Quinol derivatives as potential trypanocidal agents

Article abstract of DOI:10.1016/j.bmc.2011.12.018

Quinols have been developed as a class of potential anti-cancer compounds. They are thought to act as double Michael acceptors, forming two covalent bonds to their target protein(s). Quinols have also been shown to have activity against the parasite Trypa

Full text of DOI:10.1016/j.bmc.2011.12.018

Bioorganic & Medicinal Chemistry 20 (2012) 1607–1615
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Quinol derivatives as potential trypanocidal agents
Amy Capes a, , Stephen Patterson a, , Susan Wyllie a, Irene Hallyburton a, Iain T. Collie a,
Andrew J. McCarroll b, Malcolm F.G. Stevens c, Julie A. Frearson a, Paul G. Wyatt a, Alan H. Fairlamb a,
Ian H. Gilbert a,
⇑
a Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dundee DD1 5EH, UK
b Pharminox Ltd, Biocity, Pennyfoot St., Nottingham NG1 1GF, UK
c Centre for Biomolecular Sciences, School of Pharmacy, University of Nottingham, Nottingham NG7 2RD, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
Quinols have been developed as a class of potential anti-cancer compounds. They are thought to act as
double Michael acceptors, forming two covalent bonds to their target protein(s). Quinols have also been
shown to have activity against the parasite Trypanosoma brucei, the causative organism of human African
trypanosomiasis, but they demonstrated little selectivity over mammalian MRC5 cells in a counter-
screen. In this paper, we report screening of further examples of quinols against T. brucei. We were able
to derive an SAR, but the compounds demonstrated little selectivity over MRC5 cells. In an approach to
increase selectivity, we attached melamine and benzamidine motifs to the quinols, because these moie-
ties are known to be selectively concentrated in the parasite by transporter proteins. In general these
transporter motif-containing analogues showed increased selectivity; however they also showed reduced
levels of potency against T. brucei.
Received 23 September 2011
Revised 9 December 2011
Accepted 10 December 2011
Available online 27 December 2011
Keywords:
Inhibitors
Medicinal chemistry
Trypanosoma brucei
P2 transporter
Quinols
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
to bind preferentially to vicinal cysteines (Scheme 1).5,13,14 Quinol
protein targets have been shown to include b-tubulin, heat shock
Human African trypanosomiasis (HAT) or sleeping sickness,
caused by the parasite Trypanosoma brucei, is a major health bur-
den in sub-Saharan Africa.1 Currently, there are four licensed
drugs2 and one licensed drug combination3 for the treatment of
HAT, all of which suffer from problems such as high toxicity, inap-
propriate administration in a rural African setting, or the emer-
gence of drug resistance. HAT is invariably fatal if left untreated,
therefore the need for new drugs is urgent.1
protein 60, peroxiredoxin 1,15 Mycobacterium tuberculosis
thioredoxin C16 and the redox-regulatory protein thioredoxin
(Trx).5,13 Thioredoxin reductase (TrxR) has also been identified as
a target, although in the case of TrxR the quinol motif forms a bond
with a nucleophilic selenocysteine residue in the active site.11,12
The thioredoxin system is important in countering oxidative stress
in the cell, and inhibition of its function eventually leads to
apoptosis.13,17
Quinols (compounds containing
a
4-hydroxycyclohexa-2,
While T. brucei does not have a thioredoxin reductase/thiore-
doxin system it possesses an analogous thiol metabolism that is
only found in trypanosomatids, including Trypanosoma and
Leishmania species.18 The active metabolite of the drug melarso-
prol, melarsen oxide, is also known to bind irreversibly to vicinal
cysteines such as those in trypanothione19 as well as to other sub-
strates containing a similar arrangement of cysteines.20 Because
melarsoprol is a very potent trypanocide, compounds containing
a quinol motif may also show trypanocidal activity. We have pre-
viously reported data for seven quinol analogues against T. brucei,
and established that these compounds form stable complexes with
the anti-oxidant trypanothione, as well as inhibiting two classes of
tryparedoxin peroxidase.21 In this paper, we report the screening of
a larger library of quinols to establish a more comprehensive SAR.
These compounds showed anti-parasitic activity, but no selectivity
compared to human MRC5 cells. We then describe our approaches
to achieve selectivity.
5-dien-1-one moiety) have been investigated as potential anti-can-
cer agents.4,5 The quinol pharmacophore was discovered during
investigation of polyphenol tyrphostin kinase inhibitors; the poly-
phenols were unstable with respect to oxidation and it was noticed
that the resultant quinol oxidation products were more potent
than the parent compounds.6 Although work has continued on
the ‘parent’ phenolic compounds,7–10 the quinol pharmacophore
was identified as the active moiety, and a series of compounds
bearing this novel motif were found to be active against human co-
lon and breast cancer cell lines.11,12
The quinol moiety is a double Michael acceptor, which is able to
react with nucleophiles in various cellular proteins, and is thought
⇑
Corresponding author. Fax: +44 1382 386373.
E-mail address: i.h.gilbert@dundee.ac.uk (I.H. Gilbert).
These authors contributed equally to this work.
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.12.018

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A. Capes et al. / Bioorg. Med. Chem. 20 (2012) 1607–1615
O
O
O
S
S
SH
HS
S
HS
HO
HO
HO
R
R
R
Scheme 1. Proposed mode of action of the quinols. Cysteine residues are abbreviated to -SH.
2.2. Selectively targeting quinols to T. brucei
2. Results and discussion
2.1. Screening
One possible strategy to circumvent the poor selectivity of the
quinols for T. brucei over MRC5 cells is to selectively target them
to the trypanosome. We decided to investigate this strategy by
attaching melamine and benzamidine moieties to the quinols. T.
brucei is auxotrophic for all purines, which it scavenges from the
bloodstream of its host. In order to do this, T. brucei has a range
of nucleobase transporters in the cell membrane.27 The first such
transporter characterised was the P2 transporter.28 In addition to
the uptake of the physiological substrates adenine and adenosine,
the P2 transporter is also able to take up compounds containing
melamine and benzamidine moieties. Thus it is involved in the
selective concentration of the drugs melarsoprol, pentamidine
and berenil into trypanosomes. Melarsoprol and pentamidine are
used for treating HAT and berenil is a treatment for animal try-
panosomiasis. As the biology has been further investigated other
transporters involved in the uptake of melamine and benzamidine
moieties in trypanosomes have been discovered, such as HAPT1
and LAPT1.29 Nevertheless, melamine and benzamidine moieties
are selectively taken up into trypanosomes and this strategy has
been used to selectively target compounds to trypanosomes, with
considerable success in some cases.30
The SAR studies above were used to inform where the benzam-
idine or melamine targeting motif should be attached to the phar-
macophore. Attachment of substituents via an acetylene linker did
not give potent compounds (1 and 3), and modification of the ben-
zothiazole appeared problematic. Attaching the targeting motif to
the triazole would be feasible and result in relatively small mole-
cules; however, it would need to be attached directly to the tria-
zole, rather than through a linker (compare 8 and 9 with 12).
Similarly, attachment of the P2 motif to the R1 position of the indo-
lyl would also be synthetically feasible, although it would produce
larger molecules.
We report data for an additional 17 quinol analogues screened
against blood stream form T. brucei. This quinol collection repre-
sents a subset of known compounds22,23 in addition to two new
analogues (3 and 12). The synthesis of the latter two molecules
is described below. These latter compounds were prepared to fur-
ther investigate the SAR; their synthesis is shown in the chemistry
section. Together with our previously reported assay data,21 this al-
lows us to derive some SAR trends over several sub-series of qui-
nols. The results of this screening are shown in Table 1.
In general, the quinols showed good activity against T. brucei,
with several EC50 values in the low nanomolar range (6, 10, 15,
16, 18, 20–25), comparable in potency to pentamidine (8 nM)
and melarsoprol (33 nM) assayed under identical conditions.
However it is also apparent that there was essentially no selectiv-
ity over the human MRC5 cells, with most compounds showing a
ratio of EC50 (MRC5)/EC50 (T. brucei) of 63. The only compounds
showing a marginally higher selectivity were also the least active
analogues (11, 12). This poor selectivity ratio could be because
the compounds have so far been optimised for their activity
against human cancer cell lines. Whilst these compounds poten-
tially have value in targeting cancer cells, their selectivity is not
sufficient for treating trypanosomiasis, based on our cellular
studies. Therefore, a method for conferring selectivity on the
compounds is essential if they are to be considered as potential
trypanocides.
