[d4U]-Spacer-[HI-236] double-drug inhibitors of HIV-1 reverse-transcriptase

Article abstract of DOI:10.1016/j.bmc.2010.05.025

Four double-drug HIV NRTI/NNRTI inhibitors 15a-d of the type [d4U]-spacer-[HI-236] in which the spacer is varied as 1-butynyl (15a), propargyl-1-PEG (15b), propargyl-2-PEG (15c) and propargyl-4-PEG (15d) have been synthesized and biologically evaluated as RT inhibitors against HIV-1. The key step in their synthesis involved a Sonogashira coupling of 5-iodo d4U's benzoate with an alkynylated tethered HI-236 precursor followed by introduction of the HI-236 thiourea functionality. Biological evaluation in both cell-culture (MT-2 cells) as well as using an in vitro RT assay revealed 15a-c to be all more active than d4T. However, overall the results indicate the derivatives are acting as chain-extended NNRTIs in which for 15b-d the nucleoside component is likely situated outside of the pocket but with no evidence for any synergistic double binding between the NRTI and NNRTI sites. This is attributed, in part, to the lack of phosphorylation of the nucleoside component of the double-drug as a result of kinase recognition failure, which is not improved upon with the phosphoramidate of 15d incorporating a 4-PEG spacer.

Full text of DOI:10.1016/j.bmc.2010.05.025

Bioorganic & Medicinal Chemistry 18 (2010) 4661–4673
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
[d4U]-Spacer-[HI-236] double-drug inhibitors of HIV-1 reverse-transcriptase
a
a
a
b
Yassir Younis a, Roger Hunter a, , Clare I. Muhanji , Ian Hale , Rajinder Singh , Christopher M. Bailey ,
*
Todd J. Sullivan b, Karen S. Anderson b
a Department of Chemistry, University of Cape Town, Rondebosch 7701, Cape Town, South Africa
b Department of Pharmacology, School of Medicine, Yale University, New Haven, CT 06520, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Four double-drug HIV NRTI/NNRTI inhibitors 15a–d of the type [d4U]-spacer-[HI-236] in which the
spacer is varied as 1-butynyl (15a), propargyl-1-PEG (15b), propargyl-2-PEG (15c) and propargyl-4-
PEG (15d) have been synthesized and biologically evaluated as RT inhibitors against HIV-1. The key step
in their synthesis involved a Sonogashira coupling of 5-iodo d4U’s benzoate with an alkynylated tethered
HI-236 precursor followed by introduction of the HI-236 thiourea functionality. Biological evaluation in
both cell-culture (MT-2 cells) as well as using an in vitro RT assay revealed 15a–c to be all more active
than d4T. However, overall the results indicate the derivatives are acting as chain-extended NNRTIs in
which for 15b–d the nucleoside component is likely situated outside of the pocket but with no evidence
for any synergistic double binding between the NRTI and NNRTI sites. This is attributed, in part, to the
lack of phosphorylation of the nucleoside component of the double-drug as a result of kinase recognition
failure, which is not improved upon with the phosphoramidate of 15d incorporating a 4-PEG spacer.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 19 March 2010
Revised 5 May 2010
Accepted 6 May 2010
Available online 11 May 2010
Keywords:
HIV inhibitors
Reverse-Transcriptase
NRTI
NNRTI
Double-drug
HI-236
d4T
Antiviral
1. Introduction
chemistry research efforts towards realizing such a concept, and
in 1995 Camarasa9 published the first example of a bifunctional
HIV reverse-transcriptase (RT)1 is a multifunctional enzyme
responsible for the catalytic transformation of single-stranded
HIV viral RNA into double-stranded DNA (dsDNA). It continues to
be the principal drug-target for chemotherapy using combination
therapy HAART2 (Highly Active Antiretroviral Therapy), in which
RT inhibitors (RTIs) provide at least two of the three anti-HIV
drugs3 used. Two types of RTIs have emerged as NRTIs3 (Nucleo-
side Inhibitors), which act as competitive inhibitors of DNA poly-
merization, and NNRTIs (Non-Nucleoside Inhibitors),4 which
inhibit allosterically by binding into a hydrophobic pocket adjacent
to the DNA substrate binding site.3 The close proximity (10–15 Å)
of the two RTI sites inspired Arnold5 in 1993 and Steitz6 in 1994
to propose the double-drug concept for the first time. They postu-
lated that a molecular entity comprising one of each type of inhib-
itor joined by a spacer might be able to interact simultaneously
with the two sites on RT and in so doing provide a super-inhibitor
via additive binding.7 Biochemical support to realize such a con-
cept was subsequently provided by Anderson8a Goody8b and Wain-
berg8c who showed that binding of an NNRTI does not inhibit the
binding processes of an NRTI. These research contributions effec-
tively provided inspiration for a number of synthetic medicinal
NRTI/NNRTI anti-HIV double-drug containing a non-cleavable
spacer based on an [AZT]-spacer-[TSAO-T] or [AZT]-spacer-[HEPT]
combination joined at N-3 of the base of each drug. TSAO-T10
derivatives interact with amino acids situated in both HIV-1 RT
subunits (p51 and p66) at the dimer interface. A likely important
interaction is with the carboxyl group of Glu 138, which lies at
the entrance to the NNRTI pocket in the p51 sub-unit. Camarasa
varied both the drug combinations as well as the tether length
and did achieve some IC50 inhibition values in the 0.06–0.55 lM
range against HIV-1. However, it was concluded that the double-
drugs inhibited as extended NNRTIs with no contribution apparent
from the NRTI component. In a subsequent paper,11 the study was
broadened to accommodate other NRTI’s like d4T as well as differ-
ent attachment points on the drugs, but still without demonstrat-
ing any activity at the NRTI site. In the following ten years or so,
reports on both cleavable,12 in which the spacer is designed to be
hydrolytically labile in order to release the individual drugs, as well
as other non-cleavable double-drugs,13 also known as mixed site
inhibitors,13b appeared in the literature but similarly without evi-
dence of true synergy between the two inhibitors. For the cleavable
type, systems have become extended to different drug targets14
other than RT, whereas for the non-cleavable type, RT remains to
date as the only target15 studied for HIV in view of the uniqueness
of having the NRTI and NNRTI sites in close proximity. Challenges
* Corresponding author. Tel.: +27 21 650 2544; fax: + 27 21 650 5195.
E-mail address: Roger.Hunter@uct.ac.za (R. Hunter).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.05.025

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Y. Younis et al. / Bioorg. Med. Chem. 18 (2010) 4661–4673
O
S
N
S
S
N
N
N
spacer
O
O
N
HN
N
H
N
H
NH
Br
N
H
NH
Br
HN
1
spacer
O
2
MeO
HO
N
N
O
O
HO
5
N
O
OMe
OMe
Br
d4U-spacer-HI-23616
d4U-spacer-trovirdine derivative13a
HI-23618
Figure 1. Structures of HI-236 and d4U-spacer-PETT double-drugs.
regarding the successful realization of a truly synergistic double-
drug RTI in the non-cleavable class have been primarily twofold
as: (i) where on the drugs to attach the linker—this has suffered
from a lack of modeling data, and, (ii) the lack of phosphorylation
of the NRTI nucleoside pro-drug contained within the double-drug
in vitro. Recently, we have reported on our own efforts in the field
of non-cleavable HIV double-drugs using a d4U/HI-236 system.
Our first prototype16 with a relatively short butynyl tether against
HIV-1 (IIIB) in MT-2 cell culture using an MTT assay returned an
EC50 of 250 nM. This was only a fivefold reduction of the NNRTI
where to attach the tether, two important decisions clearly had to
be made. C-5 on the pyrimidine base was chosen as the attachment
point to the nucleoside drug as this was not only synthetically
readily accessible but was also considered to offer minimal inter-
ference with both base-pairing as well as hindrance around the
C-5 site. These views were based on the work of Ruth and Cheng,19
and the choice of the C-5 connecting group as alkynyl was consid-
ered to offer an attractive exit from the substrate binding site in
view of its directionality away from the developing DNA strand.
An alkynyl connection to the nucleoside also presented itself as
an attractive synthetic option via the well-known and versatile
Sonogashira Pd(0) cross-coupling protocol, a well-known method-
ology in the nucleoside field.11,12d,20 This left the all-important
choice of NNRTI attachment. It was significant to us that Ladurée13a
had failed to observe any appreciable activity with their trovirdine
derivative-based double-drug, which is shown against our double-
drug design together with HI-236 in Figure 1, both double-drugs
containing a non-cleavable linker.
(HI-236) EC50 and eightfold more potent than d4T (2 lM). Encour-
aged by this result we went on in a subsequent paper17 to explore
the influence of tether type and length on the anti-HIV activity of
C-2 aryl O-tethered HI-236 derivatives in order to establish the fea-
sibility of projecting a tether out of the NNRTI pocket. A 2-PEG-pro-
pynyl substituent returned an EC50 of 390 nM suggesting feasibility
of the concept. In this paper we extend these studies and report on
results pertaining to a small family of [d4U]-spacer-[HI-236] dou-
ble-drugs as well as a phosphoramidate pro-drug of one of them.
Not withstanding the different (C-5 vs N-3) attachments to the
individual nucleosides, the ‘para-like’ configuration of the tethers
attached to the piperazine nitrogens in Ladurée’s case was felt to
be extremely significant. In HI-236, the piperazine ring of the tro-
virdine derivative is replaced by a trisubstituted phenyl group in
which the C-5 (see Fig. 1 for numbering) methoxy group meta to
2. Double-drug design aspects
Regarding design aspects, d4U was chosen as the nucleoside in
view of its synthetic versatility as well as its proven activity (as a
d4T unit if not substituted at C-5 in the double-drug) in other dou-
ble-drug systems.11 A water-friendly PEG (polyethylene glycol)
spacer that could be varied in length and easily introduced via
standard substitution methodology was chosen for the tether.
The choice of HI-23618 as the NNRTI has been delineated earlier17
but essentially, apart from its potency, synthetic accessibility and
ease of manipulation, its flexibility as a second-generation NNRTI
was considered to be a crucial parameter for achieving communi-
cation between the two sites. Regarding the all-important issue of
the HI-236 thiourea tether at C-1 forges an important C–H/p inter-
action with the conserved Trp229 residue at the back of the NNRTI
pocket. This allows the C-2 phenolic methoxyl to point its methyl
group down towards the floor of the cavity where residues like
V106 reside. Modeling reported in our 2008 paper17 illustrates
such an arrangement as shown in Figure 2 (seen from either end
of the pocket) for a derivative in which the C-2 methyl is replaced
by a methoxycarbonylmethylene grouping. Importantly, it sug-
gests that a C-2 ortho-substituent to the C-1 thiourea tether allows
L 234
W 229
W 229
L 234
H 235
L 100
L 100
F 227
K 101
K 101
F 227
H 235
Y 181
Y 188
Y 181
V 106
Y 188
V 179
V 179
V 106
S
O
N
NH
H
O
MeO
N
OMe
Br
Figure 2. A C-2 O-alkylated HI-236 derivative modeled in the NNRTI pocket.17

