C-2-Aryl O-substituted HI-236 derivatives as non-nucleoside HIV-1 reverse-transcriptase inhibitors

Article abstract of DOI:10.1016/j.bmc.2008.10.048

Several novel thiourea derivatives of the NNRTI HI-236 substituted at the C-2 oxygen of the phenyl ring have been synthesized and evaluated for their inhibitory activity against HIV-1 (IIIB) replication in MT-2 cell cultures. The compounds were synthesize

Full text of DOI:10.1016/j.bmc.2008.10.048

Bioorganic & Medicinal Chemistry 16 (2008) 10270–10280
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
C-2-Aryl O-substituted HI-236 derivatives as non-nucleoside HIV-1
reverse-transcriptase inhibitors
a
a
a
a
a
Roger Hunter a, , Yassir Younis , Clare I. Muhanji , Tanith-lea Curtin , Kevin J. Naidoo , Melissa Petersen ,
*
Christopher M. Bailey b, Aravind Basavapathruni 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:
Received 8 July 2008
Accepted 20 October 2008
Available online 1 November 2008
Several novel thiourea derivatives of the NNRTI HI-236 substituted at the C-2 oxygen of the phenyl ring
have been synthesized and evaluated for their inhibitory activity against HIV-1 (IIIB) replication in MT-2
cell cultures. The compounds were synthesized in order to fine-tune the activity of HI-236 as well as to
gain insight into spatial characteristics in the pocket pertaining to the positional choice of tether in the
design of [NRTI]-tether-[HI-236] bifunctional inhibitors. Two of the thiourea derivatives bearing a buty-
nyl (6c) or hydroxyethyl tether (6n) were endowed with improved anti-HIV activity compared to HI-236.
NNRTI activity was confirmed by a cell-free RT assay on six of the derivatives in which 6c returned an IC50
of 3.8 nM compared to 28 nM for HI-236, establishing it as an improved lead for HI-236. The structure-
activity profile is discussed in terms of potential interactions in the NNRTI pocket as suggested by a dock-
ing model using AutoDock, which have a bearing on the bifunctional drug design.
Keywords:
NNRTI
HI-236
AutoDock
Thiourea
HIV
Ó 2008 Elsevier Ltd. All rights reserved.
Bifunctional inhibitors
1. Introduction
Nanni7 and coworkers, are designed to promote inhibition at both
sites cooperatively through the individual drugs in the same entity,
The HIV-1 reverse-transcriptase enzyme is responsible for con-
verting the genomic single-stranded RNA of HIV into a double-
stranded DNA and continues to be a major target for anti-HIV drug
discovery.1 Inhibitors fall into two distinct classes as nucleoside
analogues (NRTIs) and non-nucleoside analogues (NNRTIs), and
their modes of action are distinct and well documented.2 Thus,
while NRTIs act as competitive substrates at the substrate-binding
site, the NNRTIs work allosterically in an adjacent pocket, interfer-
ing with reverse transcription by altering the conformational
mobility of RT, which results in non-competitive inhibition. To
date, more than 30 structurally diverse NNRTIs have been identi-
fied, which have been comprehensively reviewed by several work-
ers.3 NNRTIs are vulnerable to HIV’s high mutation rate, and to
circumvent this, they are currently used in combination with
NRTIs. An alternative strategy that has been investigated by a num-
ber of groups over the last decade or so has been to combine an
NRTI and an NNRTI into a single bifunctional molecule.4 Such
inhibitors may be classified into two types: cleavable5 and non-
cleavable.6 The former involve NRTI–linker–NNRTI systems that
are designed to release the two drugs into the cytoplasm via enzy-
matic hydrolysis in the hope of promoting synergistic inhibitory
action. Conversely, the latter, based on an original suggestion by
on the basis that the NRTI and NNRTI drug–target sites are in close
proximity of 10–15 Å.8 The latter, ambitious strategy has produced
some inventive combinations, but true bifunctional character has
yet to be comprehensively established. In the design of such
bifunctional drugs, it is important to identify attachment points
on each drug as well as to choose an appropriate tether regarding
size and functional character.
We have been interested in developing non-cleavable bifunc-
tional NRTI/NNRTI compounds involving d4T as the NRTI and the
PETT (phenethylthiazolyl) derivative HI-236 as the NNRTI. These
compounds were selected in view of their excellent profiles as
antiviral agents, and it was decided to attach the linker on the NRTI
side of the molecule to C-5 of the pyrimidine base in view of antic-
ipated low interference with base pairing.9 On the HI-236 side, it
was decided to use the C-2 phenolic oxygen in view of studies con-
ducted by Uckun10 regarding the binding mode of HI-236 in the RT
pocket. Our recently published11 first prototype, [d4T]-butyne-
[HI-236], returned an EC50 of 250 nM against HIV-1 (IIIB) in MT-2
cell culture using an MTT assay. This result compares favourably
with Ladurée’s bifunctional,12 also based on d4T but conjugated
to a PETT derivative through a glycylsuccinyl cleavable linker,
which turned out to be inactive (Fig. 1).
PETT (phenethylthiazolyl) derivatives such as those shown in
Figure 1 were first introduced by the Eli Lilly group in 1995.13
Changing the thiazole ring to a pyridine ring as N-(5-bromo-2-
* Corresponding author. Tel.: +27 21 650 2544; fax: +27 21 689 7499.
E-mail address: Roger.Hunter@uct.ac.za (R. Hunter).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.10.048

R. Hunter et al. / Bioorg. Med. Chem. 16 (2008) 10270–10280
10271
S
N
H
N
H
N
O
H
N
O
O
N
O
O
N
Br
O
N
S
H
O
H
O
N
O
N
O
O
N
O
N
H
N
H
N
Br
HO
HO
Laduree
Hunter
[d4T]-PropynylGlycylSuccinyl-[Trovirdine]
[d4T]-Butynyl-[HI-236]
Figure 1. Structures of two recent bifunctional inhibitors based on PETT compounds with d4 T.
pyridinyl)-N0-(2-pyridylethyl)-thiourea (named as Trovirdine),
resulted in enhanced activity and a detailed structural analysis of
it carried out by the Uckun group revealed abundant sterically
usable space surrounding the pyridyl group as well as the ethyl lin-
ker.14,15 They postulated that an efficient use of this space would
lead to more potent anti-HIV agents with higher affinity for the
NNRTI binding pocket, and this resulted in several more potent
derivatives including the highly potent HI-236,10,15 with a much
superior inhibition profile in which the pyridyl ring of trovirdine
was replaced by a 2,5-dimethoxyphenyl group, Figure 2.
2. Chemistry
Synthesis of the tethered derivatives utilized phenolic amine 1
as a key intermediate that was readily available in multigram
quantities using chemistry reported by Glennon17 via a three-step
sequence from commercially available 2-hydroxy-5-methoxybenz-
aldehyde, Scheme 1. In our case, it was found it easier to isolate 1
as an N-Boc carbamate 2. Yields for the various steps in the se-
quence to carbamate 2 were high and spectroscopic data was in
accordance with literature values for all of the known compounds,
Scheme 1.
Thereafter, following benzyl group deprotection to 3, the vari-
ous O-alkylations at the C-2 phenolic oxygen could be carried
out with introduction of the thiourea moiety last for each case. A
more convergent approach involving converting amine 1 first into
the thiourea derivative, debenzylating and then carrying out the
C-2 phenolic alkylation reaction to generate a small library of tar-
gets was deemed to be unattractive in view of the anticipated
incompatibility of the thiourea functionality with the alkylation
chemistry.
Alkylation of carbamate 3 (Scheme 2 and Table 1) to afford a
library of alkylated N-Boc derivatives 4 proceeded efficiently with
bromide or tosylate electrophiles using K2CO3 in acetonitrile at
reflux for around 20 h and proved to be superior to a Mitsunobu
reaction with the corresponding alcohol. Yields of chromato-
graphed products were generally in excess of eighty percent (see
Section 4). In most cases, the electrophile was readily available
either commercially or via a one-step derivatisation.
The exceptions came for the alkynyl tethers used to probe the
question of the influence of tether length and aqueous solubility
of the bifunctional compounds. These involved alkynyl PEG (ethyl-
ene glycol) chains and were synthesized by the following sequence
shown in Scheme 3 in which the tether was developed divergently.
Thus, standard phenolic alkylation of a monobenzylated glycol
bromide afforded the alkylated product, which was committed to
Structural studies around HI-236 revealed, as with other PETT
derivatives, that the thiourea portion interacts in Wing 1 towards
the front of the pocket via H-bonding with K101.10,15 Conversely,
the C-5-methoxy group forges cooperative C–H interactions with
p
the highly conserved W229, while the phenyl ring interacts with
Y188. The methoxy group at C-2 was found to lie underneath the
ethyl linker and was considered to occupy some of the vacant
space near Y188 in Wing 2, an issue which assumes a greater
weight of importance for drug-resistant strains where there is an
increase in pocket size due to mutated residues of smaller size such
as Y181C and Y188L. The realization of the [d4T]-butynyl-[HI-236]
bifunctional prototype mentioned previously11 prompted a study
of the influence of the nature of the C-2 tether of HI-236 on its bio-
logical activity. In this paper, we present the synthesis and biolog-
ical activity of a small library of HI-236 derivatives involving
substitution of the C-2 phenolic methyl group, the objective being
not only to shed light on NNRTI attachment at the C-2 oxygen for
the bifunctional design, but also to probe the possibility of further
binding in the region of the pocket just mentioned in an attempt to
improve the activity of HI-236 even further. Tethers attached to the
C-2 phenolic oxygen were chosen to probe length, functionality
and polarity. Bearing in mind that a Sonogashira connection16
between C-5 of d4T and a terminal alkyne was used for the bifunc-
tional connection chemistry, some, but not all of the derivatives
studied were selected with an alkyne terminus.
OR
OH
OMe
2
H
N
H
N
H
N
H
N
NHBoc
NHBoc
i
N
N
N
1
S
S
Br
Br
3
4
OMe
OMe
HI-236
Trovirdine
OMe
Scheme 2. Reagents and conditions: (i) K2CO3, CH3CN, 80 °C, 20 h or NaH, DME,
80 °C, 20 h with ROTs.
Figure 2. Structures of HI-236 and Trovirdine.
OH
OBn
OBn
OH
CHO
NH2
NHBoc
NHBoc
v
i, ii, iii
iv
2
3
1
OMe
OMe
OMe
OMe
Scheme 1. Reagents and conditions: (i) BnBr, K2CO3, EtOH, reflux, 16 h; 95%; (ii) CH3NO2, NH4OAc, 70 °C, 14 h; 75%; (iii) LiAlH4, THF, reflux, 4 h; (iv) (Boc)2O, Et3N, CH3CN, rt,
overnight; 76% (2 steps); (v) H2, Pd/C, EtOH, rt, 5 h; 66%.

10272
R. Hunter et al. / Bioorg. Med. Chem. 16 (2008) 10270–10280
Table 1
O-Alkylation of 3 to afford 4 as precursors of C-2 substituted HI-236 derivatives.
R
No.
R
No.
4e
R
No.
R
No.