A few generalisations can be made from this data, which mir-
rors what has been reported in cancer cell models:
ꢀ The quinol moiety is essential for activity. Compare the phenol 7
which is inactive, with its quinol homologue 4 (PMX 464), which
showed an EC50 of 77 nM against T. brucei.21
2.3. Chemistry
ꢀ There is some room for variation around the quinol ring, as has
been reported against cancer cell lines12: for example replacing
the hydroxyl group with a methoxy group only gave a small
(fourfold) change in potency (compare 4 and 5); addition of a
chlorine atom to the quinol moiety had no effect on activity
(compare 4 and 6).
ꢀ The nature of the aromatic substituent on the quinol is impor-
tant for potency, but not selectivity. Replacing a phenyl (2) with
a 2-benzothiazole (4) led to a 10-fold increase in potency.
2.3.1. Synthesis of quinol analogues
Analogues 3 and 12 were synthesised as outlined in Scheme 2.
Compounds 3 and 12 were designed to investigate additional posi-
tions at which a benzamidine or melamine moiety could be intro-
duced into the quinol pharmacophore. The 4-ethynyl substituted
quinol 1 was prepared as previously described in the literature31
(Scheme 2).
The triazole-containing quinol 12 was prepared by the use of a
copper catalysed azide alkyne Huisgen cycloaddition reaction
(click chemistry)32 between azide 30 and alkyne 1. Azide 30 was
prepared from acid chloride 28 following a literature route.33 Ana-
logue 3 contains an alkyne linker, which was introduced in the first
step of the synthesis using a Sonogashira coupling reaction. Aryl io-
dide 31 was coupled with alkyne 1 and the resultant acid 32 was
converted to the amide 3 by reaction with aniline and PyBrop.
Compounds with
a benzothiazole group (4–6; 46–330 nM
range) or an indolyl-sulfonamide linked to the quinol (13–25,
12–170 nM range) were generally potent, confirming the previ-
ous observation that the indolyl enhances the potency of these
compounds.25,26 The compounds containing a triazole linker
with different groups at the 1-position (8–12) have a range of
potencies over three orders of magnitude. This again broadly
reflects the results against cancer cell lines.23

Table 1
Quinol analogues and their inhibitory activities against T. brucei and MRC5 cells
HO
O
X
Y
O
N
R3
R1
HO
N
S
N
N
S
R1
Ph
HN
O
OH
O
O
O
N
HO
R2O
O
R1
A
B
C
3
7
Core
Compd
Structure
EC50 T. brucei (
l
M)a (SD, n)
EC50 MRC5 (
l
M)a (SD, n)
Spec. selectb
logPc
R1
R2
R3
X
Y
A
A
n/a
1
2
3
Ethynyl
Phenyl
n/a
H
H
n/a
H
H
n/a
n/a
n/a
n/a
n/a
n/a
n/a
2.0 (0.80, 4)
0.75d
2.2 (0.0017, 2)
2.1 (0.15, 4)
1.7d
0.67 (0.088, 2)
1
2
0.3
0.76
1.3
3.3
Benzothiazoles
A
A
A
n/a
4e
5
2-Benzo[d]thiazole
2-Benzo[d]thiazole
2-Benzo[d]thiazole
n/a
H
CH3
H
H
H
Cl
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
0.077d
0.33d
0.18d
2
3
1
n/a
1.8
2.7
1.9
3.5
1.1d
6
7
0.046 (0.0067, 2)
0.055 (0.037, 4)
n/a
>50d
>50d
Triazoles
8
9
10
11
12
B
B
B
B
B
4-Methoxyphenyl
4-Methoxybenzyl
6-Benzo[d]thiazole
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
0.13d
0.16 (0.054, 4)
0.027 (0.0018, 2)
10 (6.2, 4)
0.31d
2
3
3
>5
7
0.75
1.1
1.5
0.45 (0.12, 4)
0.069 (0.017, 4)
>50 (n/a, 3)
18 (2.0, 2)
1-(b-
D
-Galactopyranosyl, 2,3,4,6-tetraacetate)
ꢁ0.30
CH2CH2CONHPh
2.7 (0.084, 2)
0.77
Indolyls
13
14
15
16
17
18
19
20
21
22
23
24
C
C
C
C
C
C
C
C
C
C
C
C
C
H
H
H
CH3
NHCO2Et
CH2CH2NHAc
CH2CH2CO2CH3
CH2CH2CH2OH
CH2CH2CO-N-morpholine
CH2CH2CO-4-(1-methylpiperazine)
CH2CH2CH2-N-morpholine
CH2CH2CH2-4-(1-methylpiperazine)
SO2CH3
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
CH
CH
N
CF
0.10d
0.17d
2
2
2
2
2
3
2
2
2
3
3
3
2
2.3
2.3
1.4
1.8
2.3
1.9
2.4
2.1
1.5
1.6
2.5
2.6
1.5
CH
CH
N
0.053d
0.11d
0.022 (0.0026, 4)
0.026 (0.0065, 4)
0.050 (0.016, 4)
0.012 (0.0019, 2)
0.16 (0.066, 14)
0.024 (0.0017, 2)
0.036 (0.0097, 4)
0.018 (0.0032, 6)
0.026 (0.00042, 2)
0.016 (0.0063, 4)
0.024 (0.0017, 4)
0.041 (0.021, 4)
0.044 (0.028, 4)
0.086 (0.029, 4)
0.036 (0.0064, 4)
0.28 (0.14, 13)
0.051 (0.015, 4)
0.067 (0.014, 4)
0.052 (0.022)
0.079 (0.043, 4)
0.050 (0.023, 4)
0.039 (0.0072, 4)
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
25
n/a is not applicable.
a
The EC50 values are the arithmetic mean of independent determinations with the standard deviations (SD) followed by the number of repeats (n) given in parentheses.
Species selectivity (Spec. select.) is defined as EC50 (MRC5)/EC50 (T. brucei).
Calculated logP values generated using the software package StarDrop by Optibrium.24
Data taken from Konig et al.21
b
c
d
e
Previously published as PMX 464.

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A. Capes et al. / Bioorg. Med. Chem. 20 (2012) 1607–1615
O
OH
MeO
OMe
a
b
HO
O
OMe
26
27
1
O
O
O
O
c
d
e
HO
HO
Ph
Ph
Ph
O
Cl
Br
N
H
Br
N
H
N
N
N
H
N3
N
28
29
30
12
HO
OH
f
g
O
Ph
HN
O
HO
O
I
O
O
31
32
3
Scheme 2. Preparation of additional quinol analogues. Reagents and conditions: (a) PIDA, MeOH, 76%; (b) (i) HC„CMgBr, THF, ꢁ78 °C; (ii) CHCl3, silica, 90%; (c) aniline,
pyridine, CH2Cl2, 0–25 °C, 1 h, 69%; (d) NaN3, DMF, 60 °C, 16 h, 66%; (e) 1, CuI, lutidine, MeCN, 0–25 °C, 16 h, 54%; (f) 1, CuI, Pd(PPh3)4, DMAc, H2O, iPr2NH, 100 °C, 10 min, 11%;
(g) aniline, PyBrop, DMF, DIPEA, 0–25 °C, 16 h, 20%.
2.3.2. Synthesis of P2 transporter motif-containing quinol
analogues
was accessed via an SNAR of the chloro-diamino-triazine 36 with
sodium azide.35 These were then reacted with the quinol alkyne
1.31 The click chemistry reactions were carried out using lutedine
and CuI under anhydrous conditions to prevent N2 loss,36 which
was found to be a problem when using standard reaction condi-
tions (CuSO4, sodium ascorbate, water/MeCN).
Two strategies were investigated for attaching the quinol phar-
macophore to the melamine and benzamidine P2 targeting motifs.
In the first approach (Scheme 3), the melamine and benzamidine
moieties were attached via a triazole ring using click chemistry.
In the second approach, the targeting moiety was introduced as a
substituent to the indolylsulphonamides (Scheme 4), because
these were amongst the most potent derivatives. In addition, the
SAR indicates a broad tolerance to substituents at the para-position
of the phenylsulfonyl ring (e.g. 16–25).