Y. Younis et al. / Bioorg. Med. Chem. 18 (2010) 4661–4673
4663
Figure 3. TMC125 in the NNRTI pocket21 showing Y181, Glu138 and Val179 compared to our modeling17 of an HI-236 derivative from Figure 2.
d4U-spacer-HI-236
O
O
NHBoc
HN
NHBoc
OMe
I
HN
spacer
O
O
O
n
O
BzO
11
O
N
+
BzO
N
O
O
OMe
7
Figure 4. Retrosynthetic analysis of the d4U-spacer-HI-236 target.
an exit possibility from the pocket and may well explain why Ladu-
ree’s ‘para-like’ arrangement on the piperazine in his d4U-spacer-
trovirdine derivative double-drug failed to accommodate the teth-
ered grouping to the nucleoside into the pocket. In this regard, one
must bear in mind the importance of the directing role that the
bromopyridyl group of the thiourea plays via tight hydrogen bond-
ing towards the front of the pocket in Wing 1 with K101.17,18a
Thus, based on these ideas and coupled with results from the
earlier work mentioned previously,16,17 we thought it feasible that
a tethered [d4U]-spacer-[HI-236] might exit the pocket into the
solvent channel close to Glu13821 (p51 sub-unit shown in Fig. 3)
and preferably closer to Tyr181 rather than Val179. On the
assumption that the NNRTI would bind first, the NRTI of the dou-
ble-drug would have to make its way to the substrate binding site
around the corner behind the hydrophobic back of the NNRTI pock-
et near to the conserved Trp229. Figure 3 depicts the NNRTI
TMC12521 bound into the NNRTI pocket and helps to clarify this
important issue.
the HI-236 thiourea functionality late, and this approach gratify-
ingly turned out to be successful. Thus, the synthesis involved syn-
thesis of two halves, a coupling step and an end-game as intimated
in the retrosynthetic analysis shown in Figure 4.
For the right-hand tethered derivatives 7, the synthesis started
with commercially available 2-hydroxy-5-methoxybenzaldehyde
1, which following a three-step sequence described by Glen-
non16,22 involving phenolic hydroxyl protection with benzyl, a
Henry aldol reaction and LAH-mediated reduction of both the dou-
ble bond and the nitro group afforded amine 2 in gram quantities,
Scheme 1. For practical reasons it was easier to isolate 2 as its
N-Boc derivative 3 via a standard Boc protection step without using
DMAP, which promoted di-Boc derivatisation. A 50% overall yield
for the four-step sequence to afford 3 could be achieved on
scale-up.
Conversion of 3 to the tethered propargylated N-Boc derivatives
7 (Scheme 2) has been described previously17 and was found to be
optimal by propargylating in the final step. Thus, hydrogenolytic
debenzylation of 3 to phenol 4 followed by alkylation with a
monobenzyl-protected PEG bromide using sodium hydride in
THF or potassium carbonate as base in acetonitrile at reflux gave
the protected PEG-alkylated phenols 5a–c. A second debenzylation
followed by tosylation of the primary hydroxyl group gave tosy-
lates 6a–c. Finally, nucleophilic displacement by an excess of prop-
argyloxide ion returned the best results overall for obtaining the
final propargylated products 7b–d, which were obtained as shown
in Scheme 2, with the 4-PEG derivative considered to be well with-
in striking distance of the substrate binding site. Each compound
7b–d was exhaustively characterized17 using 1H, 13C NMR spec-
troscopy as well as high resolution mass spectrometry (HRMS)
and/or CHN combustion analysis. 13C NMR spectroscopy was par-
ticularly useful for identifying the carbons of the PEG units to con-
firm that the correct length of tether was in place in each case,
while 1H NMR spectroscopy identified a characteristic fingerprint
for the triad of aromatic signals with well-defined coupling rela-
tionships. Finally, the propargyl group could be easily discerned
in both types of spectra. The exception in the library was the deriv-
ative without a PEG prepared by alkylation of phenol 4 with prop-
argyl bromide, in which it was found that transformation into the
3. Chemistry
Typical of the art of total synthesis, the timing of key bond con-
nections in the synthesis of the double-drugs proved to be crucial.
A completely convergent synthesis via coupling of a tethered
HI-236 alkyne to a protected derivative of 5-iodo-d4U using a
Sonogashira Pd(0) coupling as the key and final step failed to give
a significant yield of product, presumably due to interference from
the nucleophilic thiourea sulfur. Thus, it was deemed necessary to
bring the key coupling step forward in the sequence and introduce
OBn
OH
OBn
NH2
CHO
NHBoc
i, ii, iii
iv
2
3
1
OMe
OMe
OMe
Scheme 1. Reagents and conditions: (i) BnBr, K2CO3, EtOH, 80 °C; (ii) CH3NO2,
NH4OAc, 70 °C; (iii) LiAlH4, THF, 70 °C; (iv) (Boc)2O, CH3CN, rt.

4664
Y. Younis et al. / Bioorg. Med. Chem. 18 (2010) 4661–4673
O
n
O Ts
n
O Bn
n
7a
i, ii or iii
OR
O
O
O
m
NHBoc
NHBoc
NHBoc
NHBoc
i, iv
v
7a: m = 2, n = 0
7b: m = 1, n = 1
7c: m = 1, n = 2
6a: n = 1
6b: n = 2
6c: n = 4
5a: n = 1
5b: n = 2
5c: n = 4
3, R = Bn
4, R = H
OMe
OMe
OMe
OMe
7d: m = 1, n = 4
Scheme 2. Reagents and conditions: (i) H2, Pd/C, EtOH, rt; (ii) 3-butynyl-1-tosylate, K2CO3, CH3CN, 80 °C; (iii) Bn(PEG)nBr (n = 1, 2, 4), K2CO3, CH3CN, 80 °C or NaH, DME,
80 °C; (iv) TsCl, Et3N, DMAP, CH2Cl2, 0 °C–rt; (v) propargyl alcohol, NaH, THF, 70 °C.
final double-drug via Pd(0) coupling and thiourea introduction re-
sulted in an unstable product following debenzoylation, presum-
ably as a result of having the two drugs in close proximity as
well as the reactive nature of the propargylic linker. Fortunately,
the stability of the final double-drug could be improved by extend-
ing the propargyl tether by one carbon atom using 3-butynyl-1-
tosylate in the alkylation to afford intermediate compound 7a as
shown in Scheme 2.
In the event, Pd(PPh3)4 was chosen as the Pd(0) catalyst. Gener-
ally, using Pd(PPh3)4 (10 mol %) with CuI (50%) at a higher ratio
than normal with alkyne at 1.1 equiv in a deoxygenated DMF/
THF (1:2) solvent medium furnished coupled products 12a–d by
tlc within 3–4 h at rt. Following the usual extractive work-up with
aq disodium EDTA to remove metal salts, chromatography fur-
nished the requisite coupled products in around a 70% isolated
yield. Product integrity could be relatively easily discerned by
observing representative resonances for each coupling partner in
the 1H or 13C spectra. Importantly, the two NH signals could be dis-
cerned in the 1H NMR spectra (in CDCl3) at 8.50–9.00 for the
pyrimidine signal and close to 5.00 ppm for the carbamate NH.
Diagnostic peaks for key functional groups such as the triple bond
could be identified in the IR spectrum, and HRMS using electro-
spray returned correct molecular ions in each case, Scheme 4.
The end-game to the double-drug targets involved a three-step
sequence involving Boc-deprotection, thiourea coupling and ben-
zoate deprotection to the free nucleoside. Some concern was enter-
tained regarding the two deprotection steps, particularly the first
one involving trifluoroacetic acid as the standard reagent for Boc-
deprotection. In the event, following some exhaustive optimiza-
tion, it was established that exposing products 12a–d to TFA at
0 °C in DCM resulted in deprotection in about 2 h to a more polar
amine spot on TLC. In view of the anticipated water solubility of
the amine, the final step was conducted without using an aqueous
work-up. Thus, following addition of Hünig’s base (EtN(i-Pr)2) and
the complete removal of all volatiles, the residue was redissolved
in THF and the thiocarbonyl reagent 13 added according to the ori-
ginal procedure33 described by the Eli Lilly group in their work on
PETT NNRTIs. Reagent 13 could be readily prepared by reacting 2-
amino-5-bromopyridine with 1,10-thiocarbonyldiimidazole in ace-
tonitrile at room temperature for 12 h to afford a precipitate that
was used without purification. In our case, condensation between
the amine and 13 could be realized in THF or DMF at room temper-
ature overnight to afford the final double-drug targets 14 as their
50-benzoate esters in about 60% over the two steps following chro-
matography. The lower temperature for condensation with 13
compared to the 100 °C (in DMF) used33 by the Eli-Lilly group
made a significant improvement to the overall yield. Thereafter,
methoxide-catalysed benzoate deprotection in MeOH at 0 °C fur-
nished the final targets 15. Of crucial importance in this step in or-
For synthesis of the left-hand nucleoside partner, advantage
was taken of the elegant Bristol-Myers Squibb process for convert-
ing 5-methyluridine into d4T23 based on seminal work by Fox
involving uridine.24 This methodology could be carried out effi-
ciently on a several-gram-scale with a single clean-up chromatog-
raphy step at the end and was superior to other methodologies25
such as that using acetyl bromide.26 Thus, mesylation of the hydro-
xyl groups of uridine, followed by heating the product with sodium
benzoate in acetamide furnished the 20-anhydro nucleoside 8.24b
Subsequent opening using HBr (from AcBr in MeOH) to bromom-
esylate 9 followed by zinc-mediated elimination furnished the 50-
benzoate of d4U 1026a as a crystalline solid. Interestingly, it was
found that the zinc-elimination could be accelerated using 2% v/v
concd HCl in the methanol solvent, presumably as a result of disso-
lution of surface oxide exposing metal surface, Scheme 3. Finally,
iodination of 10 was accomplished using elemental iodine and ce-
ric ammonium nitrate (CAN) in acetonitrile according to the proce-
dure by Robins.27 Running the reaction at around 40 °C for about
five hours secured a high yield of the 5-iodo derivative 11 after
chromatography (82%) with minimal by-product formation involv-
ing iodination of the dideoxyribose double bond.
With the two coupling partners 7 and 11 in hand, attention was
turned towards the key Sonogashira coupling. Literature precedent
for such a reaction is well established following early work by Rob-
ins28 and others,29,30 and the topic for nucleosides has been re-
cently reviewed.31 Generally, the most encountered conditions
use either Pd(PPh3)4 and Pd(PPh3)2Cl2 as catalysts in varying
mol % (around 20%) in deoxygenated DMF as solvent with triethyl-
amine as base and with CuI in a 2:1 ratio to Pd catalyst. Excess
alkyne ensures complete conversion of nucleoside in view of
competing Glaser-type coupling of the alkyne, and one may form
a
furanopyrimidine cyclization by-product post Sonogashira
coupling depending on the catalyst used.32
O
O
O
O
HN
O
N
HN
HN
R
O
O
HO
BzO
O
BzO
N
BzO
N
N
N
O
iv
i, ii
O
iii
O
O
10, R = H
11, R = I
HO OH
8
MsO
9
MsO Br
v
Scheme 3. Reagents and conditions: (i) MsCl (3 equiv), pyr, 0 °C; (ii) NaBz (3 equiv), acetamide, 120 °C; (iii) CH3COBr (5 equiv), EtOAc/MeOH (10:1), 70 °C; (iv) Zn, concd HCl
(2% by vol), MeOH, rt; (v) CAN (0.6 eq), I2 (0.6 eq), CH3CN, 35 °C.

