), 150.0 (C-2), 144.0 (C-6), 132.2 (C-14), 124.6 (C-13), 114.8 (C-
(m, 2H, NH-w, H-5), 8.12 (m, 2H, H-7, H-8), 7.56 (d, J ¼ 8.9 Hz, 2H,
3
0
0
0
0
0
5), 85.6 (C-1, C-4 ), 79.0 (C-3 ), 68.8 (C-5), 66.7 (C-11), 63.6 (C-5 ),
H-3 ), 7.01 (d, J ¼ 8.9 Hz, 2H, H-2 ), 4.70 (s, 2H, H-e), 3.84 (t,
0
3
3
3
3
5.4 (C-17), 38.7 (C-2 ), 34.2 (C-12), 26.2 (C-9), 18.4 (C-8), -5.1
J ¼ 6.7 Hz, 2H, H-10), 3.63 (q, J ¼ 5.9 Hz, 2H, H-a), 2.87 (t,
0
þ þ
3
and ꢁ5.3 (C-7, C-7 ). HRMS m/z for [M þ H ] : calc: 679.1006
J ¼ 6.7 Hz, 2H, H-9), 2.81 (t, J ¼ 5.9 Hz, 2H, H-b), 2.68 (q,
3
found: 679.0992.
J ¼ 7.1 Hz, 4H, H-c), 1.13 (t, J ¼ 7.1 Hz, 6H, H-d), 0.89 (s, 9H, H-13),
13
0
.06 (s, 6H, H-11); C NMR (50 MHz, CDCl
3
)
d
ppm 165.5 (C-x), 162.5
0
4
.3.8. 2-(Hydroxymethyl)-5-(5-iodo-2,4-dioxo-3,4-
(C-y), 155.4 (C-1 ), 143.7 (C-3), 143.6 (C-4a), 142.0 (C-2), 138.4 (C-6),
136.9 (C-8a), 130.3 (C-4 ), 130.0 (C-3 ), 129.8 (C-8), 123.5 (C-7), 116.4
(C-5), 114.7 (C-2 ), 66.9 (C-e), 60.6 (C-10), 50.7 (C-b), 46.3 (Cec),
0
0
dihydropyrimidin-1(2H)-yl)tetrahydrofuran-3-yl (2-((4-
methoxyphenyl)thio)ethyl) carbonate (11b)
0
Compound (11b) was synthesized from (10b) (290 mg,
.43 mmol) according to the method previously described for the
40.4 (C-9), 36.3 (C-a), 25.0 (C-13), 17.4 (C-12), 10.9 (C-d), ꢁ6.3 (C-
þ þ
0
11). HRMS m/z for [M þ H ] calc: 644.2761; found: 644.2778.
synthesis of compound (9) (reaction time: 4 h). The crude product
was purified on silica gel eluted with a gradient ethyl acetate/
cyclohexane (20/80 to 55/45, v/v) to afford compound (11b)
4.4.3. 6-(2-(4-((2-((tert-Butyldimethylsilyl)oxy)ethyl)thio)
phenoxy)acetamido)-N-(2-(diethylamino)ethyl)quinoxaline-2-
carboxamide (14b)
Compound (14b) was synthesized from (4b) (540 mg,
1.90 mmol) and (13) (837.2 mg,1.72 mmol) according to the method
previously described for the synthesis of compound (14a) (reaction
time: 1 h). The crude product was purified on silica gel eluted with
a gradient of dichloromethane/methanol (95/5 to 90/10, v/v) to
ꢁ1
(
(
d
200 mg, 0.36 mmol). Yield: 83%; IR (KBr) n (cm ) 3456, 2926,1751
1
CO), 1687 (CO), 1494, 1449, 1264, 1099; H NMR (200 MHz, CDCl
3
)
3
ppm 8.87 (brs, 1H, NH), 8.27 (s, 1H, H-6), 7.41 (d, J ¼ 8.9 Hz, 2H, H-
3 3 3
1
1), 6.88 (d, J ¼ 8.9 Hz, 2H, H-12), 6.23 (dd, J ¼ 9.2, J ¼ 5.2 Hz, 1H,
0
0
0
0
0
H-1 ), 5.26 (m, 1H, H-3 ), 4.23 (m, 3H, H-8, H-4 ), 3.97 (m, 2H, H-5 ),
3
3
.81 (s, 3H, H-14), 3.07 (t, J ¼ 6.9 Hz, 2H, H-9), 2.45 (m, 2H, H-2 ).