Compounds 35 and 38 were synthesised using a copper cata-
lysed click reaction for the final step. The requisite azido benzam-
idine (34) was prepared by diazotization and subsequent
substitution of 33 with sodium azide,34 while the triazine azide
The indolyl-sulphonamide scaffold was prepared as shown in
Scheme 4, starting from the commercially available sulfonyl chlo-
ride 39 using a modification of a previously reported methodol-
ogy.22 A carboxylic acid was used as a ‘handle’ to attach the
melamine moiety by use of amide/ester coupling reactions. How-
ever, the previously reported syntheses of acid 42 involved a poor
yielding trimethyltin hydroxide mediated hydrolysis of the corre-
sponding methyl ester (19).25 Therefore, we attempted the Sono-
gashira coupling-indole forming cascade between the known aryl
iodide 41 and alkyne 1. Although it has been previously reported
that this reaction is unsuccessful22 the reaction did furnish acid
42, albeit in low yield. The hydroxyl linker P2 motif (43) required
for the ester synthesis was made as described previously.37 The lin-
ker 43 was then coupled to the indolyl carboxylic acid 42 using
either DIC or TBTU/HOAT, with the latter coupling conditions giv-
ing a cleaner product (44).
H2N
NH
H2N
NH
H2N
NH
a
b
N
N
The melamine-linker-amine moiety (45) required for the syn-
thesis of the corresponding amide analogue (46) was made in an
analogous fashion to the preparation of 43.37 The amide linkage
to the indolyl quinol was attempted using carbodiimide coupling.
However, it did not prove possible to separate the amide product
(46) from a structurally related side product of similar polarity.
To avoid the formation of side products, an alternative synthetic
strategy was used, where the amide bond was formed to give aryl
iodide 47 prior to the introduction of the quinol motif by reaction
with 1 (Scheme 4). This same strategy has previously been em-
ployed as a means to prepare a small collection of quinol-contain-
ing amides.22
N
OH
NH2
N3
O
33
34
35
H2N
N
N
NH2
N
N
N
H2N
N
NH2
H2N
N
NH2
c
d
N
N
N
N
N3
N
Cl
OH
2.4. Biological results
O
The melamine and benzamidine derived compounds (35, 38, 44,
46) were evaluated against T. brucei and MRC5 cells (Table 2).
The triazole derivatives that include a targeting motif (35 and
38) were found to have increased selectivity for T. brucei over
mammalian cells, as compared to the potent triazole analogues in
the initial screen (8–10). However, 35 and 38 show a significantly
36
37
38
Scheme 3. Preparation of triazole quinol analogues containing a benzamidine or a
melamine moiety. Reagents and conditions: (a) NaNO2, NaN3, H2SO4, H2O, 0 °C, 56%;
(b) 1, CuI, lutidine, MeCN, 4%; (c) NaN3, Et3N, DMF/H2O, 115 °C, 31%; (d) 1, CuI,
lutidine, MeCN, 3%.

A. Capes et al. / Bioorg. Med. Chem. 20 (2012) 1607–1615
1611
I
HO
HO
O
Cl
S
H2N
N
O
O
O
O
NH
S
N
O
N
N
O
O
a
c
d
S
S
O
O
NH2
N
NH2
N
O
H
O
O
OH
N
N
O
OCH3
OH
O
N
H
N
NH2
OR
39
R=CH3 40
R=H 41
42
43
44
b
I
HO
O
O
H2N
N
NH2
N
NH
NH2
N
S
O
S
O
N
N
e
O
f
g
NH2
NH2
N
N
41
46
N
N
HN
N
NH2
H
N
H
N
NH2
O
O
N
H
N
H
45
46 (purification unsuccessful)
47
Scheme 4. Preparation of indolyl quinol analogues containing a melamine moiety. Reagents and conditions: (a) 2-Iodoaniline, pyridine, THF, 25 °C, 16 h, 62%; (b) (i) KOH,
H2O, 100 °C, 1 h; (ii) HCl, H2O, 0 °C, 1 h, 95%; (c) 1, CuI, Pd(PPh3)4, DMAc, H2O, iPr2NH, 100 °C, 10 min, 14%; (d) DIC, DIPEA, DMF, 0–25 °C, 18 h, 67%; (e) 42, TBTU, HOAt, DIPEA,
i
DMF, 25 °C, 16 h, purification not successful; (f) 45, TBTU, HOAt, DIPEA, DMF, 25 °C, 16 h, 11%; (g) 1, CuI, Pd(PPh3)4, DMF, H2O, Pr2NH, 100 °C (MW), 10 min, 40%.
reduced activity compared to the analogues without a targeting
group: compounds 8 and 9 have an EC50 of 0.13 and 0.16 M,
respectively, compared to 35 and 38 at 11 and 6.2 M. The reason
for this is not known, but may be because of the targeting motif
reducing the ability of the quinol moiety to interact with its molec-
ular target(s). Interestingly, 11 and 12 also show the same pattern
of improved selectivity and decreased potency. This indicates that
it might not be the melamine or benzamidine targeting motif that
is conferring selectivity, but some additional factor(s).
The indolyl-sulphonamide derivative 46 also showed improved
selectivity compared to analogues without a targeting motif (see
compounds 13–25), although its potency is still significantly lower
than other indolyl-sulphonamide quinols (see compounds 13–25).
However it should be noted that the free acid 42 also shows im-
proved selectivity. This indicates that the melamine moiety may
not be responsible for conferring the selectivity observed for 46.
The ester linked indolyl quinol 44 also lacks selectivity. However,
it displays better potency than other P2 linked compounds, but is
still an order of magnitude less potent than the indolyl quinols
with no P2 motif (13–25).
l
l
In order to determine if analogues 35, 38, 44 and 46 are sub-
strates for the P2 transporter they were assayed against a line of
T. brucei lacking the gene which codes for the P2 transporter (Table
2).33 If the analogues are transported they would be expected to
show greater potency against wild type T. brucei than against the
P2 knock out line. Compounds 8 and 19,25 which contain the tria-
zole and indolyl quinol pharmacophore respectively, but lack a P2
motif, served as negative controls. Berenil, which is already known
to be selectively taken up by the P2 transporter was used as a po-
sitive control.40 The protocol used to assay the AT1 knock out line
is different to the HTS method used to measure the EC50 values re-
ported in Tables 1 and 2, therefore the compounds were assayed
against wild type T. brucei in parallel using the same protocol to
provide directly comparable inhibition values.
The results in Table 2 show that there is no difference in the
EC50s for analogues 35, 38, 44 and 46 in wild type T. brucei
Table 2
Activity of targeted compounds against blood stream form T. b. brucei, MRC5 cells and a P2 double knock out T. brucei cell line (T. brucei AT1 KO)
Compd T. brucei EC50
(SD, n)
(l
M)a,b
MRC5 EC50
(SD, n)
(
lM)
Spec.
T. brucei EC50
(SD)
(l
M)d,e
T. brucei AT1 KO EC50
(SD)
(l
M)d,e Cell line
selectivityf
logDh logPh
select.c
8
0.13g
11 (0.39, 4)
6.2 (0.50, 4)
0.16 (0.066, 14)
0.74 (0.027, 4)
0.35 (0.12, 7)
3.2 (0.36, 7)
0.31g
>50 (n/a, 2)
>50 (n/a, 2)
0.28 (0.14, 13)
3.3 (0.39, 4)
0.95 (0.24, 6)
18 (1.2, 6)
ND
2.4
>5
>8
1.8
4.5
2.7
5.6
—
0.095 (0.01)
5.0 (0.61)
3.8 (0.4)
0.060 (0.002)
ND
0.25 (0.026)
1.7 (0.17)
0.031 (0.0028)
0.074 (0.08)
6.0 (0.61)
3.8 (0.4)
0.080 (0.002)
ND
0.26 (0.026)
1.8 (0.17)
0.33 (0.0090)
0.78
1.2
1.0
1.3
—
1.0
1.1
11
0.76
ꢁ1.2
0.76
0.04
35
38
19
42
44
46
ꢁ0.66 ꢁ0.47
2.4
ꢁ0.30
1.5
1.4
1.8
2.4
2.1
1.8
1.3
0.00
Berenil ND
ND is not determined.
a
Determined using a 96-well HTS assay as previously described.38
b
c
d
e
f
The EC50 values are the arithmetic mean of independent determinations, with the standard deviations (SD) followed by the number of repeats (n) given in parentheses.