Y. Younis et al. / Bioorg. Med. Chem. 18 (2010) 4661–4673
4665
NHBoc
OMe
O
O
O
O
O
HN
n
HN
I
m O
m
NHBoc
n
O
O
i
+
BzO
BzO
N
N
O
O
7a: m = 2, n = 0
7b: m = 1, n =1
7c: m = 1, n =2
7d: m = 1, n =4
12a: m = 2, n = 0
12b: m = 1, n =1
12c: m = 1, n =2
12d: m = 1, n =4
11
OMe
Scheme 4. Reagents and conditions: (i) Pd(PPh3)4 (10 mol %), CuI (50 mol %), Et3N (2 equiv), DMF/THF (1:2), rt.
S
N
H
NH
Br
NHBoc
OMe
O
O
O
O
HN
HN
m O
N
m O
i, ii
n
n
O
O
RO
BzO
N
OMe
N
O
O
12a-d
Br
14a-d, R = Bz
15a-d, R = H
N
S
iii
N
H
N
13
N
Scheme 5. Reagents and conditions: (i) TFA, CH2Cl2, 0 °C; (ii) EtN(i-Pr)2 followed by 13, THF, rt/overnight; (iii) NaOMe, MeOH, 0 °C.
der to avoid degradation was the mode of isolation, for which the
best procedure turned out to be quenching with a minimal amount
of glacial acetic acid followed by direct rapid flash-chromatogra-
phy. In such a way one could minimise cleavage of the nucleoside
moiety. Once isolated, though, the double-drugs were stable
enough for biological testing and evaluation purposes. All final
double-drug products were solids that could be exhaustively char-
acterised by a full complement of spectroscopic techniques (see
Section 5). However, their recrystallization tended to promote
some ribose nucleoside cleavage, so HRMS (ES) was used to suc-
cessfully provide an accurate molecular ion in each case, Scheme 5.
The one derivative that eluded realization was the 4-PEG deriv-
ative 15d, as the product following TFA deprotection and conden-
sation with 13 was found to lack the sugar ring by NMR
spectroscopy. Since Sonogashira reactions are well known34 to pro-
ceed in the presence of unprotected hydroxyl or amino groups, it
was decided to revise the order of events in the sequence and
deprotect the Boc-protecting group of alkyne 7d first. Thereafter,
following neutralization of TFA using K2CO3 in methanol and filtra-
tion of the salts the crude amine was subjected to the Sonogashira
reaction and thiourea condensation steps to afford the 4-PEG dou-
ble-drug 14d as its 50-benzoate in 40% yield over the three steps
after chromatography. Deprotection with methoxide as usual fur-
nished the final free nucleoside double-drug 15d in a modest yield
of 52%, Scheme 6.
As a final piece of synthesis, it was decided to prepare a pronu-
cleotide of 15d as the double-drug most likely to reach the DNA
site in view of the 4-PEG spacer. The pronucleotide approach for
enhancing nucleoside activity by bypassing the first rate-determin-
ing kinase-mediated phosphorylation step is well established with
several variants on masked phosphate groupings. CycloSal35 and
phosphoramidate36 functionalities provide two of the most fre-
quently used options and we chose the latter in view of its proven
ability to enhance the activity of d4T37 as well as the likelihood of
accessing it from the nucleoside double-drug 15d in a single step.
However, when 15d was reacted with p-tolyl methoxyalaninyl
phosphorochloridate prepared38 according to a McGuigan pub-
lished procedure with N-methylimidazole as a transfer base, no
phosphoramidate could be isolated and only breakdown of the
double-drug was observed by TLC. Given the relative robustness
of the phosphoramidate grouping, it was thought that it could re-
place benzoate in the Sonogashira coupling. However, given that
we doubted its stability towards TFA, we adopted the strategy
developed for the 4-PEG double-drug 15d. Thus, Boc-deprotection
of N-Boc alkyne 7d to the free amine as described before in
Scheme 6 followed by Sonogashira coupling of the resultant amine
with the phosphoramidate of 5-iodo-d4U 16 prepared from 5-
iodo-d4U by a literature method,39 and finally coupling of the ami-
no group of the coupled product with 13 gave the target thiourea
double-drug phosphoramidate 17 as a nearly 50:50 mixture of dia-
stereomers at phosphorus in an overall yield of 20% for the three
steps, Scheme 7. Given our experience in deriving full assignments
The 1H NMR and 13C NMR spectra illustrated in Figure 5 for 15d
showing all of the required signals gave us great satisfaction.
S
N
H
NH
Br
O
O
O
O
HN
4
O
N
NHBoc
4
i, ii, iii,
O
RO
N
OMe
O
14d, R = Bz
15d, R = H
7d
OMe
iv
Scheme 6. Reagents and conditions: (i) TFA, CH2Cl2, 0 °C; (ii) 11, Pd(PPh3)4 (10 mol %), CuI (50 mol %), Et3N (2 equiv), DMF/THF (1:2), rt; (iii) 13, THF, rt; (iv) NaOMe (cat),
MeOH, 0 °C.

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Y. Younis et al. / Bioorg. Med. Chem. 18 (2010) 4661–4673
Figure 5. 400 MHz 1H NMR and 75 MHz 13C NMR spectra of 15d in DMSO-d6.
for the other double-drugs, 17 could be exhaustively characterized
using a combination of both 1D and 2D 1H, 13C and 31P NMR spec-
troscopic techniques in spite of the diastereomeric mixture.
activity to the NNRTI component. The compound was ca. twofold
less potent than HI-236 in the RT assay with IC50 values of HI-236
and 15a (38 and 61 nM respectively) both improving relative to
the cell-culture EC50 results. This was not unexpected in view of as-
pects of cell permeability and the greater possibility for degradation
in the cell-culture experiment. Lengthening the spacer resulted in a
4. Biological results and discussion
steady reduction in activity (0.25, 1.3, 1.9 and 3.1
respectively) in cell-culture. Notably, the 4-PEG derivative 15d still
retained appreciable activity (3.1 M), remarkably so for such a
large molecule. As with 15a, the RT IC50 values for 15b–d were sim-
ilarly lower than their EC50 cell-culture values, with nanomolar
activities for 15b and 15c. Disappointingly, the 4-PEG phosphoram-
lM for 15a–d
The inhibitory activities of the bifunctional compounds 15a–d
and phosphoramidate 17 together with HI-236 and d4T as controls
were measured against HIV-1 (IIIB) replication in MT-2 cell culture
using an MTT assay.40 The same compounds were also evaluated
for their in vitro activity against RT directly in a steady-state RT inhi-
bition assay using a D23/D36 primer/template in which the inhibi-
tion of incorporation of thymidine triphosphate (TTP) by each
double-drug was measured as an IC50. The results are shown in Table
l
idate 17 showed no significant improvement (3.1 to 2.9 lM) in cell
culture compared to its unphosphorylated counterpart 15d, effec-
tively indicating that triphosphorylation of pro-drugs 15a–d and
17 in cell-culture does not occur. The superior activity of the 4-
PEG double-drug nucleoside 15d in the RT assay compared with that
1 expressed in
inhibitory activity with an EC50 = 250 nM in the cell-culture assay as
nine times more potent than d4T (EC50 = 2.3 M) alone, and ca. six
times less potent than HI-236 (EC50 = 0.042 M) and thus closer in
lV units. [d4U]-Butyne-[HI-236] 15a showed a good
l
l
of its phosphoramidate 17 (1.4 lM vs 2.3 lM) suggests binding of

Y. Younis et al. / Bioorg. Med. Chem. 18 (2010) 4661–4673
4667
S
N
N
H
NH
Br
O
O
O
HN
O
O
O
4
4
NHBoc
O
P
O
i, ii, iii
N
OMe
p-TolO
O
NH
17
O
MeO2C
7d
OMe
HN
I
O
P
NH
O
O
= 16
N
p-TolO
O
MeO2C
Scheme 7. Reagents and conditions: (i) TFA, CH2Cl2, 0 °C; (ii) 16, Pd(PPh3)4 (10 mol %), CuI (50 mol %), Et3N (2 equiv), DMF/THF (1:2), rt; (iii) 13, THF, rt.
Table 1
A comparison of cell-culture versus in vitro HIV-1 RT inhibition (
l
M) for double-drugs 15a–d and 17
RT assayc
(lM)
a
b
Compound
Cell-culture EC50
(
lM)
Cell-culture CC50
(lM)
HI-236
d4T
15a
15b
15c
0.042
2.3
0.25
1.3
1.9
3.1
>1
>100
17
38
43
0.038 0.007
9.6d
0.061 0.015
0.575 0.14
0.850 0.14
1.4 0.5
15d
17
18
11
2.9
2.3 0.8
O
OH
HN
12041
N/A
O
RO
N
O
18
a
b
c
Effective concentration that inhibits viral-mediated T-cell death by 50%, determined by averaging samples of each concentration in triplicate.
Concentration that kills 50% of the T-cells, also determined by averaging triplicate samples.
Concentration of inhibitor that inhibits by 50% the steady-state thymidine incorporation into a D23/D36 primer-template as catalysed by RT.
Based on pre-steady-state kinetic analysis.
d
the 50-hydroxyl group of 15d, which is considered to likely be taking
place near to or at the substrate binding site.
that phosphoramidate 17 is hydrolysed to such a monophosphate
within the cell-culture as a by-pass of the first rate-limiting phos-
phorylation. On a positive note, our results suggest that it might be
worthwhile to consider designing extended NNRTI inhibitors con-
taining a second binding agent out of but near to the front of the
pocket. Given the plethora of X-ray structures of NNRTIs avail-
able,4a it should be possible to model certain residues for this pur-
pose, for example, Glu138 on the outside of the pocket.10 The
original double-drug suggestion by Arnold5 and Steitz6 did not
consider how the two drug-sites might communicate structurally
in practice. Regarding the kinase recognition problem just men-
tioned, targeting protected triphosphates of double-drugs would
present some serious challenges regarding their synthesis and sta-
bility. Moreover, communication between the two sites is made
difficult if the intention is for the tether to exit the NNRTI binding
pocket, since the front of the pocket points away from the sub-
strate binding site. However, the possibility that the two drugs
may communicate between the two sites by virtue of the tether
exiting the NNRTI pocket from the back end close to Trp 229 pro-
vides a more direct connection. Results on this will be communi-
cated in due course involving the diarylpyrimidine TMC-120 as
the NNRTI component of such a double-drug system.
Our results indicate that the activity of double-drugs 15a–d and
17 is mainly due to the NNRTI component, with the size of the dou-
ble-drugs suggesting that 15b–d and 17 have the nucleoside drug
outside of the NNRTI pocket. Although the data does not allow a
firm conclusion to be made regarding the possibility or nature of
NRTI binding possibilities, the relatively potent cell-culture EC50
values of 1.3
l
M and 1.9
lM for 1-PEG and 2-PEG derivatives
15b,c respectively (more active than d4T alone (2.3
lM)) strongly
suggests the possibility of some cooperative binding outside of
the pocket. If this is the case, the fact that Ladurée has shown
the 5-alkynylated analogue 1841 (Table 1) to be completely inac-
tive would support the view that the nucleoside part of the dou-
ble-drugs 15b,c may well forge cooperative interactions away
from the substrate binding site, although derivatives 15d and 17
containing the longer 4-PEG tether could well be interacting coop-
eratively near to the binding site based on anchoring from the
NNRTI pocket. Such conclusions echo those of Monneret and co-
workers based on the activities of their AZT-tether-HEPT bifunc-
tionals in which activities also suggested out-of-pocket binding
but without synergy between drug-sites, and to a lesser extent to
those of Camarasa based on her nucleoside-spacer-TSAO double-
drugs, since TSAO supposedly binds just outside of the conven-
tional NNRTI pocket. The failure to achieve synergy in the
double-drugs 15a–d as well as the other systems mentioned may
be mainly ascribed to the inability of cellular kinases to recognize
the nucleoside portion of the double-drug.39a This conclusion
unfortunately also applies to the monophosphate of 15d, assuming
5. Experimental
5.1. General procedures for synthesis
Infrared (IR) absorptions were measured on a Perkin–Elmer
Spectrum One FT-IR spectrometer. 1H NMR spectra were recorded