1
3
C NMR (50 MHz, Acetone-d
6
)
d
ppm 159.9 (C-4), 159.7 (C-13), 154.3
afford compound (14b) (520 mg, 0.85 mmol). Yield: 50%; IR (KBr) n
ꢁ
1
1
(
C-7), 150.2 (C-2), 145.3 (C-6), 133.7 (C-11), 125.1 (C-10), 114.8 (C-
(cm ) 2955, 1662(CO), 1631 (CO), 1526, 1492, 1235; H NMR
(200 MHz, CDCl ppm 9.40 (s,1H, H-3), 9.36 (brs,1H, NH-v), 8.78 (t,
0
0
0
0
1
5
5
2), 85.4 (C-1 , C-4 ), 79.0 (C-3 ), 68.1 (C-5), 66.4 (C-8), 62.0 (C-5 ),
3
) d
0
þ þ
3
4.9 (C-14), 37.9 (C-2 ), 33.8 (C-9). HRMS m/z for [M þ H ] calc:
J ¼ 5.5 Hz, 1H, NH-w), 8.44 (m, 1H, H-5), 7.98 (m, 1H, H-7), 7.86 (d,
3
3
3
3
3
0
65.0142; found: 565.0137.
J ¼ 9.0 Hz, 1H, H-8), 7.27 (d, J ¼ 8.6 Hz, 2H, H-3 ), 6.87 (d,
0
J ¼ 8.6 Hz, 2H, H-2 ), 4.66 (s, 2H, H-e), 3.82 (m, 2H, H-a), 3.67 (t,
3
4
b)
.4. Access to compounds (13), (14a, b), (15a, b), (16a, b) and (17a,
J ¼ 7.2 Hz, 2H, H-10), 3.12 (t, J ¼ 5.6 Hz, 2H, H-b), 3.00 (q,
3
3
J ¼ 7.1 Hz, 4H, H-c), 2.89 (t, J ¼ 7.2 Hz, 2H, H-9), 1.26 (t, J ¼ 7.1 Hz,
13
6
H, H-d), 0.79 (s, 9H, H-13), 0.04 (s, 6H, H-11); C NMR (50 MHz,
0
4
.4.1. 6-(2-Bromoacetamido)-N-(2-(diethylamino)ethyl)
CDCl
3
)
d
ppm 166.8 (C-x), 163.9 (C-y), 156.0 (C-1 ), 144.4 (C-4a), 144.2
0
quinoxaline-2-carboxamide hydrobromide (13)
To a solution of (12) [42] (1.2 g, 4.03 mmol, 1 eq.) dissolved in
anhydrous dichloromethane (15 mL), 2-bromoacetoyle bromide
(C-3), 142.3 (C-2), 139.9 (C-6), 137.6 (C-8a), 132.5 (C-3 ), 130.6 (C-8),
129.1 (C-4 ), 124.7 (C-7), 117.1 (C-5), 115.5 (C-2 ), 67.7 (C-e), 62.3 (C-
0
0
10), 51.6 (C-b), 47.4 (Cec), 37.4 (C-9), 36.3 (C-a), 25.9 (C-13), 18.3 (C-
þ þ
(
459
mL, 5.25 mmol, 1.3 eq.) was added dropwise. The resulting
12), 10.6 (C-d), -5.3 (C-11). HRMS m/z for [M þ H ] calc: 612.3040;
mixture was stirred at room temperature for 16 h. The precipitate
found: 612.3026.