Species selectivity (Spec. select.) is defined as EC50 (MRC5)/EC50 (T. brucei).
Determined using a 96-well manual assay as previously described.39
The EC50 values are the weighted mean of three independent experiments, the standard deviations (SD) are given in parentheses.
Cell line selectivity is defined as EC50 (AT1 KO)/EC50 (WT).
g
h
Data taken from König et al.21
Calculated using StarDrop.24

1612
A. Capes et al. / Bioorg. Med. Chem. 20 (2012) 1607–1615
compared with those for the P2 knock out strain. These analogues
show identical selectivity (ꢂ1) to the negative controls (8 and 19)
and are therefore not substrates for the P2 transporter protein. This
indicates the compounds are unlikely to be actively transported by
the parasite. Berenil shows a 10-fold selectivity for the wild type
strain, consistent with values measured in the literature for this
cell line, confirming the validity of this assay data.
Because the transporter is not fully characterised, it is open to
speculation why these motifs do not allow the active transport of
these compounds. It is possible that the indolyl quinols (44 and
46) are too large (MW >600) to be channelled through transporters
whose physiological substrates are much smaller (e.g. adenine,
MW = 135). It is known that a benzamidine moiety needs to have
an oxygen or nitrogen atom in the para-position in order to be a
substrate for the P2 transporter. However, it is not known if a
para-triazole nitrogen (as is the case in analogue 35) provides a
suitable atom to complete the recognition motif; this may explain
why 35 is not actively transported. Similarly, it has not been deter-
mined if a triazole-melamine bi-aryl system (as in 38) can act as a
substrate for the P2 transporter in an analogous fashion to a phe-
nyl-melamine system.
to the 40–60 °C boiling fraction. Analytical thin-layer chromatogra-
phy (TLC) was performed on pre-coated TLC plates (layer 0.20 mm
Silica Gel 60 with fluorescent indicator UV254) (Merck). Developed
plates were air-dried and analysed under a UV lamp (UV254/
365 nm), and where necessary, stained with
a solution of
ninhydrin or iodine on silica to aid identification. 1H, 13C NMR
and 2D-NMR spectra were recorded on a Bruker Avance DPX 500
spectrometer (1H at 500.1 MHz, 13C at 125.8 MHz). Chemical shifts
(d) are expressed in ppm recorded using the residual solvent as the
internal reference in all cases. Signal splitting patterns are de-
scribed as singlet (s), doublet (d), triplet (t), quartet (q), multiplet
(m), broad (br), or a combination thereof. Coupling constants (J)
are quoted to the nearest 0.5 Hz. LC–MS analyses were performed
with either an Agilent HPLC 1100 series connected to a Bruker Dal-
tonics MicrOTOF, or an Agilent Technologies 1200 series HPLC con-
nected to an Agilent Technologies 6130 quadrupole LC/MS, both
instruments were connected to an Agilent diode array detector.
High resolution electrospray measurements were performed on a
Bruker Daltonics MicrOTOF mass spectrometer [Caution: take
appropriate care when preparing and handling azides.].
Assuming the compounds are predominantly taken up by pas-
sive diffusion, there could be a correlation between the logP value
and the activity against T. brucei. Thus compounds 35 and 38 have
low logP values, which may correlate to poor passive diffusion
across the cell membrane. The other compounds have larger logP
values, implying they are more lipophilic and hence have greater
cellular permeability. The only exception to this is berenil, which
has a low logP value, yet is very active against T. brucei; berenil
is known to be taken up rapidly by the P2 transporter. However
this permeability hypothesis cannot explain the lack of activity of
compounds 35 and 38 against MRC5 cells. It is possible that an-
other transporter, such as HAPT1 or LAPT133 may be involved in
weak uptake of these compounds in T. brucei.
3.2.1. Screening compounds
Compounds2, 4–11, 13–18 and 20–25 were provided by Dr An-
drew McCarroll (Pharminox Ltd, Nottingham, UK) and were pre-
pared as previously described.22,23
3.2.2. Synthesis
3.2.2.1. 4,4-Dimethoxycyclohexa-2,5-dienone (27).
Com-
pound previously reported by Pelter and Elgendy31: 4-methoxy
phenol (26) (2.48 g, 20 mmol) was dissolved in methanol (40 mL)
and a solution of PIDA (6.44 g, 20 mmol) in methanol (100 mL)
was transferred by cannula over 1.66 h. The reaction mixture was
concentrated in vacuo then the crude purified by column chroma-
tography (DCM/petrol 0:100–100:0) to afford the product as a yel-
low oil (2.35 g, 76%). 1H NMR (500 MHz, CDCl3): d = 3.23 (s, 6H,
2 ꢃ CH3), 5.94 (d, 2H, J = 10.5 Hz, d, 2 ꢃ CH), 6.52 ppm (d, 2H,
J = 10.5 Hz, 2 ꢃ CH); 13C NMR (125 MHz, CDCl3) d = 50.2 (CH3),
129.7 (CH), 92.3 (C), 143.3 (CH), 184.9 ppm (C).
2.5. Summary
Screening a collection of quinols established that this inhibitor
class is very potent against T. brucei, although the counter-screen
against MRC5 cells showed they lack sufficient selectivity to be
of therapeutic value against these parasites. The strategy of syn-
thesising compounds incorporating a P2 targeting motif demon-
strated it was possible to improve their selectivity, but this
proved to be at the expense of potency. Further experiments using
P2 knock out parasites confirmed that the P2 motif-containing
compounds are not substrates for the P2 transporter; therefore
they derive their selectivity through another mechanism.
3.2.2.2. 4-Ethynyl-4-hydroxycyclohexa-2,5-dienone (1).
Com-
pound 27 (2.35 g, 15.2 mmol) was dissolved in THF (100 mL) and
cooled to ꢁ78 °C. Ethynylmagnesium bromide (60.8 mL, 0.5 M solu-
tion in THF, 30.4 mmol), was added slowly by syringe. The mixture
was allowed to warm to room temperature, and was stirred for 2 h.
The reaction mixture was diluted with ether, quenched with saturated
aqueous NH4Cl solution, and then extracted with Et2O (3 ꢃ 150 mL).
The combined organic extracts were then washed with water, brine,
dried over MgSO4, and filtered. They were then concentrated in vacuo,
and the crude was redissolved in chloroform and stirred with silica gel
overnight to afford complete deprotection of the ketone. The solution
was again filtered and concentrated then purified by column chroma-
tography (EtOAc/hexane 25:75–60:40) to afford the product as sticky
yellow oil (1.86 g, 90%). 1H NMR (500 MHz, CDCl3) d = 2.64 (s, 1H,
CH), 6.22 (d, 2H, J = 10.1 Hz, 2 ꢃ CH), 6.94 ppm (d, 2H, J = 10.1 Hz,
2 ꢃ CH); 13C NMR (125 MHz, CDCl3) d = 62.0 (CH), 74.5 (C), 80.3 (C),
127.3 (CH), 147.1 (CH), 185.2 ppm (C); LRMS (LC–MS ES+): m/z 135
(M+H+, 100); HRMS: calcd mass for C8H7O2 [M+H+]: 135.0441, found
135.0443 (ꢁ2.05 ppm).
3. Experimental section
3.1. Biology
3.1.1. Cell-based assays
The 96-well HTS assays against blood stream form T. brucei38 and
MRC5 cells were carried out as reported previously.39 The manual
assays performed on the AT1 double knock out T. brucei and wild
type T. brucei were conducted as described by Jones et al.39
3.2. Chemistry
3.2.2.3.
(29)41
Synthesis
of
3-bromo-N-phenylpropanamide
General: Chemicals and solvents were purchased from the Al-
drich Chemical Company, Fluka, ABCR, VWR, Acros, Fisher Chemi-
cals and Alfa Aesar and were used as received unless otherwise
stated. Air and moisture sensitive reactions were carried out under
an inert atmosphere of argon in oven-dried glassware. Petrol refers
.