4668
Y. Younis et al. / Bioorg. Med. Chem. 18 (2010) 4661–4673
on a Varian Mercury Spectrometer at 300 MHz or a Varian Unity
Spectrometer at 400 MHz with Me4Si as internal standard. 13C
NMR spectra were recorded at 75 MHz on a Varian Mercury Spec-
trometer or at 100 MHz on Varian Unity Spectrometer with Me4Si
as internal standard. High resolution mass spectra were recorded
on a VG70 SEQ micromass spectrometer. Melting points were
determined using a Reichert-Jung Thermovar hot-stage microscope
and are uncorrected. Analytical thin-layer chromatography (TLC)
was performed on aluminium-backed Silica-Gel 60 F254 (70–230
mesh) plates. Column chromatography was performed with Merck
Silica-Gel 60 (70–230 mesh).
and stirring mixture of phenol 4 (0.45 g, 1.68 mmol) and anhy-
drous potassium carbonate (0.93 g, 6.70 mmol) in dry acetonitrile
(20 mL). After 20 h the mixture was filtered, the solvent evapo-
rated and the residue subjected to silica-gel column chromatogra-
phy using EtOAc/petroleum ether (1/9) to give 5b as a colourless oil
(0.61 g, 81%.); IR (CHCl3): mmax 3442, 3377 (NH, OH), 3007, 2928
(C–H), 1697 (C@O), 1501 (C@O), 1227 (C–N) cmꢁ1 1H NMR
;
(400 MHz, CDCl3): d 7.31 (4H, m), 7.21 (1H, m), 6.76 (1H, d,
J = 8.7 Hz), 6.70 (1H, d, J = 2.8 Hz), 6.68 (1H, dd, J = 2.8, 8.7 Hz),
4.83 (1H, br s, NH), 4.55 (2H, s), 4.06 (2H, t, J = 4.8 Hz), 3.82 (2H,
t, J = 4.8 Hz), 3.72 (3H, s), 3.70 (2H, m), 3.62 (2H, m), 3.31 (2H, q,
J = 6.5 Hz), 2.76 (2H, t, J = 6.5 Hz), 1.38 (9H, s); 13C NMR
(100 MHz, CDCl3): d 156.0 (C@O), 153.8, 151.0, 138.2, 129.4,
128.4, 127.7, 127.6, 116.6, 113.1, 112.0, 78.8, 73.3, 70.8, 69.9,
69.5, 68.5, 55.6, 40.7, 31.0, 28.4; HRMS (EI): m/z found 445.24814
(M+). C25H35NO6 (M+) requires 445.24644.
Compounds 2,22 5a,17 6a,17 6b,17 7b,17 7c,17 and 1333 have all
been reported previously. 3-Butynyl 1-p-toluenesulfonate42 was
prepared from the alcohol by a standard tosylation procedure.
The monobenzyl PEG-Br alkylating agents for formation of 5b
and 5c were prepared by standard mono-benzylation and bromin-
ation (using PPh3 and CBr4) methods from the corresponding
glycol.
5.2.4. N-[2-(2-(11-Benzyloxy-3,6.9-trioxaundec-1-yloxy)-5-meth-
oxyphenyl)ethyl]-tert-butylcarbamate 5c
5.2. Synthesis of intermediates for the coupling partners 7 and
11
11-Benzyloxy-1-bromo-3,6.9-trioxaundecane (0.91 g, 2.62 mmol)
in dry acetonitrile (10 mL) was added dropwise over 1 h to a reflux-
ing and stirring mixture of phenol 4 (0.35 g, 1.31 mmol) and anhy-
drous potassium carbonate (0.72 g, 5.20 mmol) in dry acetonitrile
(20 mL). After 20 h the mixture was filtered, the solvent evaporated
and the residue subjected to silica-gel column chromatography
using EtOAc/petroleum ether (1/9) to give 5c as a colourless oil
(0.56 g, 80%.); IR (CHCl3): mmax 3681, 3449 (N–H), 3021, 2913 (C–
5.2.1. N-[2-(2-Benzyloxy-5-methoxyphenyl)ethyl]-tert-butylcar-
bamate 3
Di-tert-butyldicarbonate (5.22 g, 23.93 mmol) in acetonitrile
(6 mL) was added to a solutionof crude amine 2 (4.10 g, 15.95 mmol)
in acetonitrile (50 mL) and the reaction mixture was stirred at rt for
20 h. Aqueous NH4Cl (100 mL) was added and the organic material
extracted into EtOAc (3 ꢀ 100 mL). Following drying and evapora-
tion of solvent the residue was subjectedto column chromatography
employing EtOAc/petroleum ether (15/85) to give carbamate 3 as
colourless crystals (5.00 g, 88%); mp: 102–104 °C; IR (CHCl3): mmax
3449 (NH), 3007, 2935 (C–H), 1703 (C@O), 1501 (C@C), 1165 (C–
H), 1700 (C@O), 1501 (C@C) cmꢁ1 1H NMR (400 MHz, CDCl3): d
;
7.34 (4H, m), 7.21 (1H, m), 6.78 (1H, d, J = 8.8 Hz), 6.72 (1H, d,
J = 3.0 Hz), 6.70 (1H, dd, J = 3.0 Hz, 8.8 Hz), 4.90 (1H, br s, NH), 4.57
(2H, s), 4.08 (2H, t, J = 4.9 Hz), 3.83 (2H, t, J = 4.9 Hz), 3.76 (3H, s),
3.72–3.68 (10H, m), 3.64 (2H, m), 3.35 (2H, q, J = 6.7 Hz), 2.80 (2H,
t, J = 6.7 Hz), 1.43 (9H, s); 13C NMR (100 MHz, CDCl3): d 155.9
(C@O), 153.7, 151.0, 138.3, 129.3, 128.3, 127.6, 127.5, 116.6, 113.0,
111.9, 78.7, 73.1, 78.7, 70.6, 70.6, 69.8, 69.4, 68.4, 55.6, 40.6, 31.0,
28.4; HRMS (ES): m/z found 534.3036 (M++H). C29H44NO8 requires
(M++H) 534.3067.
N) cmꢁ1 1H NMR (400 MHz, CDCl3): d 7.45–7.34 (5H, m,), 6.85
;
(1H, d, J = 8.8 Hz,), 6.76 (1H, d, J = 2.8 Hz), 6.72 (1H, dd, J = 2.8,
8.8 Hz), 5.04 (2H, s,), 4.70 (1H, br s, NH), 3.78 (3H, s), 3.40 (2H, q,
J = 6.4 Hz), 2.85 (2H, t, J = 6.4 Hz), 1.44 (9H, s); 13C NMR (75 MHz,
CDCl3): d 155.9 (C@O), 153.8, 150.9, 137.4, 129.2, 128.5, 127.8,
127.2, 116.8, 113.0, 112.0, 79.0, 70.8, 55.7, 40.8, 30.9, 28.4; HRMS
(EI): m/z found 301.13383 [(M+ꢁt-butyl)+H]. C17H19NO4 requires
301.13409 [(M+ꢁt-butyl)+H]; C21H27NO4 requires C, 70.56; H,
7.61; N, 3.92. Found: C, 70.50; H, 7.62; N, 3.86.
5.2.5. N-[2-(2-(11-p-Toluenesulphonyloxy-3,6.9-trioxaundec-1-
yloxy)-5-methoxyphenyl)ethyl]-tert-butylcarbamate 6c
Triethylamine (0.18 mL, 1.36 mmol) was added to a stirring
solution in dry CH2Cl2 (5 mL) at 0 °C of the alcohol (0.50 g,
1.13 mmol) obtained from hydrogenolysis of 5c. p-Toluenesulfonyl
chloride (0.26 g, 1.36 mmol) was added together with a catalytic
amount of DMAP (20 mg). The reaction mixture was stirred at rt
for 16 h, then diluted with CH2Cl2 (80 mL) and the organic layer
washed with aq NH4Cl (25 mL), water (30 mL), dried over MgSO4
and the solvent removed under reduced pressure. Purification by
column chromatography EtOAc in petroleum ether (8/2) gave 6c
as a colorless oil (0.58 g, 84%); IR (CHCl3): mmax 3377 (NH), 2928
5.2.2. N-[2-(2-Hydroxy-5-methoxyphenyl)ethyl]-tert-butylcarba-
mate 4
The carbamate 3 (4.50 g, 12.61 mmol) was added to 10% palla-
dium-on-carbon (0.22 g, 1.26 mmol) in ethanol. Hydrogen gas was
introduced to the reaction at rt for 5 h. The palladium was filtered
from the solution through Celite and the precipitate was washed
with ethanol (3 ꢀ 100 mL). The solvent was removed under reduced
pressure and the residue was subjected to column chromatography
using EtOAc/petroleum ether (4/6) to give 4 as a colourless solid
(2.50 g, 74%); mp: 115–117 °C; IR (CHCl3): mmax 3457, 3326 (NH,
(C–H), 1707 (C@O), 1501 (C@C), 1223 (C–N), 1176 (O–SO2–) cm
ꢁ1
;
1H NMR (300 MHz, CDCl3): d 7.77 (2H, d, J = 8.4 Hz), 7.31 (2H,
d, J = 8.4 Hz), 6.76 (1H, d, J = 9.2 Hz), 6.68 (2H, m), 4.85 (1H, br s,
NH), 4.14 (2H, t, J = 4.8 Hz), 4.06 (2H, t, J = 4.8 Hz), 3.81 (2H, t,
J = 4.8 Hz), 3.73 (3H, s), 3.69–3.57 (10H, m), 3.32 (2H, q,
J = 6.5 Hz), 2.77 (2H, t, J = 6.5 Hz), 2.42 (3H, s), 1.40 (9H, s); 13C
NMR (75 MHz, CDCl3): d 155.9 (C@O), 153.8, 151.0, 144.7, 133.0,
129.7, 129.3, 127.9, 116.6, 113.0, 111.9, 78.8, 70.7 (3 ꢀ CH2O),
70.5 (CH2O), 69.8 (CH2O), 69.2 (CH2O), 68.6 (CH2O), 68.4 (CH2O),
55.6, 40.6, 31.0, 28.4, 21.5; HRMS (ES): m/z found 598.2673
(M++H), C29H44NO10S requires (M++H) 598.2686.
OH), 1686 (C@O), 1508 (C@C), 1210, 1166 (C–N) cmꢁ1 1H NMR
;
(400 MHz, CDCl3): d 6.99 (1H, br s, OH), 6.79 (1H, d, J = 8.4 Hz),
6.67 (1H, d, J = 2.8 Hz), 6.65 (1H, m), 5.00 (1H, br s, NH), 3.75 (3H,
s), 3.33 (2H, q, J = 7.1 Hz), 2.81 (2H, t, J = 7.1 Hz), 1.46 (9H, s); 13C
NMR (100 MHz, CDCl3): d 157.0 (C@O), 153.2, 148.8, 126.0, 116.5,
116.2, 112.8, 80.0, 55.8, 41.0, 31.4, 28.4; HRMS (EI): m/z found
267.14329 (M+). C14H21NO4 (M+) requires 267.14706; C14H21NO4 re-
quires C, 62.94; H, 7.92; N, 5.24. Found C, 62.82; H, 7.92; N, 5.16.
5.2.3. N-[2-(2-(5-Benzyloxy-3-oxapent-1-yloxy)-5-methoxyphen-
yl)ethyl]-tert-butylcarbamate 5b
5.2.6. N-[2-(2-(3-Butynyl-1-oxy)-5-methoxyphenyl)ethyl]-tert-
butylcarbamate 7a16
2-(2-Benzyloxy)ethoxy-1-bromoethane (0.87 g, 3.36 mmol) in
dry acetonitrile (10 mL) was added dropwise over 1 h to a refluxing
3-Butynyl-1-p-toluenesulfonate (1.34 g, 6.00 mmol), phenol 4
(0.80 g, 3.00 mmol) and anhydrous potassium carbonate (1.70 g,

