Synthesis and evaluation of phenoxymethylbenzamide analogues as anti-trypanosomal agents

Article abstract of DOI:10.1039/c4md00406j

The synthesis and anti-trypanosomal activity of a compound library based on a phenoxymethylbenzamide hit discovered in a high throughput screen is described. Several of the analogues exhibited potent activity against Trypanosoma brucei rhodesiense, a human infective strain of the trypanosome parasite, that serve as lead compounds for further optimisation. This journal is

Full text of DOI:10.1039/c4md00406j

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Synthesis and evaluation of
phenoxymethylbenzamide analogues as anti-
Cite this: DOI: 10.1039/c4md00406j
trypanosomal agents†
Alexandra Manos-Turvey,a Emma E. Watson,a Melissa L. Sykes,b Amy J. Jones,b
b
a
Jonathan B. Baell,c Marcel Kaiser,de Vicky M. Avery and Richard J. Payne
*
Received 13th September 2014
Accepted 7th October 2014
The synthesis and anti-trypanosomal activity of a compound library based on a phenoxymethylbenzamide
hit discovered in a high throughput screen is described. Several of the analogues exhibited potent activity
against Trypanosoma brucei rhodesiense, a human infective strain of the trypanosome parasite, that serve
as lead compounds for further optimisation.
DOI: 10.1039/c4md00406j
www.rsc.org/medchemcomm
Human African trypanosomiasis (HAT), also known as African are melarsoprol, eornithine and nifurtimox. All three drugs are
sleeping sickness, is a neglected tropical disease that largely associated with severe side effects and melarsoprol and eor-
affects the population of sub-Saharan Africa. Currently the nithine must be delivered intravenously.1,6,10,12–14 Eornithine
World Health Organisation (WHO) estimates that there are alone or in combination with nifurtimox is the WHO-preferred
approximately 20 000 cases of HAT, with a further 70 million HAT treatment, however this regime is not effective against
people predicted to be at risk of infection.1,2 HAT is caused by T. b. rhodesiense15–17 and is also not suitable for use in individ-
infection with one of two subspecies of parasitic trypanoso- uals co-infected with HIV as it requires a regularly functioning
matid protozoa of the Trypanosoma brucei family, Trypanosoma immune system.18,19 Consequently there is a signicant need for
brucei rhodesiense or Trypanosoma brucei gambiense. They are the development of new drugs for the more effective treatment
transmitted to humans through the bite of a tsetse y (Glossina of HAT.
genus). There are two stages of infection. Stage 1 involves the
High throughput screening (HTS) of compound libraries
invasion of the haemolymphatic system by the parasite and is against whole cell T. brucei has recently gained momentum as a
oen accompanied by bouts of fever, headaches and joint pain.3 rapid and cost effective mechanism for elucidating new inhib-
Stage 2 occurs once protozoa in the blood stream cross the itors of the protozoa that may provide new HAT drug leads.20,21
blood brain barrier (BBB) and enter the central nervous system Avery and co-workers recently reported a whole organism HTS
(CNS) and the cerebrospinal uid (CSF).2 This latter stage of of 87 926 compounds against T. b. brucei, the non-human
infection is more aggressive, resulting in higher levels of patient transmissible T. brucei subspecies.22 This screen led to a
presentation due to the onset of identiable neurological number of novel “hit” compounds that exhibited low micro-
symptoms,4 and are usually fatal if le untreated.5–7
molar inhibitory activity against T. b. brucei and limited
Despite the decline in the number of cases of HAT in recent mammalian cell toxicity, including phenoxymethylbenzamide
years,1 the widespread nature of latent infections,8,9 together hit compound 1 (Fig. 1).23,24 This compound possessed low
with diagnoses oen only in later stages of infection, means micromolar inhibition of T. b. brucei and a calculated polar
that the potential for a HAT epidemic is still present.10,11
Currently, the only drugs available for second stage treatment
aSchool of Chemistry, The University of Sydney, Sydney, NSW 2006, Australia. E-mail:
richard.payne@sydney.edu.au; Fax: +61 2 9351 3329; Tel: +61 2 93515877
bDiscovery Biology, Eskitis Institute for Drug Discovery, Griffith University, Nathan,
Queensland, Australia
cDepartment of Medicinal Chemistry, Faculty of Pharmacy and Pharmaceutical
Sciences, Monash Institute of Pharmaceutical Sciences, Monash University,
Parkville, Victoria, Australia
dSwiss Tropical and Public Health Institute, Basel, Switzerland
eUniversity of Basel, Basel, Switzerland