3-Bromopropionyl chloride (2.2 mmol, 377 mg) was
slowly added to a solution of aniline (2 mmol, 186 mg) in anhy-
drous CH2Cl2 (60 mL) at 0 °C. Pyridine (2.2 mmol, 174 mg) was
added to the resultant mixture and the reaction was warmed to
25 °C with stirring for 1 h. Workup was initiated by the addition

A. Capes et al. / Bioorg. Med. Chem. 20 (2012) 1607–1615
1613
of a NaHCO3 solution (satd aq 40 mL), the layers separated and the
CH2Cl2 layer further extracted with NaHCO3 (satd aq 40 mL), water
(2 ꢃ 40 mL) and brine (40 mL). The CH2Cl2 layer was dried over
MgSO4, filtered and the solvent removed under reduced pressure.
The crude product was purified by flash column chromatography
(EtOAc/Hexane 0:100–50:50) to give the product amide as a white
solid (158 mg, 69%) contaminated with ꢂ15% of the olefin from the
elimination of HBr. 1H NMR (500 MHz, CDCl3) d = 2.95 (t, 2H,
J = 6.5 Hz, CH2), 3.72 (t, 2H, J = 6.5 Hz, CH2), 7.13 (t, 1H, J = 7.5 Hz,
ArH), 7.28 (br s, 1H, NH), 7.34 (t, 2H, J = 7.5 Hz 2 ꢃ ArH),
7.52 ppm (d, 2H, J = 7.5 Hz, 2 ꢃ ArH); 13C NMR (125 MHz, CDCl3)
d = 27.2 (CH2), 40.7 (CH2), 120.2 (CH), 124.8 (CH), 129.1 (CH),
137.4 (C), 168.1 ppm (C); LRMS (LC–MS ES+): m/z 228 (79Br
M+H+, 100%), 230 (81Br M+H+, 100%).
irradiation at 100 °C for 10 min. After cooling the reaction mixture
was added to CH2Cl2/water (1:1, 200 mL), the layers separated and
the aqueous layer extracted with CH2Cl2 (3 ꢃ 50 mL). The com-
bined CH2Cl2 layers were dried over MgSO4, filtered and the sol-
vent removed under reduced pressure to give a brown tar, which
was purified by silica flash column chromatography (MeOH/CH2Cl2
i
0:100–20:80) to give the product as its Pr2NH salt. The salt was
partitioned between 1 N aqueous HCl/EtOAc (1:1, 10 mL), the lay-
ers separated, the EtOAc layer dried over MgSO4, filtered and evap-
orated under reduced pressure to give the free acid as a brown
solid (29 mg, 11%). 1H NMR (500 MHz, CD3OD) d = 3.52 (s, 2H,
CH2), 6.06–6.10 (m, 2H, AA0BB0, 2 ꢃ CH), 6.93–6.96 (m, 2H, AA0BB0,
2 ꢃ CH), 7.17–7.19 (m, 2H, AA0BB0, 2 ꢃ ArH), 7.28–7.31 ppm (m,
2H, AA0BB0, 2 ꢃ ArH); LRMS (LC–MS ES+): m/z 251 (MꢁOH+,
100%), 269 (M+H+, 89%).
3.2.2.4.
(30)41
Synthesis
of
3-azido-N-phenylpropanamide
.
Sodium azide (0.76 mmol, 50 mg) was added to a solu-
3.2.2.7. 2-(4-((1-Hydroxy-4-oxocyclohexa-2,5-dien-1-yl)ethynyl)-
tion of alkyl bromide 28 (0.69 mmol, 158 mg) in anhydrous DMF
(2 mL) and the reaction heated at 60 °C with stirring for 16 h.
Subsequently the reaction was diluted with EtOAc (10 mL), succes-
sively washed with NaHCO3 (satd aq 1 ꢃ 10 mL), brine (1 ꢃ 10 mL),
dried over MgSO4, filtered and the EtOAc removed under reduced
pressure. The crude product was purified by silica flash column
chromatography (EtOAc/Hexane 0:100–50:50) to give the product
azide as a white waxy solid (87 mg, 69%) contaminated with olefin
carried through from the previous reaction. 1H NMR (500 MHz,
CDCl3) d = 2.60 (t, 2H, J = 6.5 Hz, CH2), 3.72 (t, 2H, J = 6.5 Hz, CH2),
7.13 (t, 1H, J = 7.5 Hz, ArH), 7.33 (t, 2H, J = 7.5 Hz 2 ꢃ ArH), 7.45
(br s, 1H, NH), 7.51 ppm (d, 2H, J = 7.5 Hz, 2 ꢃ ArH); 13C NMR
(125 MHz, CDCl3 d = 36.8 (CH2), 47.3 (CH2), 120.2 (CH), 124.7
(CH), 129.1 (CH), 137.5 (C), 168.4 ppm (C); LRMS (LC–MS ES+): m/
z 120 (MꢁCH2CH2N3+, 30%), 163 (M+HꢁN2+, 53%), 191 (M+H+,
100%), 213 (M+Na+, 14%).
phenyl)-N-phenylacetamide(3).
added to a solution of acid 32 (0.11 mmol, 29 mg), aniline (0.1 mmol,
9 mg) and PyBrop (0.1 mmol, 47 mg) in anhydrous DMF (500 L) at
DIPEA(0.4 mmol, 52 mg)was
l
0 °C. The reaction was allowed to warm to 25 °C and stirred for 16 h
before being diluted with EtOAc (10 mL) and washed with NaHCO3
solution (satd aq 50 mL). The aqueous layer was back-extracted with
EtOAc (3 ꢃ 50 mL), the combined EtOAc layers were dried over
MgSO4, filtered and the solvent removed under reduced pressure.
The crude product was purified by silica flash column chromatogra-
phy (MeOH/CH2Cl2 0:100–20:80) to give the product as a brown solid
(7 mg, 20%). 1H NMR (500 MHz, DMSO-d6) d = 3.36 (s, 1H, OH), 3.73 (s,
2H, CH2), 6.20–6.23 (m, 2H, AA0BB0, 2 ꢃ CH), 6.85 (s, 1H, ArH), 7.08–
7.13 (m, 3H, 2 ꢃ CH & ArH), 7.35 (dd, 2H, J = 7.5, 7.5 Hz, 2 ꢃ ArH),
7.39–7.42 (m, 2H, AA0BB0, 2 ꢃ ArH), 7.46–7.48 (m, 2H, AA0BB0,
2 ꢃ ArH), 7.64 (d, 2H, J = 7.5 Hz, 2 ꢃ ArH), 10.22 ppm (br s, 1H, NH);
13C NMR (125 MHz, DMSO-d6) d = 43.0 (CH2), 61.6 (C), 83.9 (C), 86.9
(C), 119.1 (CH), 119.3 (C), 123.3 (CH), 125.7 (CH), 128.7 (CH), 129.6
(CH), 131.5 (CH), 137.4 (C), 139.1 (C), 148.5 (CH), 168.5 (C), 184.5
(C) ppm. MS (LC–MS ES+): m/z 326 (MꢁOH+, 16%), 344 (M+H+,
100%), 366 (M+Na+, 46%); HRMS (ES+): calcd for C22H18N1O3 [M+H]+
344.1281, found 344.1281 (0.15 ppm).
3.2.2.5. 3-(4-(1-Hydroxy-4-oxocyclohexa-2,5-dien-1-yl)-1H-1,2,
3-triazol-1-yl)-N-phenylpropanamide (12)36
.
Copper (I) io-
dide (0.025 mmol, 5 mg) was added to a solution of azide 29
(0.25 mmol, 48 mg) and alkyne 1 (0.3 mmol, 34 mg) in degassed
CHCl3 (2 mL) at 0 °C, followed by the slow addition of 2,6-lutidine
(0.3 mmol, 32 mg). The resultant reaction mixture was allowed to
warm to 25 °C and stirred for 16 h, before being diluted with
CH2Cl2 (2 mL) and washed with NH4Cl solution (satd aq 4 mL).