Y. Younis et al. / Bioorg. Med. Chem. 18 (2010) 4661–4673
4669
12.0 mmol) were refluxed in dry acetonitrile (30 mL). After 20 h
the mixture was filtered, the solvent evaporated and the residue
subjected to silica-gel column chromatography using EtOAc/petro-
leum ether (1/9) to give to give 7a as colourless needles (0.57 g,
61%); mp: 76–77 °C; IR (CHCl3): mmax 3455 (NH), 3309 („CH),
0.1 equiv) were added to the degassed solution under a nitrogen
atmosphere. The mixture was left stirring at rt for 4 h before adding
5% aq disodium EDTA (30 mL) and extracting with CHCl3
(3 ꢀ 30 mL). Washing with water (15 mL), drying over MgSO4, filtra-
tion and solvent evaporation under reduced pressure gave a residue
that was purified by column chromatography employing EtOAc in
petroleum ether mixtures to give the product as a yellow oil.
2413 (C„C), 1707 (C@O), 1602 (C@C) cmꢁ1 1H NMR (300 MHz,
;
CDCl3): d 6.75 (3H, m), 4.70 (1H, br s, NH), 4.05 (2H, t, J = 6.8 Hz),
3.75 (3H, s), 3.36 (2H, q, J = 6.6 Hz), 2.79 (2H, t, J = 6.6 Hz), 2.66
(2H, dt, J = 2.7, 6.8 Hz), 2.03 (1H, t, J = 2.7 Hz), 1.42 (9H, s); 13C
NMR (75 MHz, CDCl3): d 155.9 (C@O), 153.9, 150.6, 129.3, 116.8,
112.8, 112.0, 80.7, 78.9, 69.8, 66.8, 55.6, 40.7, 30.9, 28.4, 19.7;
HRMS (EI): m/z found 319.17756 (M+). C18H25NO4 (M+) requires
319.17836; C18H25NO4 requires C, 67.89; H, 7.89; N, 4.39. Found:
C, 67.10; H, 7.66; N, 3.67.
5.3.1. 5-{4-[2-(2-tert-Butoxycarbonylaminoethyl)-4-methoxyphen-
oxy]but-1-ynyl}-50-O-benzoyl-20,30-didehydro-20,30-dideoxyuridine
12a
Using 11 (70 mg, 0.20 mmol) with alkyne 7a (0.10 g,
0.30 mmol) gave 12a (95 mg, 94%): [
(CHCl3): mmax 3692, 3606, 3451 (NH), 3011, 2934 (C–H), 2243
(C„C), 1707 (C@O), 1502 (C@C) cmꢁ1 1H NMR (300 MHz, CDCl3):
a]
ꢁ14.4 (c 1.60, CHCl3); IR
D
;
5.2.7. N-[2-(2-(11-Propargyloxy-3,6.9-trioxaundec-1-yloxy)-5-
methoxyphenyl)ethyl]-tert-butylcarbamate 7d
d 8.79 (1H, br s), 8.00 (2H, d, J = 7.7 Hz), 7.61 (1H, s), 7.53 (1H, t,
J = 7.7 Hz), 7.42 (2H, t, J = 7.7 Hz), 6.91 (1H, m), 6.70 (3H, m), 6.39
(1H, dt, J = 1.7, 5.8 Hz), 5.99 (1H, dq, J = 1.4, 5.8 Hz), 5.20 (1H, m),
4.83 (1H, br s), 4.64 (1H, dd, J = 4.3, 12.5 Hz), 4.50 (1H, dd, J = 3.0,
12.5 Hz), 3.94 (2H, t, J = 6.8 Hz), 3.74 (3H, s), 3.32 (2H, m), 2.77
(2H, t, J = 6.9 Hz), 2.68 (2H, t, J = 6.8 Hz), 1.40 (9H, s); 13C NMR
(75 MHz, CDCl3): d 166.2, 161.4, 153.9, 153.9, 150.5, 149.4, 141.6,
133.5, 133.3, 132.0, 129.7, 129.2, 128.6, 127.0, 116.7, 112.9,
112.0, 100.7, 91.3, 90.7, 85.0, 78.8, 72.4, 66.7, 65.1, 55.6, 40.6,
31.0, 28.4, 20.8; FAB HRMS: m/z found 654.24300 [M+Na]+.
C34H37N3O9Na [M+Na]+ requires 654.24274.
To a stirred suspension of NaH (0.12 g, 60% in mineral oil,
3.00 mmol) in THF (10 mL) at 0 °C was added propargyl alcohol
(252 mg, 4.50 mmol) dissolved in THF (2 mL) dropwise. The mix-
ture was refluxed for 30 min and a solution of 6c (435 mg,
0.73 mmol) in THF (10 mL) then added dropwise. After refluxing
for 20 h, a solution of aq NH4Cl (20 mL) was added and the organic
material extracted into EtOAc (3 ꢀ 30 mL). The combined organic
extracts were washed with water (2 ꢀ 20 mL), dried (MgSO4) and
the solvent removed to afford a residue, which was purified by col-
umn chromatography (15% EtOAc in petroleum ether) to afford 7d
(0.290 g, 83%) as a colourless oil; IR (CHCl3):
(„CH), 3007, 2920 (C–H), 2116 (C„C), 1703 (C@O), 1501 (C@C),
1227 (C–N) cmꢁ1 1H NMR (300 MHz, CDCl3): d 6.77 (1H, d,
m
max 3449 (NH), 3304
5.3.2. 5-{6-[2-(2-tert-Butoxycarbonylaminoethyl)-4-methoxy-
phenoxy]hexa-4-oxa-1-ynyl}-50-O-benzoyl-20, 30-didehydro-20,
30-dideoxyuridine 12b
;
J = 8.8 Hz), 6.68 (2H, m), 4.88 (1H, br s, NH), 4.18 (2H, d, J = 2.1 Hz),
4.07 (2H, t, J = 4.8 Hz), 3.82 (2H, t, J = 4.8 Hz), 3.74 (3H, s), 3.72–
3.64 (12H, m, 6 ꢀ CH2O), 3.34 (2H, q, J = 6.5 Hz), 2.78 (2H, t,
J = 6.5 Hz), 2.40 (1H, t, J = 2.1 Hz), 1.41 (9H, s); 13C NMR (100 MHz,
CDCl3): d 156.0 (C@O), 153.8, 151.0, 129.3, 116.6, 113.0, 112.0,
79.7, 78.8, 74.4, 70.8 (CH2O), 70.7 (CH2O), 70.6 (2 ꢀ CH2O), 70.4
(CH2O), 69.9 (CH2O), 69.1 (CH2O), 68.5 (CH2O), 58.3, 55.6, 40.6,
31.0, 28.4; HRMS (ES): m/z found 482.2732 (M++H). C25H40NO8 re-
quires (M++H) 482.2754.
Using 11 (0.30 g, 0.68 mmol) with alkyne 7b (0.26 g,
0.75 mmol) gave 12b (0.34 g, 76%): [
(CHCl3): mmax 3449, 3384 (NH), 3007, 2928 (C–H), 2246 (C„C),
1711 (C@O), 1501 (C@C), 1169 (C-N) cmꢁ1 1H NMR (300 MHz,
a] +16.2 (c 1.0, CHCl3); IR
D
;
CDCl3): d 8.42 (1H, br s), 8.01 (2H, d, J = 7.5 Hz), 7.68 (1H, s), 7.56
(1H, m), 7.45 (2H, t, J = 7.5 Hz), 6.92 (1H, m), 6.76 (1H, d,
J = 8.7 Hz), 6.68 (2H, m), 6.40 (1H, dt, J = 1.7, 6.0 Hz), 6.00 (1H,
dq, J = 1.3, 6.0 Hz), 5.21 (1H, m), 4.94 (1H, br s), 4.69 (1H, dd,
J = 4.2, 12.5 Hz), 4.50 (1H, dd, J = 3.0, 12.5 Hz), 4.27 (2H, s), 4.07
(2H, t, J = 4.5 Hz), 3.85 (2H, m), 3.74 (3H, s), 3.33 (2H, m), 2.78
(2H, t, J = 6.9 Hz), 1.42 (9H, s); 13C NMR (75 MHz, CDCl3): d 166.2,
161.3, 156.0, 153.8, 151.0, 149.4, 142.2, 133.6, 133.5, 129.7,
129.3, 129.2, 128.6, 127.0, 116.5, 113.0, 111.9, 99.9, 90.8, 90.0,
85.1, 78.8, 77.2, 68.4, 68.2, 65.0, 59.0, 55.6, 40.6, 30.8, 28.4; HRMS
(ES): m/z found 662.2698 (M++H), C35H40N3O10 requires (M++H)
662.2714.
5.2.8. 50-Benzoyl-5-iodo-d4U 11
To a solution of 50-O-benzoyl-d4U 10 (0.88 g, 2.8 mmol)26a in
CH3CN (30 mL), were added cerium ammonium(IV) nitrate (0.92 g,
1.68 mmol) and iodine (0.43 g, 1.68 mmol). The mixture was stirred
at 35 °C for 4 h before being diluted with a solution of sodium bisul-
fite (50 mL) and extracted with EtOAc (3 ꢀ 50 mL). The organic layer
was dried over MgSO4 and the solvent reduced under vacuum.
Recrystallization from CH2Cl2/pet ether gave 11 as colourless nee-
dles (1.05 g, 85%): mp 168–169 °C (EtOAc, MeOH); 1H NMR
(300 MHz, CDCl3): d 11.78 (1H, br s, NH), 7.96 (2H, d, J = 7.9 Hz),
7.73 (1H, s), 7.67 (1H, m), 7.55 (2H, t, J = 7.9 Hz), 6.76 (1H, m), 6.54
(1H, dt, J = 1.7, 6.0 Hz), 6.12 (1H, dq, J = 1.5, 6.0 Hz), 5.18 (1H, m),
4.52 (2H, m); 13C NMR (75 MHz, CDCl3): d 165.5, 160.2, 150.1,
144.0, 133.5, 133.3, 129.2, 129.0, 128.7, 126.5, 90.0, 84.0, 69.6,
65.3; HRMS (ES): m/z found 440.9930 (M++H). C16H14IN2O5 requires
(M++H) 440.9942; C16H13IN2O5 requires C, 43.12; H, 2.97; N, 6.04.
Found C, 43.66; H, 2.98; N, 6.36.
5.3.3. 5-{9-[2-(2-tert-Butoxycarbonylaminoethyl)-4-methoxy-
phenoxy]nona-4,7-dioxa-1-ynyl}-50-O-benzoyl-20, 30-didehydro-
20, 30-dideoxyuridine 12c
Using 11 (0.20 g, 0.46 mmol) with alkyne 7c (0.20 g, 0.51 mmol)
gave 12c (0.23 g, 72%): [
3674, 3377 (NH), 3014 (C–H), 2225 (C„C), 1711 (C@O), 1501
(C@C), 1212 (C–N) cmꢁ1 1H NMR (400 MHz, CDCl3): d 8.82 (1H,
a] +32.1 (c 1.0, CHCl3); IR (CHCl3): mmax
D
;
br s), 8.02 (2H, d, J = 7.6 Hz), 7.68 (1H, s), 7.56 (1H, m), 7.46 (2H,
t, J = 7.6 Hz), 6.92 (1H, m), 6.77 (1H, d, J = 8.8 Hz), 6.69 (2H, m),
6.39 (1H, dt, J = 1.5, 6.0 Hz), 5.99 (1H, dq, J = 1.3, 6.0 Hz), 5.21
(1H, m), 4.97 (1H, br s), 4.70 (1H, dd, J = 4.2, 12.4 Hz), 4.51 (1H,
dd, J = 2.9, 12.4 Hz), 4.25 (2H, s), 4.08 (2H, t, J = 4.9 Hz), 3.84 (2H,
t, J = 4.9 Hz), 3.75 (3H, m), 3.73 (4H, m), 3.35 (2H, m), 2.80 (2H, t,
J = 6.9 Hz), 1.43 (9H, s); 13C NMR (75 MHz, CDCl3): d 166.2, 161.1,
156.0, 153.7, 151.0, 149.4, 142.2, 133.5, 133.4, 129.7, 129.3,
129.2, 128.6, 127.0, 116.5, 113.0, 112.0, 99.9, 90.7, 90.1, 85.1,
78.9, 77.2, 70.5, 69.8, 69.1, 68.4, 65.0, 58.9, 55.6, 40.6, 31.0, 28.4;
HRMS (ES): m/z found 706.2958 (M++H). C37H44N3O11 requires
(M++H) 706.2976.
5.3. General procedure for Sonogashira coupling for compounds
7a–c to afford 12a–c
A solution of 50-benzoyl-5-iodo-d4U 11 (0.5 mmol, 1 equiv) in
dry DMF (2.5 mL) was added to a stirred solution of triethylamine
(1.0 mmol, 2 equiv) and alkyne 7a–d (0.6 mmol, 1.2 equiv) in THF
(4 mL). The mixture was thoroughly degassed with nitrogen for
1 h. CuI (0.25 mmol, 0.5 equiv) and Pd(PPh3)4 (0.05 mmol,