CH2
O
4a
4i
4m
CH2
N
CH2CH2CH2
CH2
CH2
CH2O
CH2
4b
4c
4d
4f
4j
4n
4o
CH2
HO
HO
CH2
CH2
CH2O
O
Bn
4g
4h
4k
4l
CH2CH2
MeO
N
CH2
CH2
CH2CH2CH2
BnO
CH2
O
O
OH
O
OTs
O
O
n
n
NHBoc
i, ii, iii
NHBoc
NHBoc
iv
3
4i, n = 0
4j, n = 1
5a, n = 0
5b, n = 1
OMe
OMe
OMe
Scheme 3. Reagents and conditions: (i) BnPEGBr (n = 0 or 1), K2CO3, CH3CN, 80 °C, 20 h or NaH, DME, 80 °C, 20 h; (ii) H2, Pd/C, EtOH, rt, 18 h (iii) TsCl, Et3N, DMAP, CH2Cl2,
0 °C–rt, 20 h (iv) propargyl alcohol, NaH, THF, 70 °C, 5 h.
a two-step sequence involving hydrogenolytic debenzylation fol-
lowed by tosylation to afford tosylates 5a and 5b. Nucleophilic dis-
placement with propargyloxide anion then furnished the products
4i and 4j. The latter18 turned out to be superior to hydroxyl-group
alkylation with propargyl bromide. Similarly, alcohols 4n and 4o
were obtained from hydrogenolysis of their benzyl ethers obtained
from alkylation of 3 as described.
Condensation between the amine and 7 could be realized in
DMF at 100 °C as described by Bell and coworkers13 to afford the
targets 6a–o in an overall yield of around 30% for the two steps.
However, later on, it was discovered that condensation could be
carried out under much milder conditions in THF or DMF at room
temperature to increase the two-step yield to around 60%. All of
the targets were isolated by silica-gel column chromatography as
crystalline solids that were crystallized to a constant, sharp melt-
ing point to return acceptable combustion analysis data. A full
spectroscopic analysis using 1H and 13C NMR spectroscopy was
also carried out on each derivative to return acceptable data. Nota-
bly, the aromatic region in the 1H NMR spectra provided conve-
nient markers for the aromatic and heteroaromatic rings to
demonstrate that condensation had taken place. The thiourea
N–H’s could be discerned downfield as two separate resonances.
The thiourea thiocarbonyl carbon could be identified using 13C
NMR, resonating at around 179 ppm. The retention of bromine in
the pyridine ring was confirmed by 13C NMR (dC–Br ꢀ112.6 ppm)
as well as microanalytical and IR data. Table 2 summarises yields
for the two-step sequence of 4–6 in Scheme 4.
Table 1 summarises the various products of C-2 phenolic alkyl-
ation of Boc carbamate 3.
All derivatives 4a–o returned acceptable NMR spectra together
with acceptable combustion analysis data (solids) and/or HRMS
mass spectral data. Notable in the NMR were the triad of signals
for the three aromatic protons in the 1H NMR spectrum integrating
correctly against the N-Boc tert-butyl singlet. The 13C NMR spectra
returned the correct number of carbon singlets in each case.
Finally, synthesis of the target thioureas 6 was accomplished via
a Boc-deprotection, condensation sequence shown in Scheme 4.
Thus, each derivative 4 was reacted with trifluoroacetic acid
(TFA) in DCM at 0 °C for 1–3 h until TLC revealed complete depro-
tection to a polar amine spot. In view of the amine’s anticipated
water solubility, the final step was conducted without using an
aqueous work-up. Thus, following complete removal of all volatiles
including any excess TFA, the residue was redissolved in THF,
Hünig’s base (EtN(i-Pr)2) was added to liberate the free amine
and the thiocarbonyl reagent 7 added according to the original
procedure. As described by the Eli Lilly group,13 the latter could
be readily prepared by reacting 2-amino-5-bromopyridine with
1,10-thiocarbonyldiimidazole in acetonitrile at room temperature
for 12 h to afford a precipitate that was used without purification.
Attempted recrystallization of 7 from methanol resulted in a prod-
uct with imidazole substituted by methoxy (Scheme 5).
3. Biological results, modeling, and discussion
The inhibitory activity of compounds 6a–o was measured
against HIV-1 (IIIB) replication in MT-2 cell culture using an MTT
assay.19 HI-236 was included as a reference compound. The results
are shown in Table 3.
In order to lend support that these HI-236 derivatives act as
NNRTIs, six of the derivatives (6c, 6d, 6i, 6k, 6n, and 6o) covering
the most (6n) to the least active (6k) were subjected to an
in vitro steady-state RT inhibition assay, the results from which
are shown in Table 4. The results present strong support for all
of the derivatives in this study to be acting as NNRTIs since they
OR
H
N
H
N
OR
N
NHBoc
i
Br
S
N
S
S
Br
N
N
i
6
4
N
H
N
N
N
OMe
OMe
N
7
Scheme 4. Reagents and conditions: (i) CF3COOH, CH2Cl2, 0 °C, 1 h and then DIEA,
0 °C, 10 min, followed by 8, THF, rt, 2 h.
Scheme 5. Reagents and conditions: (I) 2-amino-5-bromopyridine, CH3CN, rt, 120.

R. Hunter et al. / Bioorg. Med. Chem. 16 (2008) 10270–10280
10273
Yield
Table 2
Yields for converting 4–6.
Compound
Yield
Compound
Yield
Compound
Yield
Compound
6a
6b
6c
6d
65
6e
6f
6g
6h
60
67
37
58
6i
6j
6k
6l
62
60
50
68
6m
6n
6o
53
65
51
17a
25a
69
a
DMF at 100 °C; the others involved THF at rt.
have no possibility of being phosphorylated for NRTI activity
against RT. This is also in keeping with the published findings on
HI-236. The activities ranged from 3.8 to 100 nM, and as expected
were superior to those from the cell-culture results except for 6n,
which was slightly lower. Such improvements likely reflect the
lipophilicity of the derivatives and their poor solubility in the
aqueous cell-culture medium. Notably, the ester 6k, which was
pyridinyl)-N0-[2-(4-ethoxy-3-fluoro-2-pyridinyl)ethyl]-thiourea as
template.22 The conformation of the PETT derivatives studied was
set to accommodate the well documented14,22,23 intramolecular
hydrogen bond between the hydrogen of the thiourea nitrogen
attached to the alkyl side and the nitrogen of the pyridinyl ring
to form a flat six-membered pseudo-ring. This protocol resulted
in a strong preference for one low energy conformation which
dominated 153 of the 250 possible conformations. HI-236 was
docked first, and we were able to establish parity of result with
that obtained by Uckun. The two structures are shown in Figure 3.
Thus, as established by Uckun, the inhibitor adopts the charac-
teristic butterfly-shaped orientation with the thiourea moiety
embedded in Wing 1 in which hydrogen bonding between the
thiourea hydrogen on the N-pyridyl side and the carbonyl oxygen
of K101 is clearly visible. Wing 2 accommodates the phenyl ring
of HI-236 close to the Y181 and Y188 region with the 5-methoxy
group interacting with W229. The 2-alkoxy substituent positions
into a well-containing residues Y181, Y188, F227, V106, and
V179. This feature had significance for the aims of this study. The
only difference between our structure and that of Uckun’s was
the orientation of W229 relative to Y181. In Uckun’s model, the
six-membered ring of the indole of W229 overlapped Tyr181,
whereas the model obtained from our calculations had the five-
membered ring of the indole overlapping Tyr181. This could have
effectively inactive in cell culture (12 lM), revealed a 120-fold
improvement to 100 nM in the RT assay, possibly due to ester
hydrolysis in the cell-culture medium.20 Importantly, the alkyne
6c returned a value of 3.8 nM in the RT assay, which was more
active compared to that of the alcohol 6n (19 nM), in spite of the
reverse being true in cell culture (26 nM vs 12 nM, respectively).
The most active derivative 6c was then evaluated in cell culture
against the Y181C resistant strain and retained activity much bet-
ter than HI-236 (8-fold decrease against 25 for HI-236).
Regarding a general comparison of all derivatives, the results
from the cell culture shown in Table 3 were taken to reveal impor-
tant trends within classes of different tether (polar vs lipophilic).
Four of the compounds returned activities equal to HI-236 (6b
and 6m) or greater (6c, 2-fold increase; 6n, 4-fold increase). In or-
der to assist with interpretation of the results, docking studies
were carried out using AutoDock 3.0521 based on the recently
published HIV-1 RT protein crystal structure of N-(5-chloro-2-
Table 3
Antiviral activity and cytotoxicity assaya results for 6a–o against HIV-1 (IIIB) in MT-2 cell culture.
b
c
b
c
R
EC50
(lM)
CC50
(l
M)
T.Id
R
EC50
2.0
(lM)
CC50
5.8
(lM)
T.Id
3
CH3 HI-236
0.048
0.095
0.052
0.026
0.25
1.0
21
6h
CH2
BnO
CH2
O
6a
0.1
0.1
1
6i
0.095
0.39
9.5
6.8
48
100
17
4
CH2
CH2O
CH2
CH2 6b
2
6j
CH2O
O
CH2CH2 6c
CH2CH2CH2 6d
1
38
40
74
55
18
6k
12
MeO
N
CH2
10.0
20.0
11.0
4.0
0.12
5.5
3.8
1.0
8.5
46
76
83
94
CH2 6l
6e
0.27
6m
CH2CH2CH2
0.05
CH2
N
6f
0.20
6n
CH2
0.012
0.098
CH2
HO
HO
CH2
6o
Bn 6g
0.22
a
Ref. 19; MOI was 0.1.
b
C
d
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.
In vitro therapeutic index (CC50/EC50).

10274
R. Hunter et al. / Bioorg. Med. Chem. 16 (2008) 10270–10280
Table 4
Both structures suggest the possibility of interaction with V109
or, to a lesser extent V179, towards the floor of the pocket. A coop-
erative hydrogen bond for 6o would nicely explain the much en-
Comparison of cell culture versus in vitro RT inhibition (nM) for selected derivatives
of 6.
OR
H
N
H
N
hanced activity of alcohol 6n (EC50 = 0.012 lM) with a shorter
N
(two-carbon) chain, in which presumably the distance between
hydrogen-bond donor and acceptor is optimal. 6n returned an
activity four times higher than HI-236 in cell culture, and almost
twice that in the RT assay and thus presents itself as an interesting
candidate for the problematic V106A mutation if the hydrogen
bonding postulate is correct.
Similarly, for the alkyl and alkynyl compounds 6a–f, the re-
sults show that the hydrophobic pocket is accommodating but
with the only significant cooperative interaction being with the
triple bond. In this regard, the butynyl chain of 6c, rather than
the propargyl (6b) or pentynyl substituents (6d) appears to be
optimal, with 6c indicating a 2-fold increase in activity compared
to HI-236 in cell culture and 7-fold in the RT assay. Although this
S
Br
6
OMe
R
Antiviral
activity
EC50 (nM)
RT
Antiviral
activity
Y181C EC50
(nM)
Cellular
Toxicity
Y181C CC50
(TI) (nM)
inhibition
assay IC50
(nM)
Me (HI-236)
48
28
3.8
27
1200
<200
18,000 (15)
4000 (>20)
CH2CH2 6c
CH2CH2CH2
26
250
6d
6i
classical force field does not include an explicit
charge distribution on the aromatic rings mimics this fundamen-
p p term, the
ꢁ
CH2
95
41
tally quantum effect surprisingly well. Therefore we suggest that
CH2O
O
the increased activity of 6c is an optimal cooperative
p p face-
ꢁ
to-face interaction between the aromatic residues of Y181,
Y188, and the triple bond. In addition, a much lower fold reduc-
tion in activity for 6c (8ꢂ) was observed against the Y181C24
mutant strain compared to that of HI-236 (25ꢂ), (see Table 4).