The aqueous was then back-extracted with CH2Cl2 (3 ꢃ 10 mL),
the organic layers combined, dried over MgSO4, filtered and the
solvent removed under reduced pressure. The crude product was
purified by silica flash column chromatography (MeOH/EtOAc/
Hexane 0:0:100–10:90:0) to give the product 1,2,3-triazole as an
off-white foam (44 mg, 54%). 1H NMR (500 MHz, DMSO-d6)
d = 2.99 (t, 2H, J = 7.0 Hz, CH2), 4.66 (t, 2H, J = 7.0 Hz, CH2), 6.13–
6.16 (m, 2H, AA0BB0, 2 ꢃ CH), 6.47 (s, 1H, OH), 7.05 (t, 1H,
J = 7.5 Hz, ArH), 7.12–7.15 (m, 2H, AA0BB0, 2 ꢃ CH), 7.28–7.32 (m,
2H, 2 ꢃ ArH), 7.55 (d, 2H, J = 8.0 Hz, ArH), 8.17 (s, 1H, ArH),
10.03 ppm (s, 1H, NH); 13C NMR (125 MHz, DMSO-d6) d = 36.2
(CH2) 45.7 (CH2), 65.2 (C), 119.1 (CH), 123.1 (CH), 123.3 (CH),
126.0 (CH), 128.7 (CH), 138.9 (C), 147.8 (C), 150.7 (CH), 168.0 (C),
185.2 ppm (C); LRMS (LC–MS ES+): m/z 325 (M+H+, 100%), 649
(2M+H+, 36%); HRMS (ES+): calcd for C17H16N4Na1O3 [M+Na]+
347.1115, found 347.1127 (ꢁ3.63 ppm).
3.2.2.8. Methyl 3-(4-((2-(1-hydroxy-4-oxocyclohexa-2,5-dien-1-
yl)-1H-indol-1-yl)sulfonyl)phenyl) propanoate (19)25
.
Cop-
per (I) iodide (0.1 mmol, 19 mg) and tetrakis(triphenylphosphine)
palladium(0) (0.06 mmol, 69 mg) were added to a solution of aryl
iodide 40 (1.16 mmol, 517 mg) and alkyne 1 (1.0 mmol, 134 mg)
in degassed DMAc (1.0 mL), iPr2NH (200
lL) and water (40 lL)
and the resultant mixture heated under microwave irradiation at
100 °C for 10 min. After cooling the reaction mixture was added
to CH2Cl2/H2O (1:1, 200 mL), the layers separated and the aqueous
extracted with CH2Cl2 (3 ꢃ 50 mL). The combined CH2Cl2 layers
were dried over MgSO4, filtered and the solvent removed under re-
duced pressure. The product was purified by silica flash column
chromatography (EtOAc/hexane 0:100–50:50) to give the product
as a brown solid (314 mg, 70%). 1H NMR (500 MHz, CDCl3): d
2.58 (t, 2H, J = 7.5 Hz, CH2), 2.93 (t, 2H, J = 7.5 Hz, CH2), 3.61 (s,
3H, CH3), 5.48 (s, 1H, OH), 6.32–6.29 (m, 2H, AA0BB0 2 ꢃ CH), 6.80
(s, 1H, ArH), 7.32–7.20 (m, 4H, 4 ꢃ ArH), 7.43 (d, 1H, J = 7.5 Hz,
ArH), 7.58–7.55 (m, 2H, AA0BB0 2 ꢃ CH), 7.80–7.78 (m, 2H, AA0BB0
2 ꢃ ArH), 8.00 ppm (d, 1H, J = 8.5 Hz, ArH); 13C NMR (125 MHz,
CDCl3): d 30.6 (CH2), 34.6 (CH2), 51.8 (CH3), 67.6 (C), 113.6 (CH),
115.2 (CH), 121.7 (CH), 124.6 (CH), 126.2 (CH), 126.9 (CH), 127.6
(CH), 128.3 (C), 129.3 (CH), 135.4 (C), 138.2 (C), 140.7 (C), 147.6
(CH), 147.8 (C), 172.5 (C), 185.0 ppm (C); MS (LC–MS ES+): m/z
434 (MꢁOH+, 85%), 452 (M+H+, 100%), 474 (M+Na+, 33%). HRMS
3.2.2.6.
2-(4-((1-Hydroxy-4-oxocyclohexa-2,5-dien-1-yl)ethy-
Copper (I) iodide (0.1 mmol,
nyl)phenyl)acetic acid (32)25
.
19 mg) and tetrakis(triphenylphosphine)palladium(0) (0.06 mmol,
69 mg) were added to a solution of 4-iodophenylacetic acid (31)
(1.16 mmol, 304 mg) and alkyne 1 (1 mmol, 134 mg) in degassed
(ES+): calcd for
474.0977 (1.01 ppm).
C
24H21N1Na1O6S1 [M+Na]+ 474.0982, found
dimethylacetamide (DMAc) (1 mL), iPr2NH (200
lL) and water
(40
lL) and the resultant mixture heated under microwave

1614
A. Capes et al. / Bioorg. Med. Chem. 20 (2012) 1607–1615
3.2.2.9. 4-Azidobenzimidamide sulfate (34).
Compound 33
which was further purified by recrystallisation from hot EtOAc/
(500 mg, 3.7 mmol) was dissolved in H2SO4, and the viscous yel-
low-green solution was cooled to 0 °C. NaNO2 (200 mg, 2.9 mmol)
was added in two portions and stirred at 0 °C for 30 min. NaN3
(200 mg, 3.08 mmol) was added along with one drop of water.
The reaction mixture was stirred overnight, and turned from a yel-
low solution to a thick orange suspension. The reaction mixture
was filtered and the pale orange crystals washed with H2O and
MeOH. The orange crystals were then dried (332 mg, 56%). 1H
NMR (500 MHz, DMSO-d6) d = 7.24 (d, 2H, J = 9.0 Hz, 2 ꢃ ArH),
7.73 (d, 2H, J = 9.0 Hz, 2 ꢃ ArH), 8.80 (s, 2H, NH2), 9.17 ppm (s,
2H, NH2). 13C NMR (125 MHz, DMSO-d6) d = 119.6 (CH), 124.1
(C), 130.1 (CH), 145.2 (C), 164.4 ppm (C); LRMS (LC–MS ES+): m/z
162 (M+H+, 100%); HRMS: calcd for C7H7N5 [M+H]+ 162.0774,
found 162.0768 (4.01 ppm).
petrol to give a white crystalline solid (5.54 g, 62%). 1H NMR
(500 MHz, CDCl3) d = 2.64 (t, 2H, J = 7.5 Hz, CH2), 3.00 (t, 2H,
J = 7.5 Hz, CH2), 6.78 (br s, 1H, NH), 6.86 (ddd, 1H, J = 8.0, 7.5,
1.5 Hz, ArH), 7.26–7.29 (m, 2H, AA0BB0, 2 ꢃ ArH), 7.32–7.35 (m,
1H, ArH), 7.66–7.69 ppm (m, 4H, 4 ꢃ ArH); 13C NMR (125 MHz,
DMSO-d6) d = 30.0 (CH2), 34.3 (CH2), 51.3 (CH3), 98.6 (C), 126.9
(CH), 127.1 (CH), 128.4 (CH), 128.9 (CH), 129.0 (CH), 138.2 (C),
138.5 (C), 139.6 (CH), 145.9 (C), 172.4 ppm (C); LRMS (LC–MS
ES+): m/z 446 (M+H+, 94%), 463 (M+H2O+, 100%).
3.2.2.14. Synthesis of 3-(4-(N-(2-iodophenyl)sulfamoyl)phenyl)-
propanoic acid (41)22
.
Ester 40 (1.12 mmol, 500 mg) was dis-
solved in KOH solution (10% w/v, aq 10 mL) and heated at 100 °C
for 30 min. The reaction was then cooled to 0 °C before adjusting
the mixture to pH 2 by addition of HCl (1 N, aq). After standing
at 0 °C for 1 h the resulting precipitate was collected by filtration,
washed with water (2 ꢃ 10 mL) and dried under vacuum to give
3.2.2.10. 4-(4-(1-Hydroxy-4-oxocyclohexa-2,5-dienyl)-1H-1,2,3-
triazol-1-yl)benzimidamide (35).