was filtered, then dried under reduced pressure to afford com-
ꢀ
ꢁ1
)
pound (13) (2.05 g). Yield: quant; Mp: 212 ± 1 C; IR (KBr)
n
(cm
4.4.4. N-(2-(Diethylamino)ethyl)-6-(2-(4-((2-hydroxyethyl)
2
947, 2655, 1704 (CO), 1671 (CO), 1624, 1560, 1529, 1486, 1420, 1211;
disulfanyl)phenoxy)acetamido)quinoxaline-2-carboxamide (15a)
Compound (15a) was synthesized from (14a) (890 mg,
1.38 mmol) according to the method previously described for the
synthesis of compound (9) (reaction time: 2 h 30 min). The crude
product was purified on silica gel eluted with a gradient dichloro-
methane/methanol (95/5 to 85/15, v/v) to afford compound (15a)
1
H NMR (200 MHz, MeOD)
d
ppm 9.45 (s, 1H, H-3), 8.62 (d,
4
3
3
J ¼ 2.2 Hz, 1H, H-5), 8.17 (d, J ¼ 9.2 Hz, 1H, H-8), 8.02 (dd,
4
3
J ¼ 9.2 Hz, J ¼ 2.2 Hz,1H, H-7), 4.10 (s, 2H, H-e), 3.90 (t, J ¼ 6.2 Hz,
3
13
2
H, H-a), 3.40 (m, 6H, H-b, H-c), 1.39 (t, J ¼ 7.3 Hz, 6H, H-d);
C
6
NMR (50 MHz, DMSO-d ) dppm 166.4 (C-x), 164.4 (C-y), 144.5 (C-3),
ꢁ
1
144.3 (C-4a), 142.9 (C-2), 141.9 (C-6), 137.2 (C-8a), 130.6 (C-8), 125.2
(700 mg, 1.32 mmol). Yield: 96%; IR (KBr) n (cm ) 3313, 2963, 1720
1
(
C-7), 115.9 (C-5), 49.9 (C-b), 47.2 (Cec), 34.6 (C-a), 30.7 (C-e), 8.9
3
(CO), 1645, 1531, 1489, 1449, 1246, 1208; H NMR (200 MHz, CDCl )
þ þ
(
C-d). ESI-MS: m/z 408.08 [M þ H ]
d
ppm 9.51 (s, 1H, H-3), 9.11 (brs, 1H, NH-v), 8.48 (m, 2H, NH-w, H-5),
3
4
3
8
.10 (dd, J ¼ 9.1 Hz, J ¼ 1.9 Hz, 1H, H-7), 8.00 (d, J ¼ 9.1 Hz, 1H, H-
3
0
3
0
4.4.2. 6-(2-(4-((2-((tert-Butyldimethylsilyl)oxy)ethyl)disulfanyl)
8), 7.42 (d, J ¼ 8.7 Hz, 2H, H-3 ), 6.89 (d, J ¼ 8.7 Hz, 2H, H-2 ), 4.64
3
phenoxy)acetamido)-N-(2-(diethylamino)ethyl)quinoxaline-2-
carboxamide (14a)
To a solution of (4a) (358.7 mg, 1.13 mmol, 1.1 eq.) dissolved in
anhydrous THF (8 mL), sodium hydride (63%, in oil) (118 mg,
(s, 2H, H-e), 3.83 (t, J ¼ 6.1 Hz, 2H, H-10), 3.62 (m, 3H, H-a, OH),
3
3
2.87 (t, J ¼ 6.1 Hz, 2H, H-9), 2.80 (t, J ¼ 6.1 Hz, 2H, H-b), 2.67 (q,
3
3
13
J ¼ 7.1 Hz, 4H, H-c), 1.09 (t, J ¼ 7.1 Hz, 6H, H-d); C NMR (50 MHz,
0
CDCl
3
)
d
ppm 166.8(C-x), 163.5 (C-y), 156.7 (C-1 ), 144.3 (C-3), 144.2
0
3
.10 mmol, 3 eq.) was added. After 15 min, a solution of (13)
(C-4a), 142.7 (C-2), 139.8 (C-6), 137.7 (C-8a), 131.2 (C-3 ), 130.6 (C-8),
130.4 (C-4 ), 124.8 (C-7), 117.2 (C-5), 115.6 (C-2 ), 67.7 (C-e), 60.0 (C-
0
0
(
500 mg, 1.03 mmol, 1 eq.) and triethylamine (171 L,1.23 mmol,1.2
m
eq.) dissolved in anhydrous THF (20 mL) was added. The resulting
mixture was stirred at room temperature for 1 h. The reaction
solvent was evaporated under reduced pressure and the crude
product was taken up in dichloromethane (50 mL). The mixture
was washed with brine (3 ꢂ 10 mL), dried over magnesium sulfate,
filtered, then evaporated under reduced pressure. The crude
product was purified on silica gel eluted with a gradient dichloro-
methane/methanol (95/5 to 90/10, v/v) to afford compound (14a)
10), 51.6 (C-b), 47.2 (Cec), 41.3 (C-9), 37.1 (C-a),11.5 (C-d). HRMS m/z
for [M þ H ] calc: 530.1896; found: 530.1872.