4670
Y. Younis et al. / Bioorg. Med. Chem. 18 (2010) 4661–4673
5.4. General procedure for the synthesis of thioureas 14a–d
J = 2.9 Hz), 6.77 (1H, d, J = 8.8 Hz), 6.73 (1H, dd, J = 2.9, 9.2 Hz),
6.38 (1H, dt, J = 1.6, 6.0 Hz), 5.98 (1H, m), 5.19 (1H, m), 4.67 (1H,
dd, J = 4.3, 12.5 Hz), 4.50 (1H, dd, J = 3.0, 12.5 Hz), 4.21 (2H, s),
4.03 (2H, t, J = 4.7 Hz), 3.98 (2H, q, J = 6.6 Hz), 3.82 (2H, t,
J = 4.7 Hz), 3.74 (3H, s), 3.71–3.64 (4H, m), 2.97 (2H, t, J = 6.6 Hz);
13C (75 MHz, CDCl3): d 179.2 (C@S), 166.2, 161.4, 153.6, 151.7,
151.2, 149.5, 146.6, 142.5, 141.0, 133.5, 133.4, 129.7, 129.2,
128.8, 128.6, 127.0, 117.5, 113.5, 112.7, 112.5, 111.5, 100.0, 90.8,
90.1, 85.1, 77.0, 70.6, 69.8, 69.2, 68.4, 65.1, 59.0, 55.5, 45.6, 29.8;
HRMS (ES): m/z found 820.1638 (M++H). C38H39N5O9SBr requires
(M++H) 820.1652.
Trifluoroacetic acid (0.20 mL) was added to a solution of com-
pounds 12a–c (0.2 mmol) in CH2Cl2 (3 mL) at 0 °C, and the solution
stirred for 2 h. Diisopropylethylamine (0.30 mL) was added, the
solvent evaporated in vacuo and the crude amine dried under vac-
uum for 1 h, after which it was dissolved in THF (5 mL), thiourea 13
(0.24 mmol, 1.2 equiv) added and the reaction mixture stirred at
room temperature for 20 h. Following evaporation of the solvent,
the residue was subjected directly to column chromatography
using (EtOAc in pet ether = 7:3) to give the desired products
14a–c as pale-yellow solids.
5.4.4. 5-{15-[2-(2-(5-Bromo-2-pyridinylaminothiocarbonylamino)-
ethyl)-4-methoxyphenoxy]pentadeca-4,7,10,13-tetraoxa-1-ynyl}-
50-O-benzoyl-20,30-didehydro-20,30-dideoxyuridine 14d
5.4.1. 5-{4-[2-(2-(5-Bromo-2-pyridinyl)aminothiocarbonylamino)-
ethyl)-4-methoxyphenoxy]but-1-ynyl}-50-O-benzoyl-20,30-dide-
hydro-20,30-dideoxyuridine 14a
Trifluoroacetic acid (0.20 mL) was added to a solution of alkyne
7d (0.12 g, 0.25 mmol) in CH2Cl2 (3 mL) at 0 °C, and the solution
stirred for 2 h. Anhydrous K2CO3 (0.10 g, 0.75 mmol) was added,
the mixture stirred for a further 15 min then filtered through Cel-
ite. Solvent evaporation in vacuo and drying of the residue under
vacuum for 1 h gave the crude amine, which was dissolved in
dry THF (4 mL) with DMF (2 mL) and 11 (0.10 g, 0.23 mmol) and
triethylamine (0.06 mL, 0.46 mmol) were added. The mixture was
thoroughly degassed with nitrogen for 1 h. CuI (23 mg, 0.12 mmol)
and Pd(PPh3)4 (27 mg, 0.02 mmol) were added to the degassed
solution under a nitrogen atmosphere and the mixture was left
stirring at rt for 4 h. The reaction mixture was then dissolved in
MeOH/CHCl3 (1:4) (30 mL), which was washed with portions
(15 mL) of 5% aq disodium EDTA, water (10 mL) and dried over
MgSO4. Filtration, solvent removal under reduced pressure and col-
umn chromatography of the residue employing EtOAc/MeOH/Et3N
(5/4/1) gave the coupled amine product, which was reacted with
thiourea 13 (85 mg, 0.30 mmol) in dry THF (5 mL) at room temper-
ature for 20 h. Following evaporation of solvent, the residue was
subjected directly to column chromatography using EtOAc/pet
ether (9/1) to afford the double-drug 14d as a solid (76 mg, 40%
Using 12a (100 mg, 0.16 mmol) with 13 (54 mg, 0.19 mmol)
gave 14a (72 mg, 60%); mp 95–97 °C; [
(CHCl3): mmax 3934, 3681 (NH), 2297 (C„C), 1714 (C@O), 1602
(C@C), 1418 (C@S), 1212 (C–N) cmꢁ1 1H NMR (400 MHz, CDCl3):
a]
D
ꢁ9.9 (c 0.8, CHCl3); IR
;
d 10.92 (1H, t, J = 2.7 Hz), 9.75 (1H, br s), 9.13 (1H, s), 8.03 (2H,
m), 7.91 (1H, d, J = 2.3 Hz), 7.61 (1H, s), 7.56 (1H, m), 7.45 (2H,
m), 7.28 (1H, dd, J = 2.3, 8.8 Hz), 6.96 (1H, m), 6.80 (1H, m), 6.76
(2H, m), 6.62 (1H, d, J = 8.8 Hz), 6.37 (1H, dt, J = 1.6, 6.0 Hz), 6.09
(1H, m), 5.19 (1H, m), 4.65 (1H, dd, J = 4.2, 12.3 Hz), 4.53 (1H, dd,
J = 3.8, 12.3 Hz), 3.97 (4H, m), 3.79 (3H, s), 3.03 (1H, m), 2.88 (1H,
m), 2.71 (2H, t, J = 6.3 Hz); 13C NMR (100 MHz, CDCl3): d 179.1
(C@S), 164.7, 162.2, 153.6, 151.6, 150.9, 149.3, 146.3, 141.8,
140.6, 133.3, 133.1, 129.7, 129.3, 128.8ß 128.6, 127.7, 118.3,
113.3, 112.0, 111.6, 111.2, 100.6, 91.3, 90.7, 84.9, 72.6, 66.5, 65.3,
55.5, 45.4, 30.3, 20.9; HRMS (ES): m/z found 746.1262 (M++H).
C35H33N5O7BrS requires (M++H) 746.1284.
5.4.2. 5-{6-[2-(2-(5-Bromo-2-pyridinylaminothiocarbonylamino)-
ethyl)-4-methoxyphenoxy]hexa-4-oxa-1-ynyl}-50-O-benzoyl-20,30-
didehydro-20,30-dideoxyuridine 14b
over the three steps); mp 112–115 °C; [
a
]
ꢁ24.1 (c 1.0, CHCl3);
D
Using 12b (0.20 g, 0.30 mmol) with 13 (0.11 g, 0.39 mmol) gave
IR (CHCl3): mmax 3377, 3217 (NH), 3007, 2920 (C–H), 2239 (–
C„C–), 1722, 1675 (C@O), 1501 (C@C), 1465 (C@S), 1227 (C–N)
14b (0.13 g, 60%); mp: 73–76 °C; [
(CHCl3): mmax 3601, 3391 (NH), 2957 (C–H), 2254 (–C„C–), 1718
(C@O), 1501 (C@C), 1462 (C@S) cmꢁ1 1H NMR (300 MHz, CDCl3):
a]
D
ꢁ20.2 (c 1.0, CHCl3); IR
cmꢁ1 1H NMR (400 MHz, CDCl3): d 11.09 (1H, t, J = 5.1 Hz), 8.88
;
;
(1H, br s), 8.76 (1H, br s), 8.05 (1H, d, J = 2.4 Hz), 8.02 (2H, d,
J = 7.6 Hz), 7.68 (1H, s), 7.68 (1H, dd, J = 2.4, 8.8 Hz), 7.58 (1H, m),
7.46 (2H, t, J = 7.6 Hz), 6.93 (1H, m) 6.81 (1H, d, J = 2.8 Hz), 6.78
(1H, d, J = 8.8 Hz), 6.75 (1H, dd, J = 2.8, 8.8 Hz), 6.74 (1H, d,
J = 8.8 Hz), 6.41 (1H, dt, J = 1.7, 6.0 Hz), 6.00 (1H, dq, J = 1.2,
6.0 Hz), 5.22 (1H, m,), 4.70 (1H, dd, J = 4.2, 12.5 Hz), 4.52 (1H, dd,
J = 3.0, 12.5 Hz), 4.21 (2H, s), 4.02 (4H, m), 3.83 (2H, t, J = 5.8 Hz),
3.77 (3H, s), 3.71–3.64 (12H, m), 2.99 (2H, t, J = 6.6 Hz); 13C NMR
(75 MHz, CDCl3): d 179.1 (C@S), 166.2, 161.3, 153.5, 151.8, 151.2,
149.5, 146.4, 142.3, 141.0, 133.5, 133.4, 129.6, 129.1, 128.8,
128.6, 126.9, 117.5, 113.6, 112.7, 112.4, 111.5, 100.0, 90.7, 90.1,
85.1, 76.9, 70.8, 70.6, 70.5, 70.5, 70.3, 69.8, 69.1, 68.4, 65.0, 58.9,
55.5, 45.7, 29.8; HRMS (ES): m/z found 908.2178 (M++H).
C42H47N5O11SBr requires (M++H) 908.2176.
d 10.82 (1H, m), 9.00 (1H, br s), 8.84 (1H, br s), 8.03 (1H, d,
J = 2.4 Hz), 8.00 (2H, m), 7.70 (1H, s), 7.65 (1H, dd, J = 2.4, 8.8 Hz),
7.56 (1H,, m), 7.45 (2H, m), 6.93 (1H, m), 6.87 (1H, d, J = 8.8 Hz),
6.79 (1H, d, J = 2.8 Hz), 6.77 (1H, d, J = 8.8 Hz), 6.73 (1H, dd,
J = 2.8, 8.8 Hz), 6.40 (1H, dt, J = 1.7, 6.0 Hz), 6.02 (1H, dq, J = 1.4,
6.0 Hz), 5.21 (1H, m), 4.68 (1H, dd, J = 4.3, 12.5 Hz), 4.54 (1H, dd,
J = 2.9, 12.5 Hz), 4.25 (2H, s), 4.00 (4H, m), 3.83 (2H, m), 3.75 (3H,
s), 2.98 (2H, m); 13C NMR (100 MHz, CDCl3): d 179.2 (C@S),
166.3, 161.6, 153.7, 151.8, 151.2, 149.4, 146.6, 142.6, 141.0,
133.6, 133.4, 29.7, 129.2, 128.9, 128.7, 127.0, 117.5, 113.6, 112.8,
112.5, 111.6, 100.0, 90.9, 90.0, 85.2, 77.2, 68.5, 68.3, 65.1, 59.2,
55.6, 45.7, 29.9; HRMS (ES): m/z found 776.1379 (M++H).
C36H35N5O8SBr requires (M++H) 776.1390.
5.4.3. 5-{9-[2-(2-(5-Bromo-2-pyridinylaminothiocarbonylamino)-
ethyl)-4-methoxyphenoxy]nona-4,7-dioxa-1-ynyl}-50-O-benzoyl-
20,30-didehydro-20,30-dideoxyuridine 14c
5.5. General procedure for benzoate deprotection of 14a–d to
afford 15a–d
Using 12c (0.15 g, 0.21 mmol) with 13 (76 mg, 0.27 mmol) gave
A solution of NaOMe in methanol (0.03 mL, 2 M, 0.06 mmol,
0.6 equiv) was added to a solution of the nucleoside (0.1 mmol)
in MeOH (2 mL) at 0 °C. The mixture was stirred for 30 min, acetic
acid (0.05 mL) was added, and the crude mixture was diluted with
CH2Cl2 (1 mL) and subjected directly to column chromatography
employing MeOH/CH2Cl2 (2/8) to give the desired product as a
pale-yellow solid.
14c (0.10 g, 63%); mp 88–91 °C; [
(CHCl3): mmax 3406 (NH), 3029, 2949 (C–H), 2152 (–C„C–), 1718,
1671 (C@O), 1501 (C@C), 1469 (C@S), 1126 (C–N) cmꢁ1 1H NMR
a]
ꢁ18.5 (c 1.0, CHCl3); IR
D
;
(300 MHz, CDCl3): d 11.01 (1H, t, J = 4.8 Hz), 9.29 (1H, br s), 8.94
(1H, br s), 8.04 (1H, d, J = 2.4 Hz), 8.00 (2H, d, J = 7.4 Hz), 7.67
(1H, s), 7.65 (1H, dd, J = 2.4, 8.8 Hz), 7.56 (1H, m), 7.43 (2H, t,
J = 7.4 Hz), 6.91 (1H, m), 6.80 (1H, d, J = 9.2 Hz), 6.79 (1H, d,