Compound
1
(60 mg,
0.447 mmol), compound 34 (60 mg, 0.373 mmol) and CuI (7 mg,
the product acid as a
white solid (459 mg, 95%). 1H NMR
0.037 mmol) were dissolved in dry, degassed MeCN (1 mL). 2,6-
Lutedine (52 lL, 0.447 mmol) was added at 0 °C and the reaction
(500 MHz, DMSO-d6) d = 2.58 (t, 2H, J = 7.5 Hz, CH2), 2.90 (t, 2H,
J = 7.5 Hz, CH2), 6.96–6.99 (m, 2H, 2 ꢃ ArH), 7.28–7.32 (m, 1H,
ArH), 7.42–7.44 (m, 2H, AA0BB0, 2 ꢃ ArH), 7.61–7.63 (m, 2H, AA0BB0,
2 ꢃ ArH), 7.84 (dd, 1H, J = 8..5, 1.5 Hz, ArH), 9.72 (br s, 1H, NH),
12.22 ppm (br s, 1H, COOH); 13C NMR (125 MHz, DMSO-d6)
d = 30.1 (CH2), 34.6 (CH2), 98.7 (CI), 126.8 (CH), 127.1 (CH), 128.4
(CH), 128.9 (CH), 129.0 (CH), 138.2 (C), 138.4 (C), 139.6 (CH),
146.3 (C), 173.5 ppm (C); LRMS (LC–MS ES+): m/z 432 (M+H+,
52%), 449 (M+H2O+, 100%), 880 (2M+H2O+, 48%).
mixture was stirred for 18 h under argon. The crude was concen-
trated then purified twice by semi-prep HPLC under basic condi-
tions, to afford the product as a brown powder (5 mg, 4%). 1H
NMR (500 MHz, CD3OD) d = 6.30 (d, 2H, J = 10.0 Hz, 2 ꢃ CH), 7.23
(d, 2H, J = 10.0 Hz, 2 ꢃ CH), 8.2 (d, 2H, J = 8.0 Hz, 2 ꢃ ArH), 8.20
(d, 2H, J = 8.4 Hz, 2 ꢃ ArH), 8.84 ppm (s, 1H, ArH); LRMS (LC–MS
ES+): m/z 296 (M+H+, 100%); HRMS: calcd for C15H14N5O2
[M+H]+: 296.1142, found 296.1130 (4.18 ppm).
3.2.2.15.
3-(4-((2-(1-Hydroxy-4-oxocyclohexa-2,5-dien-1-yl)-
Cop-
3.2.2.11. 6-Azido-1,3,5-triazine-2,4-diamine (37).
pound 36 (500 mg, 3.44 mmol) was suspended in DMF (3 mL),
and TEA (275 L, 2.06 mmol) was added. NaN3 (270 mg,
Com-
1H-indol-1-yl)sulfonyl)phenyl)propanoic acid (42)25
.
per (I) iodide (0.25 mmol, 48 mg) and tetrakis(triphenylphosphine)
palladium(0) (0.15 mmol, 173 mg) were added to a solution of aryl
l
4.15 mmol) was added in H2O (2 mL) and the reaction was heated
to 115 °C for 18 h. During heating a precipitate formed which was
filtered off and washed with water, methanol and DCM to afford
the product as a beige powder (160 mg, 31%). 1H NMR (500 MHz,
DMSO-d6) d = 6.91 (br s, 2H, NH2), 6.97 ppm (br s, 2H, NH2); 13C
NMR (125 MHz, DMSO-d6) d = 167.6 (C), 168.4 ppm (C); LRMS
(LC–MS ES+): m/z 153 (M+H+, 100%); HRMS: calcd for C3H4N8
[M+H]+: 153.0632, found 153.0645 (ꢁ8.45 ppm).
iodide 41 (2.9 mmol, 1.25 g) and alkyne 1 (2.5 mmol, 335 mg) in
i
degassed DMAc (2.5 mL), Pr2NH (500
l
L) and water (100
lL) and
the resultant mixture heated under microwave irradiation at
100 °C for 10 min. After cooling the reaction mixture was added
to CH2Cl2/citrate solution (10% w/v aq) (1:1, 200 mL), the layers
separated and the aqueous layer extracted with CH2Cl2
(3 ꢃ 50 mL). The combined CH2Cl2 layers were dried over MgSO4,
filtered and the solvent removed under reduced pressure to give
a brown oil, which was purified by silica flash column chromatog-
raphy (MeOH/CH2Cl2 0:100 to 20:80) to give the product as a pale
brown solid (151 mg, 14%). 1H NMR (500 MHz, CDCl3) d = 2.61 (t,
2H, J = 7.5 Hz, CH2), 2.94 (t, 2H, J = 7.5 Hz, CH2), 5.55 (br s, 1H,
OH), 6.32–6.35 (m, 2H, AA0BB0, 2 ꢃ CH), 6.80 (d, 1H, J = 1.0 Hz,
ArH), 7.21–7.24 (m, 1H, ArH), 7.28–7.33 (m, 3H, 2 ꢃ CH & ArH),
7.56–7.60 (m, 2H, AA0BB0, 2 ꢃ ArH), 7.78–7.81 (m, 2H, AA0BB0,
2 ꢃ ArH), 8.00 ppm (dd, 1H, J = 8.5, 1.0 Hz, ArH); 13C NMR
(125 MHz, DMSO-d6) d = 30.0 (CH2), 34.1 (CH2), 67.1 (C), 112.3
(CH), 115.1 (CH), 121.6 (CH), 123.9 (CH), 125.5 (CH), 126.5 (CH),
126.9 (CH), 128.0 (C), 129.1 (CH), 135.9 (C), 137.8 (C), 141.1 (C),
148.0 (C), 149.8 (CH), 173.4 (C), 185.0 ppm (C); LRMS (LC–MS
ES+): m/z 420 (MꢁOH+, 77%), 438 (M+H+, 100%), 897 (2M+Na+,
13%); HRMS (ES+): calcd for C23H19N1Na1O6S1 [M+Na]+ 460.0825,
found 460.0826 (ꢁ0.06 ppm).
3.2.2.12. 4-(1-(4,6-Diamino-1,3,5-triazin-2-yl)-1H-1,2,3-triazol-
4-yl)-4-hydroxycyclohexa-2,5-dienone (38).
Compound 1
(72 mg, 0.474 mmol), compound 26 (60 mg, 0.395 mmol) and CuI
(8 mg, 0.040 mmol) were dissolved in dry, degassed MeCN
(1 mL). 2,6-Lutedine (55 lL, 0.474 mmol) was added at 0 °C, and
the reaction mixture was stirred for 18 h under argon. The crude
was concentrated then purified twice by semi-prep HPLC under ba-
sic conditions, to afford the product as an off-white powder (4 mg,
3%). 1H NMR (500 MHz, MeOD) d = 6.29 (d, 2H, J = 10.0 Hz, 2H,
2 ꢃ CH), 7.19 (d, 2H, J = 10.0 Hz, 2 ꢃ CH), 8.75 ppm (s, 1H, CH);
13C NMR (125 MHz, CDCl3) d = 67.0 (C), 122.3 (CH), 128.3 (CH),
150.1 (C), 151.1 (CH), 162.3 (C), 169.5 (C), 187.2 ppm (C); LRMS
(LC–MS ES+): m/z 287 (M+H+, 100%); HRMS: calcd for C11H11N8O2
[M+H]+: 287.0999, found 287.0990 (3.31 ppm).
3.2.2.13. 3-(4-(N-(2-Iodophenyl)sulfamoyl)phenyl)propanoate
3.2.2.16. 3-((4,6-Diamino-1,3,5-triazin-2-yl)amino)propyl 3-(4-
(40)22
.
2-Iodoaniline (20 mmol, 4.38 g) was added to a solu-
((2-(1-hydroxy-4-oxocyclohexa-2,5-dien-1-yl)-1H-indol-1-yl)
tion of methyl 3-(4-(chlorosulfonyl)phenyl)propanoate (39)
(20 mmol, 5.25 g) in anhydrous THF/pyridine (1:1, 16 mL) and
the reaction mixture stirred at 25 °C for16 h before removal of
the solvent under reduced pressure. The resultant crude product
was redissolved in CHCl3 (50 mL), washed with citrate solution
(10% w/v aq 3 ꢃ 50 mL), dried over MgSO4, filtered and the CHCl3
removed under reduced pressure to give an orange semi-solid
sulfonyl)phenyl)propanoate (44).