þ þ
4.4.5. N-(2-(Diethylamino)ethyl)-6-(2-(4-((2-hydroxyethyl)thio)
phenoxy)acetamido)quinoxaline-2-carboxamide (15b)
Compound (15b) was synthesized from (14b) (440 mg,
0.72 mmol) according to the method previously described for the
synthesis of compound (9) (reaction time: 5 h). The crude product
was purified on silica gel eluted with a gradient of dichloro-
methane/methanol (95/5 to 85/15, v/v) to afford compound (15b)
ꢁ1
(
330 mg, 0.51 mmol). Yield: 50%; IR (KBr)
855, 1682 (CO), 1655 (CO), 1517, 1491, 1238, 1090; H NMR
200 MHz, CDCl ppm 9.64 (s, 1H, H-3), 8.66 (brs, 1H, NH-v), 8.46
n (cm ) 3343, 2927,
1
2
ꢁ1
(
3
)
d
(330 mg, 0.66 mmol). Yield: 92%; IR (KBr) n (cm ) 3281, 2966, 1713

678
R. El Aissi et al. / European Journal of Medicinal Chemistry 101 (2015) 668e680
1
0
0
3
(
d
CO), 1656 (CO), 1525, 1491, 1352, 1222; H NMR (200 MHz, CDCl
3
)
2H, H-11), 4.14 (m, 1H, H-4 ), 3.88 (m, 2H, H-5 ), 3.58 (q, J ¼ 6.0 Hz,
3
3
3
ppm 9.59 (s, 1H, H-3), 8.80 (brs, 1H, NH-v), 8.55 (t, J ¼ 4.9 Hz, 1H,
2H, H-a), 3.06 (t, J ¼ 6.0 Hz, 2H, H-12), 2.75 (t, J ¼ 6.0 Hz, 2H, H-b),
3
3 3 3
NH-w), 8.45 (m, 1H, H-5), 8.10 (m, 2H, H-7, H-8), 7.41 (d, J ¼ 8.8 Hz,
2.64 (q, J ¼ 7.1 Hz, 4H, H-c), 2.47 (dd, J ¼ 14.0 Hz, J ¼ 5.1 Hz,1H, H-
3
0
3
0
0
0
2
4
H, H-3 ), 6.94 (d, J ¼ 8.8 Hz, 2H, H-2 ), 4.68 (s, 2H, H-e), 3.70 (m,
2 ), 2.08 (m, 1H, H-2 ), 1.06 (t, J ¼ 7.1 Hz, 6H, H-d), 0.90 (s, 9H, H-9),
3
3
0
0
13
H, H-10, H-a), 3.05 (t, J ¼ 6.0 Hz, 2H, H-9), 2.84 (t, J ¼ 6.0 Hz, 2H,
0.14 (s, 3H, H-7 or H-7 ), 0.13 (s, 3H, H-7 or H-7 ); C NMR (50 MHz,
3
3
13
00
H-b), 2.73 (q, J ¼ 7.0 Hz, 4H, H-c), 1.15 (t, J ¼ 7.0 Hz, 6H, H-d);
C
CDCl
(C-10), 150.4 (C
14), 139.7 (C-18), 137.7 (C-20a), 134.1 (C-2 ), 130.7 (C-20), 127.6 (C-
3
)
d
ppm 166.6 (C-x), 163.4 (C-y), 160.4 (C-4), 156.6 (C-4 ), 154.2
0
NMR (50 MHz, CDCl
1
(
6
1
4
3
)
d
ppm 166.5 (C-x), 163.6 (C-y), 156.2 (C-1 ),
2
O), 144.4 (C-15), 144.2 (C-16a),143.6 (C-6), 142.9 (C-
0
0
44.5 (C-3), 144.4 (C-4a), 142.8 (C-2), 139.5 (C-6), 137.8 (C-8a), 133.4
C-3 ), 130.7 (C-8), 128.0 (C-4 ), 124.6 (C-7), 117.3 (C-5), 115.6 (C-2 ),
0
0
0
0
0
0
0
0
0
1 ), 124.5 (C-19), 117.2 (C-17), 115.7 (C-3 ), 85.4 (C-1 ), 85.3 (C-4 ),