The PEG derivatives predictably returned lower activities, but
not overly so given their bulk. Thus 6j, with 10 atoms in the sub-
6k
12,000
100
MeO
HO
CH2
6n
12
98
19
24
CH2
CH2
6o
HO
stituent, returned an activity of 0.39 lM as only 8-fold less potent
than HI-236. By comparison, the benzyl derivative 6h is too bulky
to be satisfactorily accommodated. Finally, the nitrile derivative
6m indicated some level of cooperation in the pocket being as
active as HI-236, in spite of its five-atom substituent. Once again,
been due to differences in the static protein structure used in our
docking calculations as compared with the structure of the HIV-1
RT binding pocket used in the studies by Uckun. Attention was
then focused on docking two of the derivatives, chosen as the ester
6k and the alcohol 6o. The parity of 6k and 6o with our docked
HI-236 in terms of overall topology in the pocket was first investi-
gated, as illustrated in Figure 4 for 6k. The elongation of the 2-alk-
oxy substituent can be seen but the positioning of the rest of the
PETT structure essentially stayed the same, revealing that EC50 dif-
ferences are likely to be based on interactions in the Wing 2 region
as postulated. Alcohol 6o gave a similar result.
Examination of the way in which the 2-alkoxy substituents of
6k and 6o positioned in the hydrophobic region of Wing 2 as
shown in Figures 5 and 6 taken from each end of the pocket for
both cases, gave insights into the possible interactions responsible
for fluctuation of biological activity.
p p and/or hydrogen bonding possibilities appear to be likely
ꢁ
explanations.
In summary, it is important to note that of the 15 com-
pounds tested in cell culture, two (6c and 6n) were more active
than HI-236 and two (6b and 6m) were as active, while this
picture improved in the RT assay in which of the six derivatives
tested, two (6c and 6n) were more active and two were as ac-
tive as HI-236 (6d and 6o). Such results endorse the conclusions
drawn from the study by Uckun15 that the PETT derivatives
have unused available pocket volume with good potential for
drug-development, and have identified tethered butynyl deriva-
tive 6c as an advanced lead. Further fine-tuning is worthwhile
pursuing on developing side chains that can cope with mutated
L 100
K 101
V 179
Wing 2
Y 181
Wing 1
L 234
W 229
Y 188
H 235
V 106
Uckun’s HI-236 docked into the HIV pocket
Our HI-236 docked by AutoDock 3.05
Figure 3. HI-236 docked into the HIV pocket.

R. Hunter et al. / Bioorg. Med. Chem. 16 (2008) 10270–10280
10275
Wing 1
Wing 2
Figure 4. The similarities in orientation between the AutoDock models of HI-236 and 6k.
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
Y 188
V 106
V 179
V 179
V 106
Figure 5. Docking of ester 6k.
L 234
W 229
H 235
L 234
W 229
F 227
L 100
L 100
F 227
K 101
K 101
H 235
V 106
Y 181
Y 188
Y 188
Y 181
V 179
V 106
V 179
Figure 6. Docking of alcohol 6o.
residues contained in resistant strains,25 and the modelling re-
sults suggest that this might be possible to achieve by adding
further substitution at the C-3 position of the aromatic ring
ortho to the C-2 O-tether. In addition, the study has generated
important insights regarding the choice of the C-2 oxygen as
the attachment point for the tether in the bifunctional com-
pounds, and the likelihood of a tether at this position providing
a route from the pocket to the NRTI binding site. In this regard,
a comprehensive study of elongated alkylated bifunctional dou-
ble-drugs in order to shed light on the origin of biological activ-
11
ity for the prototype in Figure 1
forthcoming paper.
will be communicated in a

10276
R. Hunter et al. / Bioorg. Med. Chem. 16 (2008) 10270–10280
4.3. Procedure for tosylates 5a and 5b
4. Experimental
4.1. Docking aspects
The phenol 3 was O-alkylated with the appropriate PEGbromide
as its monobenzyl ether according to the procedure below for for-
mation of the alkylated derivatives 4. The alkylated products were
subjected to hydrogenolysis in ethanol using Pd–C (10 mol%) at
atmospheric pressure for 18 h. Following filtration through Celite,
the alcohols were purified by column chromatography in yields
in excess of 80% before being tosylated with p-toluenesulfonyl
chloride (1.5 equiv) in dichloromethane using triethylamine
(2 equiv) and DMAP (cat). Following a conventional work-up, the
product was isolated by column chromatography. Solid products
were generally recrystallized from ethyl acetate/hexane mixtures.
The binding conformations of HI-236 (1) and its ester (6k) and
alcohol (6o) derivatives bound to HIV-1 Reverse Transcriptase (RT)
were modelled using AutoDock 3.0521 based on the published
HIV-1 RT protein crystal structure of N-(5-chloro-2-pyridinyl)-N0-
[2-(4-ethoxy-3-fluoro-2-pyridinyl)ethyl]-thiourea.22 Each ligand
was built in Gaussview,26 and optimised using Gaussian 98 to relax
bond lengths and angles that were not varied in the docking
simulation. Polar hydrogen atoms were added to the protein and
Kollman united-atom partial charges assigned using the AutoDock-
Tools package. For each ligand, Gasteiger-Marsili27 partial charges
were assigned, as implemented in AutoDockTools. Autodock 3.05
utilises an empirical scoring function21 to calculate binding free
energies, which incorporates five energy terms, including a Len-
nard–Jones 12-6 term and a directional 12-10 hydrogen bond term,
for which the default parameters distributed with AutoDockTools
were used. Electrostatic interactions were calculated using a dis-
tance-dependent dielectric constant.28 Atomic solvation parame-
ters and fragmental volumes were assigned using the AddSol
utility, from which the desolvation contribution to the binding free
energy is calculated. A 61 ꢂ 61 ꢂ 61 grid map was used in all dock-
ing calculations, with a grid spacing of 0.375 Å. Given the known
location of the NNRTI binding site, the grid was centred on the
coordinates for the equivalent atom of PETT-lig corresponding to
the default selected root atom of each ligand investigated. Docked
conformations were generated using the Lamarckian genetic algo-
rithm (LGA) with an initial population size of 150 structures.
Translation, quaternion and torsional step sizes were set to 2 Å,
5.0° and 5.0° respectively for HI-236 and 0.1 Å, 1.0° and 1.0° for
ligands 9k and 9o. Further parameters were set to their default
values. A total of 250 runs were performed for each ligand, and
the resulting conformations clustered using a root mean-squared
deviation criterion of 0.5 Å in x, y, z positional coordinates. Bond
rotation from the pyridinyl ring across to the thiourea moiety
was disallowed, and the conformation was set to accommodate
the well documented14,21,22 intramolecular hydrogen bond be-
tween the hydrogen of one of the thiourea nitrogens and the nitro-
gen of the pyridinyl ring to form a flat six-membered pseudo-ring.
This protocol resulted in a strong preference for one low energy
conformation which dominated 153 of the 250 possible conforma-
tions. Although using a static protein structure in the docking
simulation and limiting the rotation of certain bonds, the binding
conformation of HI-236 in this study compared well with that
obtained by Uckun.15
4.3.1. N-(tert-Butoxycarbonyl)-2-[5-methoxy-2-(2-p-toluene-
sulfonyloxyethoxy)phenyl]ethylamine (5a)
76% Yield as a colourless oil; IR (CHCl3) mmax 3690, 3451, 1706,
;
1367, 1164 cmꢁ1 1H NMR (400 MHz, CDCl3) d: 7.75 (2H, d,
J = 8.2 Hz), 7.28 (2H, d, J = 8.2 Hz), 6.62 (3H, m), 4.77 (1H, br s),
4.30 (2H, t, J = 4.7 Hz), 4.05 (2H, t, J = 4.7 Hz), 3.68 (3H, s), 3.22
(2H, q, J = 6.8 Hz), 2.65 (2H, t, J = 6.8 Hz), 2.38 (3H, s), 1.38 (9H,
s); 13C NMR (100 MHz, CDCl3) d: 155.9, 154.1, 150.2, 145.0,
132.9, 129.9, 129.3, 127.8, 116.7, 112.0, 111.9, 78.8, 68.4, 66.4,
55.5, 40.5, 30.9, 28.4, 21.5; EI-HRMS: m/z; found: 465.18130
(M+). C23H31NO7S (M+) requires 465.18212.
4.3.2. N-(tert-Butoxycarbonyl)-2-{5-methoxy-2-[2-(2-p-tolu-
enesulfonyloxyethoxy)ethoxy]phenyl}ethylamine (5b)
83% Yield as a colourless oil; IR (CHCl3) mmax 3693, 3453, 1706,
1367, 1168 cmꢁ1 1H NMR (400 MHz, CDCl3) d: 7.76 (2H, d,
;
J = 7.9 Hz), 7.28 (2H, d, J = 7.9 Hz), 6.73 (1H, d, J = 8.8 Hz), 6.68
(2H, m), 4.80 (1H, br s), 4.17 (2H, t, J = 4.7 Hz), 3.98 (2H, t,
J = 4.7 Hz), 3.75 (4H, m), 3.73 (3H, s), 3.29 (2H, q, J = 6.7 Hz), 2.74
(2H, t, J = 6.7 Hz), 2.39 (3H, s), 1.39 (9H, s); 13C NMR (100 MHz,
CDCl3) d: 155.9, 153.9, 150.9, 144.8, 133.1, 129.8, 129.3, 127.9,
116.6, 113.0, 112.0, 78.8, 70.0, 69.3, 68.8, 68.3, 55.6, 40.6, 31.0,
28.4, 21.5; EI-HRMS: m/z; found: 509.20675 (M+). C25H35NO8S
(M+) requires 509.20834.
4.4. Procedure for the N-Boc O-alkylated derivatives 4
The alkylating agent as a tosylate or bromide (2.0 mmol) in dry
acetonitrile (5 mL) was added dropwise over 1 h to a refluxing and
stirring mixture of the phenol 3 (1.0 mmol) and anhydrous potas-
sium carbonate (4.0 mmol) in dry acetonitrile (15 mL). The reac-
tion was refluxed for 20 h. The mixture was filtered, the
acetonitrile evaporated, and the residue subjected to silica-gel col-
umn chromatography (10% EtOAc/pet ether) to afford the product
generally as a colourless solid. Entries 4n and 4o were derived via
hydrogenolysis of their corresponding benzyl ethers, while entries
4i and 4j were obtained via substitution of tosylates 5a and 5b
respectively using propargyloxide ion in refluxing THF (5 h). Yields
cited in the text refer to the final step in each case.