Y. Younis et al. / Bioorg. Med. Chem. 18 (2010) 4661–4673
4671
5.5.1. 5-{4-[2-(2-(5-Bromo-2-pyridinylaminothiocarbonylamino)-
ethyl)-4-methoxyphenoxy]but-1-ynyl}-20,30-didehydro-20,30-
dideoxyuridine 15a16
Compound 14a (83 mg, 0.11 mmol) gave 15a (43 mg, 60%); mp
121–122 °C; [a]D +22.4 (c 1.10, CHCl3); IR (CHCl3): mmax 3934, 3688
(1H, dd, J = 3.2, 8.8 Hz), 6.38 (1H, dt, J = 1.7, 6.0 Hz), 5.91 (1H, dq,
J = 1.4, 6.0 Hz), 5.02 (1H, t, J = 5.2 Hz), 4.79 (1H, m), 4.28 (2H, s),
4.00 (2H, t, J = 4.9 Hz), 3.81 (2H, q, J = 6.6 Hz), 3.71 (2H, t,
J = 4.9 Hz), 3.66 (3H, s), 3.60–3.47 (14H, m), 2.87 (2H, t,
J = 6.6 Hz); 13C NMR (75 MHz, DMSO-d6): d 179.1 (C@S), 161.4,
153.0, 152.2, 150.6, 149.6, 145.6, 144.6, 141.1, 135.2, 128.4,
125.5, 116.8, 114.3, 113.3, 111.6, 111.5, 97.5, 89.4, 88.5, 87.5,
78.2, 69.8, 69.7, 69.6, 69.6, 69.4, 68.9, 68.4, 68.2, 61.7, 58.0, 55.1,
44.5, 29.0; HRMS (ES): m/z found 804.1932 (M++H).
C35H43N5O10SBr requires (M++H) 804.1914.
(NH), 2304 (–C„C), 1696 (C@O), 1606 (C@C), 1425 (C@S) cmꢁ1; 1H
NMR (400 MHz, CDCl3): d 11.05 (1H, t, J = 4.2 Hz), 8.70 (1H, br s),
7.99 (1H, d, J = 2.6 Hz), 7.97 (1H, s), 7.56 (1H, dd, J = 2.6, 8.8 Hz),
6.97 (1H, m), 6.80 (1H, m), 6.77 (2H, m), 6.66 (1H, d, J = 8.8 Hz),
6.37 (1H, dt, J = 1.5, 5.8 Hz), 5.86 (1H, m), 4.93 (1H, m), 4.05 (2H,
t, J = 5.9 Hz), 3.95 (2H, m), 3.80 (2H, m), 3.78 (3H, s), 2.98 (2H,
m), 2.84 (2H, t, J = 5.8 Hz); 13C NMR (100 MHz, CDCl3): d 178.9,
162.2, 153.4, 151.5, 150.9, 149.1, 146.6, 143.6, 140.8, 135.6,
129.3, 128.9, 118.0, 113.4, 112.8, 111.4, 110.8, 100.3, 91.3, 90.4,
87.7, 72.5, 66.8, 62.9, 55.6, 45.7, 30.2, 21.0; HRMS (ES): m/z found
642.1008 (M++H). C28H29N5O6BrS requires (M++H) 642.1022.
5.6. 20,30-Didehydro-20,30-dideoxy-5-iodouridine (5-iodo-d4U)
To a solution of 11 (0.80 g, 1.82 mmol) in dry MeOH (15 mL),
was added a solution of NaOMe in methanol (0.90 mL, 2 M,
1.80 mmol) at 0 °C. The mixture was stirred at rt for 2 h before
being diluted with aq NH4Cl (25 mL) and then extracted using
CHCl3/MeOH (4:1) (3 ꢀ 40 mL). Drying (MgSO4), evaporation of
solvent and purification of the residue on silica-gel chromatogra-
phy using EtOAc as eluent gave 20,30-didehydro-20,30-dideoxy-5-
iodouridine as a colourless solid (0.45 g, 74%); mp 176–177 °C;
1H NMR (300 MHz, DMSO-d6): d 11.58 (1H, br s), 8.23 (1H, s),
6.78 (1H, m), 6.40 (1H, dt, J = 1.7, 6.0 Hz), 5.94 (1H, dq, J = 1.4,
6.0 Hz), 5.01 (1H, t, J = 4.9 Hz), 4.83 (1H, m), 3.62 (2H, m); 13C
NMR (75 MHz, DMSO-d6): d 160.3, 150.3, 145.7, 135.2, 125.7,
89.1, 87.4, 68.6, 61.5.
5.5.2. 5-{6-[2-(2-(5-Bromo-2-pyridinylaminothiocarbonylamino)-
ethyl)-4-methoxyphenoxy]hexa-4-oxa-1-ynyl}-20,30-didehydro-
20,30-dideoxyuridine 15b
Compound 14b (100 mg, 0.14 mmol) gave 15b (59 mg, 69%);
mp 76–79 °C; [
3594, 3391 (NH, OH), 3022, 2928 (C–H), 2239 (–C„C–), 1715,
1697 (C@O), 1501 (C@C), 1465 (C@S), 1216 (C–N) cmꢁ1 1H NMR
a]
+49.1 (c 1.0, CHCl3); IR (CHCl3): mmax 3681,
D
;
(300 MHz, CDCl3): d 11.09 (1H, t, J = 5.1 Hz), 9.44 (1H, br s), 9.09
(1H, br s), 8.14 (1H, s), 8.00 (1H, d, J = 2.4 Hz), 7.65 (1H, dd,
J = 2.4, 8.7 Hz), 6.97 (1H, m), 6.83 (1H, d, J = 8.7 Hz), 6.75 (3H, m),
6.35 (1H, dt, J = 1.7, 6.0 Hz), 5.85 (1H, m), 4.93 (1H, m), 4.38 (2H,
s), 4.06–3.78 (8H, m), 3.75 (3H, s), 3.36 (1H, br s), 2.97 (2H, t,
J = 6.6 Hz); 13C NMR (100 MHz, CDCl3): d 178.8 (C@S), 161.8,
153.7, 151.8, 151.1, 149.9, 146.4, 144.7, 141.0, 135.1, 128.9,
126.0, 117.5, 113.8, 112.9, 112.6, 111.6, 99.3, 90.3, 89.4, 87.7,
77.5, 68.4, 68.4, 62.8, 59.3, 55.6, 45.8, 30.0; HRMS (ES): m/z found
672.1139 (M++H). C29H31N5O7SBr requires (M++H) 672.1128.
5.7. 20,30-Didehydro-20,30-dideoxy-5-iodouridine-50-[p-tolylmeth-
oxyalaninyl phosphate] 16
p-Tolyl methoxylalaninyl phosphorochloridate38 (1.77 g, 6.42
mmol) and 20,30-didehydro-20,30-dideoxy-5-iodouridine (0.72 g,
2.14 mmol) were dissolved in THF (25 mL) and N-methylimidazole
(1.02 mL, 12.84 mmol) was added with vigorous stirring. After 24 h
at rt the solvent was removed under vacuum. The residue was dis-
solved in CHCl3 (100 mL) and washed with hydrochloric acid solu-
tion (1 M, 2 ꢀ 30 mL), aq NaHCO3 (2 ꢀ 30 mL), and then water
(3 ꢀ 30 mL). The organic layer was dried over MgSO4 and the sol-
vent evaporated under vacuum. Purification of the residue by chro-
matography on silica-gel eluting with 3% MeOH in CHCl3 gave 16
(0.45 g, 35%) as a 1:1 mixture of diastereoisomers and as a colour-
less solid; mp 40–44 °C; IR (CHCl3): mmax 3384 (NH), 3007 (C–H),
5.5.3. 5-{9-[2-(2-(5-Bromo-2-pyridinylaminothiocarbonylamino)-
ethyl)-4-methoxyphenoxy]nona-4,7-dioxa-1-ynyl}-20,30-
didehydro-20,30-dideoxyuridine 15c
Compound 14c (80 mg, 0.11 mmol) gave 15c (37 mg, 54%); mp
130–134 °C; [
(NH, OH), 2928 (C–H), 2246, 2123 (–C„C–), 1700 (C@O), 1462
(C@S), 1223 (C–N) cmꢁ1 1H NMR (300 MHz, DMSO-d6): d 11.50
a]D +41.1 (c 1.0, DMSO); IR (DMSO): mmax 3442, 3260
;
(1H, br s),1.05 (1H, t, J = 5.0 Hz),10.52 (1H, br s), 8.11 (1H, d,
J = 2.6 Hz), 8.10 (1H, s), 7.93 (1H, dd, J = 2.6, 8.7 Hz), 7.12 (1H, d,
J = 8.7 Hz), 6.92 (1H, d, J = 8.7 Hz), 6.82 (1H, d, J = 3.0 Hz), 6.80
(1H, m), 6.77 (1H, dd, J = 3.0, 8.7 Hz), 6.40 (1H, dt, J = 1.7, 6.0 Hz),
5.93 (1H, dq, J = 1.4 Hz, 6.0 Hz), 4.94 (1H, t, J = 5.0 Hz), 4.81 (1H,
m), 4.31 (2H, s), 4.03 (2H, t, J = 4.9 Hz), 3.84 (2H, q, J = 6.6 Hz),
3.74 (2H, t, J = 4.9 Hz), 3.68 (3H, s), 3.66–3.58 (6H, m, H-50), 2.90
(2H, t, J = 6.6 Hz); 13C NMR (75 MHz, DMSO-d6): d 179.1 (C@S),
161.3, 153.1, 152.1, 150.6, 149.5, 145.6, 144.5, 141.0, 135.2,
128.4, 127.4, 116.8, 114.3, 113.4, 111.6, 111.4, 97.5, 89.4, 88.5,
87.5, 78.1, 69.5, 68.9, 68.5, 68.2, 61.7, 58.0, 55.1, 44.5, 28.9; HRMS
(ES): m/z found 716.1389 (M++H), C31H35N5O8SBr requires (M++H)
716.1390.
1743, 1704 (C@O), 1505 (C@C), 1245 (P@O) cmꢁ1 1H NMR
;
(400 MHz, CDCl3): d (some peaks are split due to diastereoisomers
at P) 8.91 (1H, br s), 7.92, 7.90 (1H, s), 7.11 (4H, m), 6.94, 6.92 (1H,
m), 6.38, 6.32 (1H, dt, J = 1.7, 6.0 Hz), 5.94, 5.86 (1H, dq, J = 1.3,
6.0 Hz), 5.05 (1H, m), 4.46–3.82 (4H, m), 3.72, 3.71 (3H, s), 2.31
(3H, s), 1.41, 1.36 (3H, d, J = 7.6 Hz); 13C NMR (75 MHz, CDCl3): d
(values bearing an asterisk are given as an average of peaks, split
*
*
*
due to diastereoisomers and/or C–P coupling) 173.9 , 159.9 ,
*
*
*
*
*
*
150.3, 148.2 , 144.6 , 134.6, 133.7 , 133.0 , 126.9 , 120.0 , 90.2 ,
*
*
*
*
*
*
*
85.3 , 69.0 , 66.6 , 52.4 , 50.0 , 21.0 , 20.8 .
31P NMR (CDCl3): d 3.7, 3.5 (1:1); HRMS (ES): m/z found
592.0356 (M++H). C20H24N3O8PI requires (M++H) 592.0346.
5.8. 5-{15-[2-(2-(5-Bromo-2-pyridinylaminothiocarbonylamino)-
ethyl)-4-methoxyphenoxy]pentadeca-4,7,10,13-tetraoxa-1-ynyl}-
20,3-didehydro-20,30-dideoxy-5-iodouridine-50-50-(p-methylphenyl
methoxyalaninyl phosphate)-20,30-didehydro-20,30-dideoxyuridine
17
5.5.4. 5-{15-[2-(2-(5-Bromo-2-pyridinylamino thiocarbonylami-
no)ethyl)-4-methoxyphenoxy]pentadeca-4,7,10,13-tetraoxa-1-
ynyl}-20,30-didehydro-20,30-dideoxyuridine 15d
Compound 14d (70 mg, 0.08 mmol) gave 15d (32 mg, 52%); mp
63–65 °C; [
(NH, OH), 2246, 2123 (C„C), 1660 (C@O), 1469 (C@S), 1226 (C–
N) cmꢁ1 1H NMR (400 MHz, DMSO-d6): d 11.60 (1H, br s) 11.09
(1H, t, J = 4.8 Hz), 10.62 (1H, br s), 8.12 (1H, s), 8.09 (1H, d,
J = 2.4 Hz), 7.93 (1H, dd, J = 2.4, 8.8 Hz), 7.09 (1H, d, J = 8.8 Hz),
6.89 (1H, d, J = 8.8 Hz), 6.81 (1H, d, J = 3.2 Hz), 6.78 (1H, m), 6.75
a] + 53.2 (c 1.0, DMSO); IR (DMSO): mmax 3442, 3283
D
Trifluoroacetic acid (0.20 mL) was added to a solution of alkyne
7d (92 mg, 0.19 mmol) in CH2Cl2 (2 mL) at 0 °C, and the solution
stirred for 2 h. Anhydrous K2CO3 (80 mg, 0.57 mmol) was added,
the mixture was stirred for a further 15 min and then filtered
through Celite, The solvent was then evaporated in vacuo, the
;