0
0
7.7 (C-e), 60.3 (C-10), 51.7 (C-b), 47.3 (Cec), 38.5 (C-9), 37.0 (C-a),
79.1 (C-3 ), 69.5 (C-5), 67.7 (C-e), 67.2 (C-11), 63.6 (C-5 ), 51.4 (C-b),
46.9 (Cec), 38.6 (C-2 ), 37.2 (C-a), 34.1 (C-12), 26.2 (C-9), 18.4 (C-8),
11.6 (C-d), -5.1 and ꢁ5.3 (C-7, C-7 ). HRMS m/z for [M þ H ] calc:
þ þ
0
1.4 (C-d). HRMS m/z for [M þ H ] calc: 498.2159; found:
0
þ þ
98.2159.
9
92.2545; found: 992.2508.
4.4.6. 2-(((tert-Butyldimethylsilyl)oxy)methyl)-5-(5-iodo-2,4-
dioxo-3,4-dihydropyrimidin-1(2H)-yl)tetrahydrofuran-3-yl(2-((4-
2-((2-((2-(diethylamino)ethyl)carbamoyl)quinoxalin-6-yl)amino)-
4.4.8. 2-((4-(2-((2-((2-(Diethylamino)ethyl)carbamoyl)quinoxalin-
6-yl)amino)-2-oxoethoxy)phenyl)disulfanyl)ethyl (2-
(
2
-oxoethoxy)phenyl)disulfanyl)ethyl) carbonate (16a)
To a solution of (15a) (300 mg, 0.57 mmol, 1 eq.) dissolved in
(hydroxymethyl)-5-(5-iodo-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-
yl)tetrahydrofuran-3-yl) carbonate (17a)
anhydrous THF (5 mL), DMAP (83 mg, 0.68 mmol, 1.2 eq.) and (7)
448.5 mg, 0.71 mmol, 1.3 eq.) were added, successively. After
stirring for 16 h at room temperature, the reaction solvent was
evaporated under reduced pressure and the crude product was
taken up in dichloromethane (15 mL). The organic layer was
washed with an aqueous saturated sodium bicarbonate solution
Compound (17a) was synthesized from (16a) (280 mg,
0.27 mmol) according to the briefly modified method described for
the synthesis of compound (9) (reaction time: 16 h). After evapo-
ration of the reaction solvent, the crude product was dissolved in
methanol (40 mL) then filtered. The filtrate was concentrated under
reduced pressure and was purified on silica gel with a gradient of
dichloromethane/methanol (95/5 to 90/10, v/v) to afford com-
(
(
10 mL) and brine (2 ꢂ 5 mL), dried over magnesium sulfate, filtered
ꢁ
1
and evaporated under reduced pressure. The crude product was
purified on silica gel eluted with a gradient dichloromethane/
methanol (95/5 to 85/15, v/v) to afford compound (16a) (456 mg,
0
(
pound (17a) (170 mg, 0.184 mmol). Yield: 68%; IR (KBr)
n
(cm
)
1