4.2. General procedures for synthesis
Microanalyses were obtained with a Fisons EA 110 CHN Ele-
mental Analyser. Infrared (IR) absorptions were measured on a
Perkin-Elmer Spectrum One FT-IR spectrometer. 1H NMR spectra
were recorded on a Varian Mercury Spectrometer at 300 MHz
and a Varian Unity Spectrometer at 400 MHz with Me4Si as inter-
nal standard. 13C NMR spectra were recorded at 75 MHz on a Var-
ian Mercury Spectrometer or at 100 MHz on a Varian Unity
Spectrometer with Me4Si as internal standard. High resolution
mass spectra were recorded on a VG70 SEQ micromass spectrom-
4.4.1. N-(tert-Butoxycarbonyl)-2-(5-methoxy-2-propoxyphe-
nyl)ethylamine (4a)
95% Yield, mp 52–54 °C; IR (CHCl3)
mmax 3684, 3452, 3002, 2977,
1706, 1502 cmꢁ1 1H NMR (400 MHz, CDCl3) d: 6.76 (1H, d,
;
J = 9.2 Hz), 6.70 (2H, m), 4.78 (1H, br s), 3.88 (2H, t, J = 6.4 Hz),
3.75 (3H, s), 3.35 (2H, q, J = 6.6 Hz), 2.78 (2H, t, J = 6.6 Hz), 1.80
(2H, m), 1.42 (9H, s), 1.04 (3H, t, J = 7.4 Hz); 13C NMR (100 MHz,
CDCl3) d: 155.9, 153.5, 151.3, 129.5, 116.8, 112.3, 112.0, 79.8,
70.2, 55.7, 40.9, 30.9, 28.4, 22.8, 10.7; EI-HRMS: m/z; found:
309.19401 (M+). C17H27NO4 (M+) requires 309.19400. Anal. Found:
C, 66.13; H, 8.80; N, 3.91. C17H27NO4 requires; C, 65.99; H, 8.80; N,
4.53.
eter. Melting points were determined using
a Reichert-Jung
Thermovar hot-stage microscope and are uncorrected. Analytical
thin-layer chromatography (TLC) was performed on silica-gel 60
F254 (Merck). Column chromatography was performed with Merck
silica-gel 60 (70–230 mesh). HI-23610 was synthesized by the same
sequence as for derivatives 6 and returned acceptable NMR data.

R. Hunter et al. / Bioorg. Med. Chem. 16 (2008) 10270–10280
10277
4.4.2. N-(tert-Butoxycarbonyl)-2-(5-methoxy-2-propargyloxy-
phenyl)ethylamine (4b)
NMR (CDCl3, 100 MHz) d: 155.9, 153.9, 151.0, 137.4, 128.6, 128.0,
127.9, 127.3, 116.8, 113.0, 112.0, 79.0, 70.9, 55.7 (OCH3), 40.8
(C-1), 30.9 (C-2), 27.8 (OC(CH3)3); EI-HRMS: m/z; found:
301.13383 [(M+ꢁtert-butyl) + H]. C21H27NO4 requires 301.13409
[(M+ ꢁ tert-butyl) + H]. Anal. Found: C, 70.50; H, 7.62; N, 3.86.
C21H27NO4 requires C, 70.56; H, 7.61; N 3.92.
75% Yield, mp 49–50 °C; IR (CHCl3): mmax 3691, 3454, 3308,
3022, 2124, 1707, 1501 cmꢁ1 1H NMR (300 MHz, CDCl3): d 6.90
;
(1H, d, J = 9.3 Hz), 6.72 (2H, m), 4.65 (3H, d, J = 2.4 Hz), 3.75 (3H,
s), 3.35 (2H, q, J = 6.8 Hz), 2.79 (2H, t, J = 6.8 Hz), 2.47 (1H, t,
J = 2.4 Hz), 1.42 (9H, s); 13C NMR (75 MHz, CDCl3): d 155.9, 154.3,
149.8, 129.6, 116.7, 113.6, 112.0, 79.0, 79.0, 56.7, 55.6, 40.6, 30.9,
28.4; EI-HRMS: m/z; found: 305.16244 (M+). C17H23NO4 (M+)
requires 305.16271. Anal. Found: C, 66.90; H, 7.54; N, 4.54.
C17H23NO4 requires C, 66.86; H, 7.59; N, 4.59.
4.4.8. N-(tert-Butoxycarbonyl)-2-[2-(2-benzyloxyethyl-1-oxy)-
5-methoxyphenyl]ethylamine (4h)
85% Yield; mp 56–58 °C; IR (CHCl3) mmax 3673, 3449, 3000,
2398, 1701, 1501 cmꢁ1 1H NMR (300 MHz, CDCl3) d: 7.40 (5H,
;
m), 6.79 (1H, d, J = 8.8 Hz), 6.70 (2H, m), 4.78 (1H, br s), 4.63 (2H,
s), 4.10 (2H, t, J = 4.8 Hz), 3.82 (2H, t, J = 4.8 Hz), 3.75 (3H, s), 3.37
(2H, q, J = 6.5 Hz), 2.80 (2H, t, J = 6.5 Hz), 1.42 (9H, s); 13C NMR
(75 MHz, CDCl3) d: 155.9, 153.7, 151.0, 138.1, 129.2, 128.4, 127.7,
127.6, 116.6, 112.9, 111.9, 78.7, 73.2, 68.7, 68.4, 55.5, 40.7, 30.9,
28.4; EI-HRMS: m/z; found: 401.22044 (M+). C23H31NO5 (M+)
requires 401.22022. Anal. Found: C, 68.90; H, 7.80; N, 3.37.
C23H31NO5 requires C, 68.80; H, 7.78; N, 3.49.
4.4.3. N-(tert-Butoxycarbonyl)-2-[2-(3-butynyl-1-oxy)-5-
methoxyphenyl]ethylamine (4c)
61% Yield, mp 76–77 °C; IR (CHCl3): mmax 3455, 3309, 2413,
1707, 1602 cmꢁ1 1H NMR (300 MHz, CDCl3): d 6.75 (3H, m), 4.70
;
(1H, br s), 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, 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; EI-HRMS: m/z; found: 319.17756
(M+). C18H25NO4 (M+) requires 319.17836. Anal. Found: C, 67.10; H,
7.66; N, 3.67. C18H25NO4 requires C, 67.89; H, 7.89; N, 4.39.
4.4.9. N-(tert-Butoxycarbonyl)-2-[5-methoxy-2-(2-propargyl-
oxyethoxy)phenyl]ethylamine (4i)
74% Yield from tosylate 5a with propargyloxide; colourless oil;
IR (CHCl3): mmax 3674, 3452, 3307, 2121, 1703 cmꢁ1 1H NMR
;
4.4.4. N-(tert-Butoxycarbonyl)-2-[5-methoxy-2-(4-pentynyl-1-
oxy)phenyl]ethylamine (4d)
(300 MHz, CDCl3): d 6.78 (1H, d, J = 7.8 Hz), 6.69 (2H, m), 4.79
(1H, br s), 4.26 (2H, d, J = 2.3 Hz), 4.10 (2H, m), 3.88 (2H, m), 3.75
(3H, s), 3.35 (2H, q, J = 6.7 Hz), 2.80 (2H, t, J = 6.7 Hz), 2.45 (1H, t,
J = 2.3 Hz), 1.42 (9H, s); 13C NMR (75 MHz, CDCl3) d: 156.0, 153.9,
150.9, 129.4, 116.7, 113.0, 112.0, 79.6, 78.9, 74.7, 68.4, 68.3, 58.5,
55.7, 40.8, 31.0, 28.4; EI-HRMS: m/z; found: 349.18937 (M+).
C19H27NO5 (M+) requires 349.18892.
93% Yield; mp 69–71 °C; IR (CHCl3) mmax 3696, 3452, 3307,
2980, 2344, 1707, 1502, 1218, 1164 cmꢁ1 1H NMR (300 MHz,
;
CDCl3) d: 6.79 (1H, d, J = 9.6 Hz), 6.70 (2H, m), 4.72 (1H, br s),
4.02 (2H, t, J = 6.5 Hz), 3.75 (3H, s), 3.34 (2H, q, J = 6.5 Hz), 2.78
(2H, t, J = 6.5 Hz), 2.41 (2H, td, J = 2.7, 6.5 Hz), 1.99 (2H, m), 1.96
(1H, t, J = 2.7 Hz), 1.43 (9H, s); 13C NMR (75 MHz, CDCl3) d: 155.9,
153.7, 151.0, 129.0, 116.7, 112.5, 112.0, 83.4, 79.3, 68.9, 66.9,
55.7, 40.8, 30.9, 28.4, 28.4, 15.4. Anal. Found: C, 68.44; H, 8.16;
N, 4.20. C19H27NO4 requires; C, 68.53; H, 8.28; N, 3.87.
4.4.10. N-(tert-Butoxycarbonyl)-2-{5-methoxy-2-[2-(2-propar-
gyloxyethoxy)ethoxy] phenyl}ethylamine (4j)
91% Yield from tosylate 5b with propargyloxide; colourlesss oil;
IR (CHCl3): mmax 3693, 3607, 3453, 3308, 3012, 2980, 2934, 2120,
1706, 1502; 1H NMR (400 MHz, CDCl3): d 6.74 (1H, d, J = 8.6 Hz),
6.66 (2H, m), 4.90 (1H, br s), 4.17 (2H, d, J = 2.4 Hz), 4.05 (2H, t,
J = 4.9 Hz), 3.80 (2H, t, J = 4.9 Hz), 3.71 (3H, s), 3.70 (4H, m), 3.31
(2H, q, J = 6.7 Hz), 2.76 (2H,t, J = 6.7 Hz), 2.42 (1H, t, J = 2.4 Hz),
1.39 (9H, s); 13C NMR (100 MHz, CDCl3): d 156.0, 153.8, 151.0,
129.4, 116.6, 113.1, 112.0, 79.6, 78.7, 74.6, 70.5, 69.8, 69.1, 68.5,
58.3, 55.6, 40.7, 31.0, 28.4; EI-HRMS: m/z; found: 393.21448
(M+). C21H31NO6 (M+) requires 393.21514.
4.4.5. N-(tert-Butoxycarbonyl)-2-[2-(2-butynyl-1-oxy)-5-
methoxyphenyl]ethylamine (4e)
92% Yield; mp 53–55 °C; IR (CHCl3) mmax 3681, 3449, 3014,
2246, 1703, 1501, 1205 cmꢁ1 1H NMR (300 MHz, CDCl3) d: 6.90
;
(1H, d, J = 9.6 Hz), 6.71 (2H, m), 4.65 (1H, br s), 4.60 (2H, q,
J = 2.3 Hz), 3.76 (3H, s), 3.35 (2H, q, J = 6.0 Hz), 2.79 (2H, t,
J = 6.0 Hz), 1.83 (3H, t, J = 2.3 Hz), 1.43 (9H, s); 13C NMR (75 MHz,
CDCl3) d: 155.9, 154.0, 150.1, 129.4, 116.5, 113.6, 111.9, 83.3,
78.6, 74.5, 57.3, 55.6, 40.6, 30.6, 28.4, 3.6. Anal. Found: C, 67.39;
H, 7.97; N, 4.12. C18H25NO4 requires C, 67.69; H, 7.89; N, 4.23.