4672
Y. Younis et al. / Bioorg. Med. Chem. 18 (2010) 4661–4673
crude amine dried under vacuum for 1 h, dissolved in dry THF
(3 mL) with DMF (2 mL), and 5-iodouridine-50-[p-methylphenyl
methoxyalaninyl phosphate] 16 (0.10 g, 0.17 mmol), followed by
triethylamine (0.05 mL, 0.34 mmol) were added, The mixture was
thoroughly degassed with nitrogen for 1 h. CuI (17 mg, 0.09 mmol)
and Pd(PPh3)4 (20 mg, 0.02 mmol) were then added to the de-
gassed solution under a nitrogen atmosphere. The mixture was left
stirring at rt for 2 h, after which a mixture of MeOH/CHCl3 (1:4,
30 mL) was added and washed with portions (2 ꢀ 10 mL) of 5%
aq disodium EDTA, water (10 mL) and then dried over MgSO4. Fil-
tration and solvent evaporation under reduced pressure gave a
crude product, which was flashed through a silica-gel column
employing EtOAc/MeOH/Et3N (5/4/1) as eluent. The coupled amine
product was dissolved in dry THF (3 mL), thiourea 13 (65 mg,
0.23 mmol) was added and the mixture stirred at room tempera-
ture for 20 h. Following evaporation of solvent, the residue was
subjected directly to column chromatography using EtOAc/MeOH
(9/1) to give 17 as a pale-yellow solid (36 mg, 20% over the three
50-radiolabeled primer/template prior to mixing with appropriate
concentrations of inhibitor and allowed to incubate for a minimum
of 15 additional minutes on ice. DMSO concentrations were kept
constant at less than 2%. DMSO alone was added as a no inhibitor
control for each set of experiments. Reactions were initiated by the
addition of 5 lM dTTP and 10 mM MgCl2 and were quenched after
15 min at 37 °C with 0.3 M EDTA. All concentrations represent final
concentrations after mixing. Reaction products were subjected to
20% denaturing polyacrylamide gel-electrophoresis and quanti-
tated on a Bio-Rad Molecular Imager FX. Product formation was
plotted as a function of inhibitor concentration and fitted to a
hyperbola to generate IC50 curves. IC50 values are defined as the
concentration of inhibitor that inhibits steady-state single nucleo-
tide incorporation by 50%.
Acknowledgments
We thank the National Research Foundation of South Africa, the
Third World Organization for Women in Science and the University
of Cape Town for financial support. We also thank the National
Institutes of Health (GM49551) for support to K.S.A.
steps); mp 51–54 °C; [
3391 (NH), 3007, 2928 (C–H), 1707 (C@O), 1505 (C@C), 1462
(C@S), 1259 (P@O), 1223 (C–N) cmꢁ1 1H NMR (300 MHz, CDCl3):
a
]
D
+4.4 (c 1.0, CHCl3); IR (CHCl3): mmax
;
d (some peaks are split due to diastereoisomers at P) 11.11 (1H,
t, J = 4.9 Hz, NH), 9.11 (1H, br s, NH), 9.05, 9.00 (1H, s, NH), 8.03
(1H, d, J = 2.4 Hz), 7.79, 7.78 (1H, s), 7.66, 7.63 (1H, dd, J = 2.4,
8.8 Hz), 7.08 (4H, s), 6.96, 6.94 (1H, m), 6.80 (1H, d, J = 3.0 Hz),
6.78 (1H, d, J = 8.8 Hz), 6.74 (1H, d, J = 8.8 Hz), 6.72 (1H, dd,
J = 3.0, 8.8 Hz), 6.37, 6.27 (1H, dt, J = 1.7, 6.0 Hz), 5.91, 5.81 (1H,
dq, J = 1.4, 6.0 Hz), 5.02 (1H, m), 4.41–3.97 (10H, m), 3.81 (2H, t,
J = 4.9 Hz), 3.75 (3H, s), 3.68, 3.67 (3H, s), 3.70–3.57 (12H, m),
2.97 (2H, t, J = 6.6 Hz), 2.28 (3H, s), 1.37, 1.33 (3H, d, J = 6.3 Hz);
13C NMR (75 MHz, CDCl3): d (values bearing an asterisk are given
as an average of peaks, split due to diastereoisomers and/or C-P
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.05.025.
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*
*
*
*
*
*
148.3 , 146.5, 143.1 , 141.0, 134.7, 133.8, 130.1 , 128.8, 126.7 ,
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