3368, 2926, 1712 (CO), 1677 (CO), 1618, 1525, 1489, 1261; H NMR
(200 MHz, MeOD) ppm 9.44 (s, 1H, H-12), 8.61 (m, 1H, H-14), 8.42
(s, 1H, H-6), 8.12 (m, 2H, H-16, H-17), 7.54 (d, J ¼ 8.9 Hz, 2H, H-2 ),
d
ꢁ
1
3
00
.446 mmol). Yield: 78%; IR (KBr)
n
(cm ) 2928, 1747 (CO), 1685
CO),1605,1523,1489,1256; H NMR (200 MHz, CDCl ppm 9.63 (s,
H, H-15), 8.88 (brs, 1H, NH-v), 8.43 (m, 3H, NH-w, NH-3, H-17),
1
3
00
3
3
3
)
d
7.09 (d, J ¼ 8.9 Hz, 2H, H-3 ), 6.19 (dd, J ¼ 8.3 Hz, J ¼ 5.9 Hz, 1H,
3
0
0
1
8
H-1 ), 5.15 (d, J ¼ 5.7 Hz, 1H, H-3 ), 4.80 (s, 2H, H-e), 4.34 (t,
3 3
3
4
3
0
.24 (dd, J ¼ 9.1 Hz, J ¼ 2.3 Hz, 1H, H-19), 8.07 (d, J ¼ 9.1 Hz, 1H,
J ¼ 6.2 Hz, 2H, H-8), 4.10 (m, 1H, H-4 ), 3.89 (t, J ¼ 6.1 Hz, 2H, H-a),
3 3
3
00
0
H-20), 8.05 (s, 1H, H-6), 7.55 (d, J ¼ 8.8 Hz, 2H, H-2 ), 7.01 (d,
3.80 (m, 2H, H-5 ), 3.47 (t, J ¼ 6.1 Hz, 2H, H-b), 3.37 (q, J ¼ 7.2 Hz,
3
3
00
3
3
0
0
J ¼ 8.8 Hz, 2H, H-3 ), 6.29 (dd, J ¼ 9.1 Hz, J ¼ 5.2 Hz, 1H, H-1 ),
4H, H-c), 3.03 (t, J ¼ 6.2 Hz, 2H, H-9), 2.36 (m, 2H, H-2 ), 1.38 (t,
3
0
3
3
13
5
2
.13 (d, J ¼ 5.5 Hz, 1H, H-3 ), 4.70 (s, 2H, H-e), 4.37 (t, J ¼ 6.4 Hz,
J ¼ 7.2 Hz, 6H, H-d); C NMR (50 MHz, MeOD)
dppm 168.4 (C-x),
0
0
00
H, H-11), 4.20 (m, 1H, H-4 ), 3.92 (m, 2H, H-5 ), 3.63 (m, 2H, H-a),
165.5 (C-y), 161.3 (C-4), 158.0 (C-4 ), 154.3 (C-7), 150.6 (C-2), 145.6
(C-6),144.2 (C-13a),143.7 (C-12),142.3 (C-11),141.3 (C-15),137.8 (C-
3
3
3
.00 (t, J ¼ 6.4 Hz, 2H, H-12), 2.74 (t, J ¼ 6.1 Hz, 2H, H-b), 2.68 (q,
3 3
3
0
00 00
17a), 131.4 (C-2 ), 130.2 (C-1 ), 129.5 (C-17), 125.3 (C-16), 116.5 (C-
J ¼ 7.1 Hz, 4H, H-c), 2.53 (dd, J ¼ 14.1 Hz, J ¼ 5.2 Hz, 1H, H-2 ),
0
3
00 0 0 0
14), 115.6 (C-3 ), 85.6 (C-1 ), 85.4 (C-4 ), 78.9 (C-3 ), 67.7 (C-5), 67.4
2
.07 (m,1H, H-2 ), 1.10 (t, J ¼ 7.1 Hz, 6H, H-d), 0.93 (s, 9H, H-9), 0.17
0
0
13
0
0
(
s, 3H, H-7 or H-7 ), 0.15 (s, 3H, H-7 or H-7 ); C NMR (50 MHz,
(C-e), 65.4 (C-8), 61.6 (C-5 ), 51.2 (C-b), 37.7 (C-2 ), 36.9 (C-9), 34.5
00
þ þ
CDCl
3
)
d
ppm 166.5 (C-x), 163.4 (C-y), 160.3 (C
4
O), 156.7 (C-4 ), 154.2
(C-a), 7.9 (C-d). HRMS m/z for [M þ H ] calc: 910.1401; found:
(
C
10O),150.3 (C
2
O),144.5 (C-15),144.2 (C-16a),143.6 (C-6),143.0 (C-
910.1403.