4.4.11. N-(tert-Butoxycarbonyl)-2-(2-methoxycarbonylmethyl-
oxy-5-methoxyphenyl)ethylamine (4k)
4.4.6. N-(tert-Butoxycarbonyl)-2-(2-allyloxy-5-methoxyphenyl)
ethylamine (4f)
98% Yield; mp 66–68 °C; IR (CHCl3) mmax 3681, 3449, 1758, 1704,
1501, 1227 cmꢁ1;1H NMR (300 MHz, CDCl3): d 6.67 (3H, m), 4.84
(1H, br s), 4.61 (2H, s), 3.78 (3H, s), 3.74 (3H, s), 3.40 (2H, q,
J = 6.5 Hz), 2.90 (2H, t, J = 6.5 Hz), 1.41 (9H, s);13C NMR (75 MHz,
CDCl3) d: 169.6, 156.0, 154.3, 150.1, 129.5, 116.9, 112.4, 112.0,
78.8, 66.0, 55.6, 52.1, 40.7, 30.9, 28.4. Anal. Found: C, 60.37; H,
7.36; N, 3.91. C17H25NO6 requires C, 60.16; H, 7.42; N; 4.13.
99% Yield; mp 54–56 °C; IR (CHCl3)
mmax 3682, 3449, 3201, 1502,
1703, 1210 cmꢁ1 1H NMR (300 MHz, CDCl3) d: 6.78 (1H, d,
;
J = 9.0 Hz), 6.71 (2H, m), 6.05 (1H, ddt, J = 5.1 Hz, 10.5 Hz, 17.3 Hz),
5.39 (1H, dq, J = 1.6 Hz, 17.3 Hz), 5.26 (1H, dq, J = 1.6 Hz, 10.5 Hz),
4.71 (1H, br s), 4.50 (2H, dt, J = 1.6 Hz, 5.1 Hz), 3.76 (3H, s), 3.40
(2H, q, J = 6.5 Hz), 2.80 (2H, t, J = 6.5 Hz), 1.43 (9H, s);13C NMR
(75 MHz, CDCl3) d: 156.0, 153.7, 150.8, 133.6, 129.2, 117.1, 116.7,
112.9, 112.0, 79.0, 69.5, 55.7, 40.8, 30.9, 28.4. Anal. Found: C,
66.41; H, 8.21; N, 4.40. C17H25NO4 requires C, 66.43; H, 8.20; N, 4.56.
4.4.12. N-(tert-Butoxycarbonyl)-2-(2-cyanomethoxy-5-methoxy-
phenyl)ethylamine (4l)
96% Yield; mp 98–102 °C; IR (CHCl3) mmax 3681, 3449, 2240,
1703, 1501, 1226 cmꢁ1 1H NMR (300 MHz, CDCl3) d: 6.89 (1H, d,
;
4.4.7. N-(tert-Butoxycarbonyl)-2-(2-benzyloxy-5-methoxy-
phenyl)ethylamine (4g)
J = 9.6 Hz), 6.74 (2H, m), 4.73 (2H, s), 4.61 (1H, br s), 3.76 (3H, s),
3.35 (2H, q, J = 6.8 Hz), 2.80 (2H, t, J = 6.8 Hz), 1.43 (9H, s); 13C
NMR (75 MHz, CDCl3) d: 155.9, 155.4, 148.9, 130.1, 117.1, 115.4,
114.0, 112.2, 79.2, 55.6, 54.9, 40.5, 31.4, 28.4. Anal. Found: C,
62.87; H, 7.09; N, 8.30. C16H22N2O4 requires C, 62.73; H, 7.24; N,
9.14.
89% Yield; mp 103-104°C; IR (CHCl3): mmax 3691, 3453, 1707,
1503 cmꢁ1 1H NMR (CDCl3, 400 MHz): 7.41 (5H, m), 6.84 (1H, d,
;
J = 8.1 Hz), 6.71 (2H, m), 5.03 (2H, s), 4.70 (1H, br s), 3.76 (3H, s),
3.37 (2H, q, J = 6.4 Hz), 2.83 (2H, t, J = 6.4 Hz), 1.42 (9H, s); 13C

10278
R. Hunter et al. / Bioorg. Med. Chem. 16 (2008) 10270–10280
4.4.13. N-(tert-Butoxycarbonyl)-2-[2-(3-cyanopropyl-1-oxy)-5-
methoxyphenyl]ethylamine (4m)
153.3, 151.7, 151.5, 146.8, 141.1, 128.5, 117.7, 113.2, 112.6,
112.2, 111.5, 70.2, 55.6, 45.8, 30.0, 22.8, 10.7. Anal. Found: C,
50.77; H, 5.21; N, 9.50; S, 7.14%. C18H22BrN3O2S requires C,
50.95; H, 5.23; N, 9.90; S, 7.55.
96% Yield; mp 69–71 °C; IR (CHCl3) mmax 3681, 3449, 3014,
2246, 1703 cmꢁ1 1H NMR (300 MHz, CDCl3) d: 6.74 (3H, m), 4.73
;
(1H, br s), 4.03 (2H, t, J = 5.8 Hz), 3.75 (3H, s), 3.32 (2H, q,
J = 6.8 Hz), 2.77 (2H, t, J = 6.8 Hz), 2.60 (2H, t, J = 7.0 Hz), 2.14 (2H,
m), 1.42 (9H, s); 13C NMR (75 MHz, CDCl3) d: 155.9, 154.0, 150.6,
128.9, 119.3, 116.8, 112.5, 112.0, 79.5, 66.3, 55.7, 40.8, 31.2, 28.4,
25.7, 14.4; EI-HRMS: m/z; found: 334.1924 (M+). C18H26N2O4
(M+) requires 334.1893. Anal. Found: C, 63.62; H, 7.82; N, 8.06.
C18H26N2O4 requires C, 64.65; H, 7.84; N, 8.38.
4.5.2. N-(5-Bromo-2-pyridinyl)-N0-[2-(5-methoxy-2-propargyl-
oxyphenyl)ethyl]-thiourea (6b)
Using DMF/100 °C, (17%); mp 121–122ꢀC; IR (CHCl3)
m
max 3691,
3416 (NH), 3307 („CH), 3174, 1602, 1137 cmꢁ1
;
1H NMR
(400 MHz, CDCl3) d: 11.13 (1H, br s, NH), 8.29 (1H, br s, NH), 8.12
(1H, d, J = 2.4 Hz), 7.69 (1H, dd, J = 2.4, 8.8 Hz), 6.95 (1H, d,
J = 8.8 Hz), 6.82 (1H, d, J = 3.1 Hz), 6.76 (1H, dd, J = 3.1, 8.8 Hz),
6.59 (1H, d, J = 8.8 Hz), 4.67 (1H, d, 1H, J = 2.2 Hz), 4.01 (1H, q,
J = 6.7 Hz), 3.76 (3H, s), 3.01 (2H, t, J = 6.7 Hz), 2.47 (1H, t,
J = 2.2 Hz); 13C NMR (75 MHz, CDCl3) d: 179.1 (C@S), 154.2, 151.7,
150.0, 146.7, 141.1, 129.2, 117.6, 113.5, 113.3, 112.6, 111.5, 79.0,
75.3, 56.8, 55.5, 45.7, 29.8. Anal. Found: C, 51.23; H, 4.50; N, 8.62%.
C18H18BrN3O2S requires C, 51.44; H, 4.32; N, 10.00.
4.4.14. N-(tert-Butoxycarbonyl)-2-[2-(2-hydroxyethoxy)-5-
methoxyphenyl]ethylamine (4n)
93% Yield via hydrogenolysis of 4h; mp 90–91 °C; IR (CHCl3)
mmax 3681, 3615, 3449, 3021, 2405, 1700, 1501, 1165 cmꢁ1;1H
NMR (400 MHz, CDCl3) d: 6.74 (3H, m), 4.87 (1H, br s), 4.00 (4H,
m), 3.88 (1H, br s), 3.75 (3H, s), 3.31 (2H, q, J = 6.7 Hz), 2.78 (2H,
t, J = 6.7 Hz), 1.42 (9H, s); 13C NMR (100 MHz, CDCl3) d: 156.5,
153.9, 151.4, 128.9, 117.1, 112.6, 112.0, 79.7, 70.6, 61.6, 55.9,
41.0, 32.6, 28.6; EI-HRMS: m/z; found: 311.17278 (M+).
C16H25NO5 (M+) requires 311.17327. Anal. Found: C, 61.70; H,
8.03; N, 4.14. C16H25NO5 requires C, 61.72; H, 8.09; N, 4.50.
4.5.3. N-(5-Bromo-2-pyridinyl)-N0-{2-[2-(3-butynyl-1-oxy)-5-
methoxyphenyl]ethyl}-thiourea (6c)
Using DMF/100 °C, (25%); mp 155–156 °C; IR (CHCl3)
mmax 3691,
3415, 3308 („CH), 3176, 3048, 2360 (C„C), 1591, 1511, 1138 cmꢁ1
;
1H NMR (300 MHz, CDCl3) d: 11.13 (1H, br s, NH), 8.44 (1H, br s, NH),
8.10 (1H, d, J = 2.4 Hz), 7.68 (1H, dd, J = 2.4, 8.7 Hz), 6.76 (3H, m), 6.61
(1H, d, J = 8.7 Hz), 4.03 (4H, m), 3.75 (3H, s), 3.00 (2H, t, J = 6.6 Hz),
2.68 (2H, td, J = 2.6, 6.9 Hz), 2.04 (1H, t, J = 2.6 Hz); 13C NMR
(75dMHz, CDCl3) d: 179.2 (C@S), 153.8, 151.6, 150.9, 146.9, 141.2,
128.9, 117.8, 113.1, 112.8, 112.7, 111.6, 80.8, 69.9, 66.9, 55.6, 45.8,
30.0, 19.8. Anal. Found: C, 52.01; H, 4.46; N, 9.00%. C19H20BrN3O2S
requires C, 52.54; H, 4.64; N, 9.67.
4.4.15. N-(tert-Butoxycarbonyl)-2-[2-(3-hydroxypropyl-1-oxy)-
5-methoxyphenyl]ethylamine (4o)
100% and 95% Yields in the alkylation and hydrogenolysis steps
respectively; mp 44–46 °C; IR (CHCl3)
mmax 3681, 3623, 3449, 2399,
1700, 1505, 1205 cmꢁ1 1H NMR (300 MHz, CDCl3) d: 6.80 (1H, d,
;
J = 9.6 Hz), 6.70 (2H, m), 4.78 (1H, br s), 4.06 (2H, t, J = 5.9 Hz),
3.87 (2H, t, J = 5.9 Hz), 3.75 (3H, s), 3.34 (2H, q, J = 6.6 Hz), 2.76
(2H, t, J = 6.6 H), 2.37 (1H, br s), 2.03 (2H, quin, J = 5.9 Hz), 1.41
(9H, s); 13C NMR (100 MHz, CDCl3) d: 155.9, 153.6, 151.1, 128.9,
116.8, 112.4, 112.0, 79.0, 66.9, 65.9, 55.7, 40.7, 31.0, 29.9, 28.4.