0.154 mmol), compound 43 (45 mg, 0.103 mmol) and DIPEA
(20 L, 0.113 mmol) were dissolved in DMF (0.5 mL). The reaction
mixture was cooled to 0 °C and DIC (24 L, 0.154 mmol) was
Compound 4237 (28 mg,
l
l
added. The reaction mixture was stirred under argon for 18 h,
and then purified directly by semi-prep HPLC to afford the product
as a beige powder (42 mg, 67%). 1H NMR (500 MHz, MeOD) d = 1.78

A. Capes et al. / Bioorg. Med. Chem. 20 (2012) 1607–1615
1615
(dd, 2H, J = 6.5 Hz, CH2), 2.64 (t, 2H, J = 7.5 Hz, CH2), 2.94 (t, 2H,
J = 7.5 Hz, CH2), 3.27 (t, 2H, J = 7.0 Hz, CH2), 4.07 (t, 2H, J = 6.0 Hz,
CH2), 6.27 (d, 2H, J = 10.0 Hz, 2 ꢃ CH), 6.94 (s, 1H, CH), 7.24 (t,
1H, J = 7.5 Hz, ArH), 7.34 (d, 2H, J = 8.5 Hz, 2 ꢃ ArH), 7.51 (d, 1H,
J = 8.0 Hz, ArH), 7.58 (d, 2H, J = 10.0, 2 ꢃ CH), 7.84 (d, 2H,
J = 8.5 Hz, 2 ꢃ ArH), 8.20 ppm (d, 1H, J = 8.5 Hz, ArH); 13C NMR
(125 MHz, CDCl3) d = 29.8 ppm (CH2), 31.6 (CH2), 38.3 (CH2), 35.7
(CH2), 63.6 (CH2), 68.7 (C), 114.2 (CH), 116.6 (CH), 122.2 (C),
125.2 (CH), 126.7 (CH), 127.9 (CH), 128.4 (CH), 130.0 (CH), 130.1
(CH), 138.0 (C), 140.2 (C), 142.2 (C), 149.1 (CH), 151.2 (C), 167.5
(C), 168.2 (C), 174.1 (CNH2), 187.4 ppm (C); LRMS (LC–MS ES+):
m/z 153 (M+H+, 100%); HRMS: calcd for C29H29N7O6S [M+H]+:
604.1973, found 604.1982 (ꢁ1.55 ppm).
analyses and for assistance with performing other NMR and MS
analyses; Mr. Daniel James, for data management; Mrs. Lorna
Campbell and Ms. Bhavya Rao for cell screening assays; and Profes-
sor Mike Barrett, University of Glasgow for kindly providing the
AT1 knock out cell line.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.12.018.
References and notes
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3.2.2.17. N-(3-((4,6-Diamino-1,3,5-triazin-2-yl)amino)propyl)-3-
(4-(N-(2-iodophenyl)sulfamoyl)phenyl)
(47).
propanamide
Acid 41 (539 mg, 1.25 mmol), amine 4537 (344 mg,
1.875 mmol), TBTU (602 mg, 1.875 mmol) and HOAt (255 mg,
1.875 mmol) were dissolved in anhydrous DMF (20 mL) and cooled
to 0 °C, before the addition of DIPEA (485 mg, 3.75 mmol). Subse-
quently, the reaction mixture was stirred at 25 °C for 18 h followed
by purification of half of the material by reverse phase HPLC to af-
ford the product as a white solid (42 mg, 11%). 1H NMR (500 MHz,
CD3OD) d = 1.67 (tt, 2H, J = 7.0, 7.0 Hz, CH2), 2.52 (t, 2H, J = 7.5 Hz,
CH2), 3.00 (t, 2H, J = 7.5 Hz, CH2), 3.19 (t, 2H, J = 7.0 Hz, CH2), 3.28
(t, 2H, J = 7.0 Hz, CH2), 6.92–6.95 (m, 1H, ArH), 7.31–7.37 (m, 3H,
3 ꢃ ArH), 7.42 (dd, 1H, J = 8.0, 1.5 Hz, ArH), 7.63–7.65 (m, 2H,
AA0BB0, 2 ꢃ ArH), 7.76 ppm (dd, 1H, J = 8.0, 1.5 Hz, ArH); 13C NMR
(125 MHz, CD3OD) d = 30.4 (CH2), 32.5 (CH2), 37.8 (CH2), 38.2
(CH2), 38.9 (CH2), 96.5 (C), 127.9 (CH), 128.7 (CH), 129.1 (CH),
130.1 (CH), 130.2 (CH), 139.4 (C), 139.6 (C), 140.9 (CH), 148.2 (C),
165.8 (C), 174.6 ppm (C); LRMS (LC–MS ES+): m/z 597 (M+H+,
100%). HRMS (ES+): calcd for C21H26I1N8O3S1 [M+H]+ 597.0888,
found 597.0890 (ꢁ0.29 ppm).
3.2.2.18. N-(3-((4,6-Diamino-1,3,5-triazin-2-yl)amino)propyl)-3-
(4-((2-(1-hydroxy-4-oxocyclohexa-2,5-dien-1-yl)-1H-indol-1-yl)
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sulfonyl)phenyl)propanamide (46).
Copper (I) iodide
(0.007 mmol, 1.3 mg) and tetrakis(triphenylphosphine) palla-
dium(0) (0.0042 mmol, 5 mg) were added to a solution of aryl io-
dide 47 (0.07 mmol, 42 mg) and alkyne 1 (0.0812 mmol, 11 mg)
i
in degassed DMF (332
l
L), Pr2NH (140
l
L) and water (28
l
L) and
the resultant mixture heated under microwave irradiation at
100 °C for 10 min. After cooling the reaction mixture was directly
purified by reverse phase HPLC to give the product as a white solid
(17 mg, 40%). 1H NMR (500 MHz, CD3OD) d = 1.56 (tt, 2H, J = 7.0,
7.0 Hz, CH2), 2.45 (t, 2H, J = 7.5 Hz, CH2), 2.94 (t, 2H, J = 7.5 Hz,
CH2), 3.11 (t, 2H, J = 7.0 Hz, CH2), 3.15 (t, 2H, J = 7.0 Hz, CH2),
6.25–6.29 (m, 2H, AA0BB0, 2 ꢃ CH), 6.93 (d, 1H, J = 0.5 Hz, ArH),
7.22–7.25 (m, 1H, ArH), 7.31–7.36 (m, 3H, 3 ꢃ ArH), 7.49–7.51
(m, 1H, ArH), 7.55–7.58 (m, 2H, AA0BB0, 2 ꢃ ArH), 7.83–7.86 (m,
2H, AA0BB0, 2 ꢃ ArH), 8.19 ppm (dd, 1H, J = 8.5, 1.0 Hz, ArH); 13C
NMR (125 MHz, CD3OD) d = 30.4 (CH2), 32.5 (CH2), 37.7 (CH2),
37.9 (CH2), 38.6 (CH2), 68.7 (C), 114.2 (CH), 116.6 (CH), 122.6
(CH), 125.2 (CH), 126.7 (CH), 127.8 (CH), 128.4 (CH), 130.2 (CH),
137.9 (C), 140.1 (C), 142.2 (C), 149.3 (C), 151.3 (CH), 174.4 (C),
187.4 ppm (C). Note, two quaternary 13C resonances are not visible.
MS (LC–MS ES+): m/z 603 (M+H+, 100%). HRMS (ES+): calcd for
29H31N8O5S1 [M+H]+ 603.2133, found 603.2138 (ꢁ0.84 ppm).
37. Chollet, C.; Baliani, A.; Wong, P. E.; Barrett, M. P.; Gilbert, I. H. Bioorg. Med.
Chem. 2009, 17, 2512.
C
38. Spinks, D.; Shanks, E. J.; Cleghorn, L. A.; McElroy, S.; Jones, D.; James, D.;
Fairlamb, A. H.; Frearson, J. A.; Wyatt, P. G.; Gilbert, I. H. ChemMedChem 2009, 4,
2060.
Acknowledgments
39. Jones, D. C.; Hallyburton, L.; Stojanovski, K. D.; Read, J. A.; Frearson, J. A.;
Fairlamb, A. H. Biochem. Pharmacol. 2010, 80, 1478.
40. Lanteri, C. A.; Stewart, M. H.; Brock, J. M.; Alibu, V. P.; Meshnick, S. R.; Tidwell,
R. R.; Barrett, M. P. Mol. Pharmacol. 2006, 70, 1585.
We would like to acknowledge the MRC (A.C.) for a PhD stu-
dentship and the Wellcome Trust for funding (WT 07938, WT
077705 and WT 083481). The authors would like to thank Mrs.
Gina MacKay for performing high resolution mass spectrometry
41. Srinivasan, R.; Uttamchandani, M.; Yao, S. Q. Org. Lett. 2006, 8, 713.