00
1
1
7
4
1
4), 139.7 (C-18), 137.8 (C-20a), 131.3 (C-2 ), 130.7 (C-20), 130.1 (C-
0
0
00
0
0
), 124.5 (C-19), 117.2 (C-17), 115.7 (C-3 ), 85.5 (C-1 ), 85.4 (C-4 ),
4.4.9. 2-((4-(2-((2-((2-(Diethylamino)ethyl)carbamoyl)quinoxalin-
6-yl)amino)-2-oxoethoxy)phenyl)thio)ethyl(2-(hydroxymethyl)-5-
(5-iodo-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)tetrahydrofuran-
3-yl) carbonate (17b)
0
0
9.2 (C-3 ), 69.4 (C-5), 67.7 (C-e), 65.6 (C-11), 63.6 (C-5 ), 51.5 (C-b),
7.0 (Cec), 38.7 (C-2 ), 37.2 (C-a), 36.9 (C-12), 26.2 (C-9), 18.4 (C-8),
0
0
þ þ
1.6 (C-d), ꢁ5.1 and ꢁ5.3 (C-7, C-7 ). HRMS m/z for [M þ H ] calc:
1
024.2266; found: 1024.2224.
Compound (17b) was synthesized from (16b) (130 mg,
0.13 mmol) according to the briefly modified method described for
4
.4.7. 2-(((tert-Butyldimethylsilyl)oxy)methyl)-5-(5-iodo-2,4-
dioxo-3,4-dihydropyrimidin-1(2H)-yl)tetrahydrofuran-3-yl (2-((4-
2-((2-((2-(diethylamino)ethyl)carbamoyl)quinoxalin-6-yl)amino)-
the synthesis of compound (9) (time: 16 h). The reaction solvent
was evaporated under reduced pressure and the crude product was
dissolved in methanol (30 mL), then filtered. The filtrate was
concentrated under reduced pressure and purified on silica gel
eluted with a gradient dichloromethane/methanol (95/5 to 90/10,
v/v) to afford compound (17b) (49 mg, 0.056 mmol). Yield: 43%; IR
(
2-oxoethoxy)phenyl)thio)ethyl) carbonate (16b)
Compound (16b) was synthesized from (15b) (270 mg,
.54 mmol) and (7) (429.2 mg, 0.68 mmol) according to the method
0
ꢁ1
1
previously described for the synthesis of compound (16a) (reaction
time: 16 h). The crude product was purified on silica gel eluted with
a gradient dichloromethane/methanol (95/5 to 85/15, v/v) to afford
(KBr)
NMR (200 MHz, MeOD)
8.42 (s,1H, H-6), 8.13 (m, 2H, H-16, H-17), 7.49 (d, J ¼ 8.8 Hz, 2H, H-
n
(cm ) 3338,1713 (CO),1678 (CO),1618,1525,1491,1263; H
d
ppm 9.45 (s, 1H, H-12), 8.63 (m, 1H, H-14),
3
ꢁ
1
00
3
00
3
3
compound (16b) (410 mg, 0.41 mmol). Yield: 76%; IR (KBr)
n
(cm
)
2 ), 7.07 (d, J ¼ 8.8 Hz, 2H, H-3 ), 6.15 (dd, J ¼ 8.2 Hz, J ¼ 5.8 Hz,
3
1
0
0
2
(
(
929, 1721 (CO), 1686 (CO), 1619, 1523, 1491, 1257; H NMR
200 MHz, CDCl ppm 9.56 (s, 1H, H-15), 9.01 (brs, 1H, NH-v), 8.37
m, 3H, NH-w, NH-3, H-17), 8.18 (dd, J ¼ 9.1 Hz, J ¼ 1.2 Hz, 1H, H-
1H, H-1 ), 5.12 (d, J ¼ 5.7 Hz, 1H, H-3 ), 4.79 (s, 2H, H-e), 4.29 (m,
3
0
3
)
d
2H, H-8), 4.04 (m, 1H, H-4 ), 3.88 (t, J ¼ 5.9 Hz, 2H, H-a), 3.80 (m,
3
3
4
0
2H, H-5 ), 3.33 (m, 6H, H-b, H-c), 3.13 (t, J ¼ 6.1 Hz, 2H, H-9), 2.29
3
00
0
3
13
1
9), 8.00 (m, 2H, H-20, H-6), 7.40 (d, J ¼ 8.0 Hz, 2H, H-2 ), 6.94 (d,
(m, 2H, H-2 ),1.37 (t, J ¼ 7.1 Hz, 6H, H-d); C NMR (50 MHz, MeOD)
3
00
3
3
0
00
J ¼ 8.0 Hz, 2H, H-3 ), 6.24 (dd, J ¼ 8.9 Hz, J ¼ 5.1 Hz, 1H, H-1 ),
d
ppm 168.4 (C-x), 165.4 (C-y), 161.2 (C-4), 157.4 (C-4 ), 154.2 (C-7),
3
0
3
5