Anal. Found: C, 62.97; H, 8.35; N, 4.13. C17H27NO5 requires C,
62.75; H, 8.36; N, 4.30.
4.5.4. N-(5-Bromo-2-pyridinyl)-N0-{2-[5-methoxy-2-(4-pentynyl-
1-oxy)phenyl]ethyl}-thiourea (6d)
Using THF/rt, (69%); mp 140–141 °C; IR (CHCl3) mmax 3684, 3415
(NH), 3308 („CH), 3152, 3018, 2956, 2245 (C„C), 1512, 1468 (C@S),
1226 (C–N) cmꢁ1; 1H NMR (400 MHz, CDCl3) d: 11.18 (1H, br s, NH),
8.83 (1H, br s, NH), 8.10 (1H, d, J = 2.4 Hz), 7.68 (1H, dd, J = 2.4 Hz,
8.8 Hz), 6.81 (1H, d, J = 9.0 Hz), 6.81 (1H, d, J = 3.0 Hz), 6.75 (1H,
dd, J = 3.0, 9.0 Hz), 6.70 (1H, d, J = 8.8 Hz), 4.01 (4H, m), 3.75 (3H,
s), 2.98 (2H, t, J = 6.8 Hz), 2.41 (2H, td, J = 2.4, 6.8 Hz), 2.02 (2H, m),
1.96 (1H, t, J = 2.4 Hz); 13C NMR (100 MHz, CDCl3) d: 179.1 (C@S),
153.5, 151.7, 151.2, 146.7, 141.1, 128.5, 117.7, 113.2, 112.6, 112.3,
111.5, 83.5, 68.9, 66.9, 55.6, 45.7, 30.0, 28.4, 15.4. Anal. Found: C,
53.54; H, 4.78; N, 7.81; S, 5.62%. C20H22BrN3O2S requires C, 53.58;
H, 4.95; N, 9.37; S, 7.15.
4.5. Procedure for the synthesis of thioureas 6a–o
Trifluoroacetic acid (0.2 mL) was added to a solution of the
N-Boc carbamate 4 (1 mmol) in CH2Cl2 (2 mL) at 0 °C, and the solu-
tion stirred for 2 h. Diisopropylethylamine (0.4 mL) was added, the
solvent evaporated in vacuo and the crude amine dried under vac-
uum for 1 h. Thiocarbonyl reagent 7 (1.3 mmol) was added to the
crude amine in DMF (5 mL) and the mixture stirred at 100 °C for
16 h. The mixture was then poured into ice-cold water (5 mL)
and stirred for 30 min. The precipitate formed was filtered and
washed with cold water (2ꢂ 5 mL) or alternatively extracted into
ethyl acetate and the residue purified by column chromatography
using ethyl acetate/light petroleum mixtures as eluent. Alterna-
tively, the condensation reaction with 7 could be carried out in
THF (5 mL) at room temperature for 20 h. Following evaporation
of solvent, the residue was subjected directly to column chroma-
tography to afford thioureas 6.
4.5.5. N-(5-Bromo-2-pyridinyl)-N0-{2-[2-(2-butynyl-1-oxy)-5-
methoxyphenyl]ethyl}-thiourea (6e)
Using THF/rt, (60%); mp 149–150 °C; IR (CHCl3) mmax 3690,
3410 (NH), 3018, 2239 (C„C), 1510, 1469 (C@S), 1207 (C–N)
cmꢁ1 1H NMR (400 MHz, CDCl3) d: 11.12 (1H, br s, NH), 8.58
;
(1H, br s, NH), 8.11 (1H, d, J = 2.4 Hz), 7.68 (1H, dd, J = 2.4 Hz,
8.8 Hz), 6.93 (1H, d, J = 8.8 Hz), 6.81 (1H, d, J = 3.2 Hz), 6.75 (1H,
dd, J = 3.2 Hz, 8.8 Hz), 6.65 (1H, d, J = 8.8 Hz), 4.61 (2H, q,
J = 2.4 Hz), 4.01 (2H, m), 3.75 (3H, s), 3.00 (2H, t, J = 6.8 Hz), 1.83
(3H, t, J = 2.4 Hz); 13C NMR (100 MHz, CDCl3) d: 179.1 (C@S),
153.9, 151.6, 150.4, 146.8, 141.1, 129.1, 117.5, 113.6, 113.1,
112.6, 111.6, 83.4, 74.5, 57.5, 55.6, 45.8, 29.8, 3.7. Anal. Found:
C, 52.51; H, 4.53; N, 8.95; S, 6.31%. C19H20BrN3O2S requires C,
52.54; H, 4.64; N, 9.67; S, 7.38.
4.5.1. N-(5-Bromo-2-pyridinyl)-N0-[2-(5-methoxy-2-propyl-1-
oxyphenyl)ethyl]-thiourea (6a)
Using THF/rt, (65%); mp 162–163 °C; IR (CHCl3)
m
max 3696, 3413
(NH), 3167, 2963 (C–H), 1512, 1472 (C@S), 1212 (C–N) cmꢁ1
;
1H
NMR (400 MHz, CDCl3) d: 11.18 (1H, br s, NH), 8.95 (1H, br s,
NH), 8.08 (1H, d, J = 2.4 Hz), 7.67 (1H, dd, J = 2.4 Hz, 8.8 Hz), 6.81
(1H, d, J = 3.2 Hz), 6.78 (1H, d, J = 8.8 Hz), 6.74 (1H, dd, J = 3.2,
8.8 Hz), 6.72 (1H, d, J = 8.8 Hz), 4.02 (2H, m), 3.87 (2H, t,
J = 6.4 Hz), 3.75 (3H, s), 2.99 (2H, t, J = 6.4 Hz), 1.80 (2H, m), 1.04
(3H, t, J = 7.4 Hz); 13C NMR (100 MHz, CDCl3) d: 179.0 (C@S),
4.5.6. N-(5-Bromo-2-pyridinyl)-N0-[2-(2-allyloxy-5-methoxy-
phenyl)ethyl]-thiourea (6f)
Using THF/rt, (67%); mp 153–154 °C; IR (CHCl3)
m
max 3681, 3413
1H NMR
(N–H), 3041, 1509 (C@C), 1422 (C@S), 1212 (C–N) cmꢁ1
;

R. Hunter et al. / Bioorg. Med. Chem. 16 (2008) 10270–10280
10279
(400 MHz, CDCl3) d: 11.11 (1H, br s, NH), 8.13 (1H, br s, NH), 8.10
(1H, d, J = 2.4 Hz), 7.69 (1H, dd, J = 2.4, 8.7 Hz), 6.82 (1H, d,
J = 2.9 Hz), 6.80 (1H, d, J = 9.0 Hz), 6.74 (1H, dd, J = 2.9, 9.0 Hz),
6.55 (1H, d, J = 8.7 Hz), 6.05 (1H, ddt, J = 5.1, 10.5, 17.3 Hz), 5.39
(1H, dq, J = 1.6, 17.3 Hz), 5.26 (1H, dq, J = 1.6, 10.5 Hz), 4.49 (2H,
dt, J = 1.6, 5.1 Hz), 4.01 (2H, m), 3.76 (3H, s), 3.01 (2H, t,
J = 6.6 Hz); 13C NMR (100 MHz, CDCl3) d: 179.1 (C@S), 153.5,
151.5, 151.0, 146.9, 141.1, 133.6, 128.7, 117.7, 117.0, 113.0,
112.8, 112.6, 111.5, 69.5, 55.6, 45.8, 30.0. Anal. Found: C, 51.32;
H, 4.62; N, 9.56%. C18H20BrN3O2S requires C, 51.18; H, 4.78; N, 9.95.
J = 2.1 Hz); 13C NMR (75 MHz, CDCl3) d: 179.1 (C@S), 153.6,
151.6, 151.2, 146.8, 141.1, 128.8, 117.7, 113.2, 112.8, 112.6,
111.5, 79.6, 74.6, 70.6, 70.0, 69.2, 68.4, 58.5, 55.6, 45.7, 30.0; HRMS
(EI) m/z; found: 507.0821 (M+). C22H26BrN3O4S (M+) requires
507.0827. Anal. Found: C, 52.02; H, 4.10; N, 7.87; S, 6.24%.
C22H26BrN3O4S requires C, 51.97; H, 5.15; N, 8.26; S, 6.31.
4.5.11. N-(5-Bromo-2-pyridinyl)-N0-[2-(2-methoxycarbonylm-
ethyloxy-5-methoxyphenyl)ethyl]-thiourea (6k)
Using THF/rt, (50%); mp 161–164 °C; IR (CHCl3) mmax 3681
(N–H), 3015, 1519, 1469 (C@S), 1212 (C–N) cmꢁ1
;
1H NMR
4.5.7. N-(5-Bromo-2-pyridinyl)-N0-[2-(2-Benzyloxy-5-methoxy-
phenyl)ethyl]-thiourea (6g)
(400 MHz, CDCl3) d: 11.12 (1H, br s, NH), 8.32 (1H, br s, NH),
8.11 (1H, d, J = 2.4 Hz), 7.69 (1H, dd, J = 2.4, 9.1 Hz), 6.83 (1H, d,
J = 2.4 Hz), 6.71 (2H, m), 6.61 (1H, d, J = 9.1 Hz), 4.62 (2H, s), 4.05
(2H, m), 3.78 (3H, s), 3.74 (3H, s), 3.06 (2H, t, J = 6.8 Hz); 13C
NMR (100 MHz, CDCl3 ppm) d: 179.2 (C@S), 169.6, 154.2, 151.5,
150.3, 146.9, 141.2, 129.2, 117.8, 113.0, 112.7, 112,6, 111.6, 66.3,
55.5, 52.2, 45.7, 29.9; HRMS (ES) m/z; found: [M+H]+, 454.0437.
Calcd. for C18H21BrN3O4S (M+H), 454.0436.
Using THF/rt, (37%); mp 141–142 °C as a yellow solid; IR
(CHCl3) mmax 3691, 3416, 1602, 1505, 1138 cmꢁ1
;
1H NMR
(300 MHz, CDCl3) d: 11.18 (1H, br s, NH), 8.80 (1H, br s, NH),
8.03 (1H, d, J = 2.6 Hz), 7.65 (1H, dd, J = 2.6, 8.7 Hz), 7.37 (5H,
m, ArH), 6.86 (1H, d, J = 9.0 Hz), 6.84 (1H, d, J = 3.2 Hz), 6.74
(1H, dd, J = 3.2, 9.0 Hz), 6.67 (1H, d, J = 8.7 Hz), 5.02 (2H, s), 4.03
(2H, m), 3.75 (3H, s), 3.03 (2H, t, J = 6.6 Hz); 13C NMR (75 MHz,
CDCl3) d: 179.0 (C@S), 153.6, 151.6, 151.1, 146.7, 141.1, 137.3,
128.8, 128.5, 127.8, 127.2, 117.7, 113.2, 112.9, 112.6, 111.5,
70.7, 55.6, 45.7, 30.0; HRMS (EI) m/z; found: 471.0594 (M+),
C22H22BrN3O2S (M+) requires 471.0616. Anal. Found: C, 55.48; H,
4.68; N, 8.59; S, 6.17%. C22H22BrN3O2S requires C, 55.94; H,
4.69; N, 8.90; S, 6.79.
4.5.12. N-(5-Bromo-2-pyridinyl)-N0-[2-(2-cyanomethyloxy-5-
methoxyphenyl)ethyl]-thiourea (6l)
Using THF/rt, (68%); mp 155–156 °C; IR (CHCl3) mmax 3681,
3413, 3022 (NH), 2239 (C„N), 1505, 1469 (C@S), 1216 cmꢁ1 1H
;
NMR (400 MHz, CDCl3) d: 11.20 (1H, br s, NH), 8.72 (1H, br s,
NH), 8.10 (1H, d, J = 2.4 Hz), 7.70 (1H, dd, J = 2.4 Hz, 8.8 Hz), 6.92
(1H, d, J = 8.8 Hz), 6.85 (1H, d, 3.2 Hz), 6.78 (1H, dd, J = 3.2,
8.8 Hz), 6.69 (1H, d, J = 8.8 Hz), 4.75 (2H, s), 3.99 (2H, q,
J = 6.8 Hz), 3.76 (3H, s) 3.01 (2H, t, J = 6.8 Hz); 13C NMR
(100 MHz, CDCl3) d: 179.4 (C@S), 155.4, 151.6, 149.0, 146.7,
141.3, 129.8, 117.9, 115.4, 114.0, 113.3, 112.8, 111.9, 55.6, 54.9,
45.6, 29.6. Anal. Found: C, 48.38; H, 4.04; N, 12.38; S; 6.76%.
C17H17BrN4O2S requires C, 48.46; H, 4.07; N, 13.30; S, 7.61.
4.5.8. N-(5-Bromo-2-pyridinyl)]-N0-{2-[2-(2-Benzyloxyethyl-1-
oxy)-5-methoxyphenyl]ethyl}-thiourea (6h)
Using THF/rt, (58% yield); mp 110–111 °C; IR (CHCl3) mmax 3692,
3416 (NH), 3175, 3016, 1506, 1475 (C@S) cmꢁ1; 1H NMR (400 MHz,
CDCl3) d: 11.09 (1H, br s, NH), 8.38 (1H, br s, NH), 8.04 (1 H, d,
J = 2.4 Hz), 7.66 (1H, dd, J = 2.4 Hz, 8.8 Hz), 7.31 (5H, m, ArH),
6.81 (1H, d, J = 8.8 Hz), 6.80 (1H, d, J = 3.2 Hz), 6.74 (1H, dd,
J = 3.2 Hz, 8.8 Hz), 6.58 (1H, d, J = 8.8 Hz), 4.63 (2H, s), 4.11 (2H, t,
J = 4.8 Hz), 4.03 (2H, m), 3.84 (2H, t, J = 4.8 Hz), 3.75 (3H, s), 3.01
(2H, t, J = 6.6 Hz); 13C NMR (100 MHz, CDCl3) d: 179.0 (C@S),
153.6, 151.8, 151.3, 146.6, 141.0, 138.1, 128.8, 128.5, 127.7,
127.7, 117.7, 113.4, 112.8, 112.9, 111.6, 73.4, 68.9, 68.5, 55.6,
45.7, 30.0. Anal. Found: C, 56.07; H, 4.86; N, 7.37; S, 5.80%.
C24H26BrN3O3S requires C, 55.82; H, 5.07; N, 8.14; S, 6.21.
4.5.13. N-(5-Bromo-2-pyridinyl)]-N0-{2-[2-(3-cyanopropyl-1-
oxy)-5-methoxyphenyl]ethyl}-thiourea (6m)
Using THF/rt, (53%); mp 165–166 °C; IR (CHCl3)
(NH), 3167, 2964 (C–H), 2246 (C„N), 1505, 1465 (C@S), 1239
(C–N) cmꢁ1 1H NMR (400 MHz, CDCl3) d: 11.26 (1 H, br s, NH),
mmax, 3413
;
9.34 (1H, br s, NH), 8.08 (1H, d, J = 2.4 Hz), 7.67 (1H, dd,
J = 2.4 Hz, 8.8 Hz), 6.82 (1H, d, J = 3.2 Hz), 6.81 (1H, d, J = 8.8 Hz),
6.77 (1H, d, J = 8.8 Hz), 6.74 (1H, dd, J = 3.2 8.8 Hz), 3.99 (4H, m),
3.74 (3H, s), 2.98 (2H, t, J = 6.8 Hz), 2.62 (2H, t, J = 7.0 Hz), 2.14
(2H, m); 13C NMR (75 MHz, CDCl3) d: 179.0 (C@S), 153.7, 151.7,
150.6, 146.5, 141.1, 128.4, 119.2, 117.7, 113.4, 112.7, 112.2,
111.5, 66.0, 55.5, 45.6, 29.9, 25.6, 14.4. Anal. Found: C, 50.61; H,
4.78; N, 12.37; S, 6.92%. C19H21BrN4O2S requires C, 50.78; H,
4.71; N, 12.47; S, 7.13.
4.5.9. N-(5-Bromo-2-pyridinyl)]-N0-{2-[5-methoxy-2-(2-propar-
gyloxyethoxy)phenyl]ethyl}-thiourea (6i)
Using DMF / rt, (62%); mp 128–130 °C; IR (CHCl3) mmax 3693,
3416, 3307, 3165, 2961, 1506, 1475, 1223 cmꢁ1
;
1H NMR
(300 MHz, CDCl3) d: 11.19 (1H, br s, NH), 9.24 (1H, br s, NH), 8.07
(1H, d, J = 2.6 Hz), 7.66 (1H, dd, J = 2.6, 8.7 Hz), 6.81–6.77 (3H, m),
6.72 (1H, dd, J = 2.9, 9.2 Hz), 4.25 (2H, d, J = 2.4 Hz), 4.09 (2H, t,
J = 4.8 Hz), 4.01 (2H, q, J = 6.6 Hz), 3.88 (2H, t, J = 4.8 Hz), 3.74 (3H,
s), 2.99 (2H, t, J = 6.6 Hz), 2.45 (1H, t, J = 2.4 Hz); 13C NMR (75 MHz,
CDCl3) d: 178.9 (C@S), 153.6, 151.7, 151.1, 146.6, 141.0, 128.8,
117.6, 113.4, 112.9, 112.6, 111.5, 79.5, 74.7, 68.4, 68.2, 58.5, 55.5,
45.6, 29.9; HRMS (EI) m/z; found: 463.0578 (M+). C20H22BrN3O3S
(M+) requires 463.0565. Anal. Found: C, 51.76; H, 4.72; N, 8.66; S,
6.86%. C20H22BrN3O3S requires C, 51.73; H, 4.78; N, 9.05; S, 6.91.
4.5.14. N-(5-Bromo-2-pyridinyl)]-N0-{2-[2-(2-hydroxyethoxy)-
5-methoxyphenyl]ethyl}-thiourea (6n)
Using THF/ rt, (65%); mp 162–163 °C; IR (CHCl3) mmax 3623, 3413,
3181 (NH, OH), 2928, 1505 (C–H), 1472 (C@S) 1227 (C–N) cmꢁ1; 1H
NMR (400 MHz, CDCl3) d: 11.16 (1H, br s, NH), 8.47 (1H, br s, NH),
8.12 (1H, d, J = 2.4 Hz), 7.70 (1H, dd, J = 2.4, 8.8 Hz), 6.81 (1H, d,
J = 2.8 Hz), 6.77 (1H, d, J = 8.8 Hz), 6.73 (1H. dd, J = 2.8, 8.8 Hz), 6.66
(1H, d, J = 8.8 Hz), 3.99 (6H, m), 3.77 (3H, s), 3.03 (2H, t, J = 7.2 Hz),
2.66 (1H, br s); 13C NMR (75 MHz, CDCl3) d: 179.4 (C@S), 153.7,
151.6, 151.3, 146.7, 141.3, 128.3, 117.4, 113.4, 112.8, 112.5, 111.7,
70.3, 61.5, 55.6, 45.7, 30.3. Anal. Found: C, 47.96; H, 4.61; N, 9.77;
S, 7.01%. C17H20BrN3O3S requires C, 47.89; H, 4.75; N, 9.86; S, 7.52.
4.5.10. N-(5-Bromo-2-pyridinyl)-N0-{2-[5-methoxy-2-(2-(2-
propargyloxyethoxy)ethoxy)phenyl]ethyl}-thiourea (6j)
Using DMF/rt, (60%); mp 91–92 °C; IR (CHCl3) mmax 3691, 3416,
3307, 3166, 2935, 1506, 1475, 1266 cmꢁ1 1H NMR (300 MHz,
;
CDCl3) d: 11.11 (1H, br s, NH), 8.67 (1H, br s, NH), 8.07 (1H, d,
J = 2.4 Hz), 7.67 (1H, dd, J = 2.4, 8.7 Hz), 6.81–6.78 (2H, m), 6.73
(1H, dd, J = 3.0, 8.7 Hz), 6.67 (1H, d, J = 8.7 Hz), 4.21 (2H, d,
J = 2.1 Hz), 4.07 (2H, t, J = 4.9 Hz), 4.01 (2H, q, J = 6.6 Hz), 3.85
(2H, t, J = 4.9 Hz), 3.73 (7H, m), 2.99 (2H, t, J = 6.6 Hz), 2.42 (1H, t,
4.5.15. N-(5-Bromo-2-pyridinyl)]-N0-{2-[2-(3-hydroxypropyl-1-
oxy)-5-methoxyphenyl]ethyl}-thiourea (6o)
Using THF/rt, (51%), mp 163–165 °C; IR (CHCl3) mmax 3681
(N–H), 3406 (O–H) 3014, 1509, 1472 (C@S), 1216 (C–N) cmꢁ1 1H
;

10280
R. Hunter et al. / Bioorg. Med. Chem. 16 (2008) 10270–10280
NMR (400 MHz, CDCl3) d: 11.15 (1H, br s, NH), 8.46 (1H, br s, NH),
8.10 (1H, d, J = 2.4 Hz), 7.68 (1H, dd, J = 2.4, 8.8 Hz), 6.81 (1H, d,
J = 8.8 Hz), 6.81 (1H, d, J = 2.9 Hz), 6.74 (1H, dd, J = 2.9, 8.8 Hz),
6.62 (1H, d, J = 8.8 Hz), 4.05 (2H, t, J = 5.9 Hz), 4.00 (2H, m), 3.87
(2H, q, J = 5.9 Hz), 3.76 (3H, s), 2.98 (2H, t, J = 6.6 Hz), 2.05 (2H,
quin, J = 5.9 Hz), 2.04 (1H, br s, OH); 13C NMR (101 MHz, CDCl3
ppm) d: 179.1 (C@S), 153.5, 151.6, 151.3, 146.8, 141.2, 128.4,
117.7, 113.2, 112.7, 112.4, 111.6, 65.9, 60.1, 55.6, 45.8, 32.3, 30.0.
Anal. Found: C, 49.37; H, 4.88; N, 9.08; S, 6.60%. C18H22BrN3O4S
requires C, 49.09; H, 5.04; N, 9.54; S, 7.28.