.07 (d, J ¼ 5.5 Hz, 1H, H-3 ), 4.67 (s, 2H, H-e), 4.26 (t, J ¼ 6.0 Hz,
150.5 (C-2), 145.4 (C-6), 144.1 (C-13a), 143.5 (C-12), 141.2 (C-11),

R. El Aissi et al. / European Journal of Medicinal Chemistry 101 (2015) 668e680
679
0
0
00
1
1
(
3
40.2 (C-15), 137.6 (C-17a), 133.5 (C-2 ), 130.1 (C-17), 127.1 (C-1 ),
(see conditions A and B) and the amount of released IUdR was
determined from the standard curve.
00 0 0
25.2 (C-16), 116.4 (C-14), 115.3 (C-3 ), 85.5 (C-1 ), 85.2 (C-4 ), 78.7
0
0
C-3 ), 67.5 (C-e), 67.2 (C-5, C-8), 61.5 (C-5 ), 51.1 (C-b), 47.5 (Cec),
7.5 (C-2 ), 34.5 (C-a), 33.6 (C-9), 7.9 (C-d). HRMS m/z for
M þ H ] calc: 878.1680; found: 878.1640.
Difference between the initial concentration of the prodrug and
the concentration of released IUdR give access to the effective
concentration of the prodrug (¼ sum of concentrations of all spe-
cies other than IUdR, present in the reaction mixture and resulting
from the prodrug cleavage) at each time analysis. These species are
noted (11a* and 17a*). The intersection of the two curves
concentration ¼ f (time) then gave access to the effective half-life
time of the prodrug (11a* and 17a*), which also corresponds to
the half-formation time of IUdR. Note that the half-formation times
are the concentrations for which (11a or 17a) and IUdR are equal to
half of the initial concentration of the prodrug (11a and 17a).
0
þ þ
[
4
4
.5. Material for analytical experiments
.5.1. HPLC material and conditions
The analytical reversed phase-high pressure liquid chromatog-
raphy (RP-HPLC) measurements were performed on a system
consisting of HP1100 (Hewlett Packard, Les Ulis, France). The sep-
aration was carried out on a C18 column (Agilent Zorbax, 80 Å,
4
.6 mm ꢂ 150 mm, 5
mM) using the following conditions:
-
-
initial concentration of (11a) ¼ 0.9 mM
initial concentration of (17a) ¼ 0.55 mM
-
For standard curve preparation and conjugates (11a,b) cleavage
and stability studies: gradient time 30 min, flow
¼
rate ¼ 0.5 mL/min, eluent A (ammonium formate buffer, pH 4),
Appendix A. Supplementary data
eluent B (acetonitrile); gradient: 75/25 (A/B, v/v) for 0e3.5 min,
7
7
5/25 / 50/50 (A/B, v/v) for 3.5e7 min, 50/50 (A/B, v/v) for
e15 min, 50/50 / 75/25 (A/B, v/v) for 15e20 min, 75/25 (A/B,
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2015.06.055.
v/v) for 20e30 min,
l
¼ 285 nm (Conditions A).
-
For conjugates (17a,b) cleavage and stability studies: gradient
time ¼ 20 min, flow rate ¼ 0.5 mL/min, eluent A (ammonium
formate buffer, pH 4), eluent B (acetonitrile); gradient: 75/25 (A/
B, v/v) for 0e3.5 min, 75/25 / 50/50 (A/B, v/v) for 3.5e7 min,
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.5.3. IUdR dosage
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.5.4. Half-life determination
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