References and notes
1. (a) Aquaro, S.; Svicher, V.; Schols, D.; Pollicita, M.; Antinori, A.; Balzarini, J.;
Perno, C. F. J. Leukoc. Biol. 2006, 80, 1103; (b) Meadows, D. C.; Gervay-Hague, J.
ChemMedChem 2006, 1, 16; (c) Stevens, M.; De Clercq, E.; Balzarini, J. Med. Res.
Rev. 2006, 26, 595.
2. (a) Pauwels, R. Antiviral Res. 2006, 71, 77; (b) De Clercq, E. J. Med. Chem. 2005,
48, 1297.
3. (a) D’Cruz, O. J.; Uckun, F. M. J. Enzyme. Inhib. Med. Chem. 2006, 21, 329; (b)
Waters, L.; John, L.; Nelson, M. Int. J. Clin. Prac. 2006, 61, 105; (c) Zhou, Z.; Lin,
X.; Madura, J. D. Infect. Disord.: Drug Targets 2006, 6, 391; (d) Balzarini, J. Curr.
Top. Med. Chem. 2004, 4, 921; (e) Pauwels, R. Curr. Opin. Pharmacol. 2004, 4,
437; (f) De Clercq, E. Chem. Biodiversity 2004, 1, 44; (g) Campiani, G.; Ramunno,
A.; Maga, G.; Nacci, V.; Fattorusso, C.; Catalanotti, B.; Morelli, E.; Novellino, E.
Curr. Pharm. Des. 2002, 8, 615.
4.6. Anti-HIV evaluation
4. Muhanji, C. I.; Hunter, R. Curr. Med. Chem. 2007, 14, 1207.
5. (a) Pedersen, L.; Jørgensen, P. T.; Nielsen, S.; Hansen, T. H.; Nielsen, J.; Pedersen,
E. B. J. Med. Chem. 2005, 48, 1211; (b) Velázquez, S.; Lobatón, E.; De Clercq, E.;
Koontz, D. L.; Mellors, J. W.; Balzarini, J.; Camarasa, M. J. J. Med. Chem. 2004, 47,
3418; (c) Gavriliu, D.; Fossey, C.; Ciurea, A.; Delbederi, Z.; Sugeac, E.; Ladurée,
D.; Schmidt, S.; Laumond, G.; Aubertin, A. M. Nucleosides Nucleotides Nucleic
Acids 2002, 21, 505.
6. (a) Pontikis, R.; Dollé, V.; Guillaumel, J.; Dechaux, E.; Note, R.; Nguyen, C. H.;
Legraverend, M.; Bisagni, E.; Aubertin, A. M.; Grierson, D. S.; Monneret, C. J.
Med. Chem. 2000, 43, 1927; (b) Velázquez, S.; Tuñón, V.; Jimeno, M. L.;
Chamorro, C.; De Clercq, E.; Balzarini, J.; Camarasa, M-J. J. Med. Chem. 1999, 42,
5188; (c) Renoud-Grappin, M.; Fossey, C.; Fontaine, G.; Ladurée, D.; Aubertin, A.
M.; Kirn, A. Antiviral Chem. Chemother. 1998, 9, 205.
The following reagents were obtained through the NIH AIDS
Research and Reference Reagent Program, Division of AIDS, NIAID,
NIH: MT-2 cells and Nevirapine-Resistant HIV-1 (N119) from Dr.
Douglas Richman and HTLV-IIIB/H9 from Dr. Robert Gallo.
Antiviral activity and cellular toxicity were determined using
the MTT colorimetric method.19 MT-2 cells29 at a concentration
of 1 ꢂ 105 cells per millilitre were infected with wild-type HIV-
IIIB30 or Y181C mutant virus31 at a multiplicity of infection (MOI)
of 0.1. Infected and mock-infected cells were incubated in growth
medium (RPMI 1640, 10% dFBS, kanamycin) for 5 days with vary-
ing concentrations of each compound being tested in triplicate in
a 96-well plate. MTT, a cell-permeable tetrazolium dye was then
added to each well. After 5 h, acidified isopropanol was added to
lyse the cells and stop the reaction. The plates were gently shaken
overnight, and the absorbance measured at 595 nm on a plate
reader. The average of these triplicate samples was then plotted
versus inhibitor concentration to generate dose–response curves.
The 50% effective concentration (EC50) and 50% cytotoxic concen-
tration (CC50) of the compounds were defined as the concentra-
tions required to inhibit viral replication and to reduce the
number of viable cells by 50%, respectively.
7. Nanni, R. G.; Ding, J.; Jacobo-Molina, A.; Hughes, S. H.; Arnold, E. Perspect. Drug
Discovery Des. 1993, 1, 129.
8. (a) Shuker, S.; Hajduk, P.; Meadow, R.; Fesik, S. Science 1996, 274, 1531; (b) Ren,
J.; Esnouf, R.; Garman, E.; Somers, D.; Ross, C.; Kirby, I.; Keeling, J.; Darby, G.;
Jones, Y.; Stuart, D.; Stammers, D. Nat. Struct. Mol. Biol. 1995, 2, 293; (c) Jacobo-
Molina, A.; Arnold, E. Biochemistry 1991, 30, 6351; (d) Jacobo-Molina, A.; Ding,
J.; Nanni, R. J.; Clark, A. D.; Lu, X., Jr.; Tantillo, C.; Williams, R. L.; Kamer, G.;
Ferris, A. L.; Clark, P.; Hizi, A.; Hughes, S. H.; Arnold, E. Proc. Natl. Acad. Sci. U.S.A.
1993, 90, 6320.
9. Ruth, J. L.; Cheng, Y. C. J. Biol. Chem. 1982, 257, 10261.
10. Mao, C.; Sudbeck, E. A.; Venkatachalam, T. K.; Uckun, F. M. Bioorg. Med. Chem.
Lett. 1999, 9, 1593.
11. Hunter, R.; Muhanji, C. I.; Hale, I.; Bailey, C. M.; Basavapathruni, A.; Anderson,
K. S. Bioorg. Med. Chem. Lett. 2007, 17, 2614.
12. Sugeac, E.; Fossey, C.; Laduree, D.; Schmidt, S.; Laumond, G.; Aubertin, A. M. J.
Enzyme Inhib. Med. Chem. 2003, 18, 175.
13. Bell, F. W.; Cantrell, A. S.; Högberg, M.; Jaskunas, S. R.; Johansson, N. G.; Jordan,
C. L.; Kinnick, M. D.; Lind, P.; Morin, Jr. J. M.; Noréen, R.; Öberg, B.; Palkowitz, J.
A.; Parrish, C. A.; Pranc, P.; Sahlberg, C.; Ternansky, R. J.; Vasileff, R. T.; Vrang, L.;
West, S. J.; Zhang, H.; Zhou, X.-X. J. Med. Chem. 1995, 38, 4929.
14. Vig, R.; Mao, C.; Venkatachalam, T. K.; Tuel-Ahlgren, L.; Sudbeck, E. A.; Uckun, F.
M. Bioorg. Med. Chem. 1998, 6, 1789.
15. Mao, C.; Sudbeck, E. A.; Venkatachalam, T. K.; Uckun, F. M. Biochem. Pharmacol.
2000, 60, 1251.
16. Agrofoglio, L. A.; Gillaizeau, I.; Saito, Y. Chem. Rev. 2003, 103, 1875.
17. Glennon, R. A.; Liebowitz, S. M.; Leming-Doot, D.; Rosecrans, J. A. J. Med. Chem.
1980, 23, 990.
18. Arnott, G.; Hunter, R.; Mbeki, L.; Mohamed, E. Tetrahedron Lett. 2005, 46, 4023.
19. Pannecouque, C.; Daelemans, D.; De Clercq, E. Nat. Protoc. 2008, 3, 427.
20. Barreiro, G.; Kim, J. T.; Guimaraes, C. R. W.; Bailey, C. M.; Domaoal, R. A.; Wang,
L.; Anderson, K. S.; Jorgensen, W. L. J. Med. Chem. 2007, 50, 5324.
21. Morris, G. M.; Goodsell, D. S.; Halliday, R. S.; Huey, R.; Hart, W. E.; Belew, R. K.;
Olson, A. J. J. Comput. Chem. 1998, 19, 1639.
22. Ren, J.; Diprose, J.; Warren, J.; Esnouf, R. M.; Bird, L. E.; Ikemizu, S.; Slater, M.;
Milton, J.; Balzarini, J.; Stuart, D. I.; Stammers, D. K. J. Biol. Chem. 2000, 275, 5633.
23. (a) Venkatachalam, T. K.; Sudbeck, E.; Uckun, F. M. J. Mol. Struct. 2005, 751, 41;
(b) Sudbeck, E. A.; Jennissen, J. D.; Venkatachalam, T. K.; Uckun, F. M. Acta
Crystallogr. Sect. C: Cryst. Struct. Commun. 1999, C55, 2122.
4.7. Steady-state IC50 determination
6 nM RT (active sites based on pre-steady-state active site
determination) was pre-incubated for at least 15 min with 1 lM
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 with
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 sub-
jected to 20% denaturing polyacrylamide gel-electrophoresis and
quantitated 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
nucleotide incorporation by 50%.
´
24. Udier-Blagovic,M.;Tirado-Rives,J.;Jorgensen,W.L.J. Am.Chem. Soc. 2003,125,6016.
25. Chen, C-L.; Venkatachalam, T. K.; Waurzyniak, B.; Chelstrom, L.; Uckun, F. M.
Arzneimittel-Forschung 2001, 51, 574.
26. GaussView, Version 3.09. Dennington, R. D.; Keith, T.; Millam, J.; Eppinnett, K.;
Hovell, W. L.; Gilliland, R. Semichem, Inc., Shawnee Mission, KS, 2003.
27. (a) Marsili, M.; Gasteiger, J. Croat. Chem. Acta 1981, 53, 601; (b) Gasteiger, J.;
Marsili, M. Tetrahedron 1980, 36, 3210.
Acknowledgments
28. Mehler, E. L.; Solmajer, T. Protein Eng. 1991, 4, 903.
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.
29. (a) Haertle, T.; Carrera, C. J.; Wasson, D. B.; Sowers, L. C.; Richmann, D. D.;
Carson, D. A. J. Biol. Chem. 1988, 263, 5870; (b) Harada, S.; Koyanagi, Y.;
Yamamoto, N. Science 1985, 229, 563.
30. (a) Popovic, M.; Read-Connole, E.; Gallo, R. Lancet 1984, ii, 1472; (b) Popovic,
M.; Sarngadharan, M. G.; Read, E.; Gallo, R. C. Science 1984, 224, 497; (c) Ratner,
L.; Haseltine, W.; Patarca, R.; Livak, K. J.; Starcich, B.; Josephs, S. F.; Doran, E. R.;
Rafalski, J. A.; Whitehorn, E. A.; Baumeister, K.; Ivanoff, L.; Petteway, S. R., Jr.;
Pearson, M. L.; Lautenberger, J. A.; Papas, T. S.; Ghrayab, J.; Chang, N. T.; Gallo,
R. C.; Wong-Stall, F. Nature 1985, 313, 277.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2008.10.048.
31. Richman, D.; Shih, C-K.; Lowy, I.; Rose, J.; Prodanovic, P.; Goff, S.; Griffin, J.
Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 11241.