† Electronic supplementary information (ESI) available: Detailed experimental
procedures, characterisation data and original NMR spectra. See DOI:
10.1039/c4md00406j
Fig. 1 Activity and physicochemical parameters of the phenoxy-
methylbenzamide lead
manipulation.
1
revealing regions identified for SAR
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Table 1 Inhibitory activity of 1, 8 and 11–38 against T. b. brucei
together with selectivity indices (SI)
surface area indicative of a molecule that may be able to cross
the BBB, an important consideration for the treatment of
T. brucei infections that have progressed into the CNS (Fig. 1).25
Herein, we describe the rst detailed examination of the
structure–activity relationships for 1, to allow for optimization
of anti-trypanosomal activity.
T. b. brucei
IC50 [mM]
Compound
R1
X
R2
Sel. indexa (SI)
8
OEt
O
>50
—
At the outset, investigation of the SAR of 1 was proposed by
division of the structure into three key regions: the masked
hydroquinone moiety, the central aromatic core and the car-
boxamide piperidyl ring (Fig. 1). Initial studies then centred on
modication of the two outer regions, namely the masked
hydroquinone and the 3-methylpiperidyl moiety.
Synthesis of a 50 analogue compound library began from
methyl(4-bromomethyl)benzoate 2,26 which was reacted with a
range of 4-substituted phenols, or 4-ethoxyaniline, which aer
base hydrolysis afforded free acids 3–10 in good yields (see
Scheme 1 for the general synthesis of a selection of the analogue
library). Reaction of 3–10 with a range of amines, using HATU as
the coupling reagent and N,N-diisopropylethylamine as the
base in DMF, then generated the benzamide library members in
moderate to good yields.
1
OEt
OMe
nPr
O
O
O
NH
1.15
4.60
>50
36
18
—
27
11
12
13
OEt
3.13
14
15
16
17
18
Me
nPr
OMe
OEt
OEt
O
O
O
O
9.20
10.0
3.75
0.49
1.93
9
8
22
84
43
NH
19
20
21
22
Me
Et
OMe
OEt
O
O
O
O
>50
>50
>50
>50
—
—
—
—
23
24
OEt
OEt
O
NH
2.70
2.90
31
29
The target compound library was then subjected to a
preliminary screen against non-pathogenic T. b. brucei to assess
the anti-protozoa activity as well as against the human cell line
HEK293 as a measure of selectivity (Table 1). These assays were
performed using the HTS assay previously described by Avery
and co-workers.27 Unfortunately, a number of the compounds
(not shown in Table 1) proved to be insoluble under the assay
conditions and thus IC50 values could not be obtained in these
cases (for full results table, see ESI†). A number of interesting
structure–activity trends were elucidated from these prelimi-
nary screening results. Notably compound 8, a fragment of the
original lead 1, was found to be inactive (IC50 > 50 mM), thus
conrming the importance of the piperidyl ring for anti-trypa-
nosomal activity. In general, compounds possessing a 4-alkoxy
substituted masked hydroquinone moiety possessed more
potent activity when compared with 4-alkyl substitution. Of all
the compounds bearing a piperidyl functionality (11–24), those
also containing an ethoxy-substituted masked hydroquinone
possessed the most potent anti-trypanosomal activity. This
included analogues 13, 17, 18, 23 and 24 (IC50 ¼ 0.49–3.13 mM),
with compound 17 exhibiting slightly increased potency and
selectivity [IC50 ¼ 0.49 mM, selectivity index (SI) ¼ 84] relative to
25
26
27
28
Et
O
O
O
NH
14.8
4.17
4.20
20.6
6
20
20
4
nPr
OEt
OEt
29
30
31
Et
OMe
OEt
O
O
O
>50
>50
>50
—
—
—
32
33
34
35
Et
O
O
O
NH
4.84
18.0
2.88
4.64
17
5
29
18
OMe
OEt
OEt
36
OEt
O
>50
—
37
38
OEt
OEt
O
NH
1.73
3.51
48
24
a
SI ¼ (IC50 against HEK293)/(IC50 against T. b. brucei).
initial hit 1. Interestingly, incorporation of a 4-methyl piperidyl
substituent in 19–22 led to a complete loss in activity (IC50 > 50
mM). This observation was mirrored in analogues 29–31, con-
taining a 4-methylpiperazine moiety. Other substitutions of the
methylpiperidyl ring appeared to be tolerated with the mor-
pholino series 25–28 and aniline-based analogues 32–35 pos-
sessing signicant activity (IC50 ¼ 4.17–20.6 mM), albeit less
potent than the homologues bearing a piperidyl moiety. Inclu-
sion of a smaller N-isopropylamide substituent in 36 in place of
the 3-piperidyl substituent in 1 led to loss of activity. However,
inclusion of a N,N-diisopropylamide substituent led to recovery
Scheme 1 Synthesis of phenoxymethylbenzamide 1 and analogues
11–38.
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Table 2 Active alkyl-linked analogues 39–41 against T. b. brucei
functionality, we were next interested in investigating the
requirements of the central aryl ring for anti-trypanosomal
activity. To this end, analogues 39–41 were synthesised in which
the central aryl ring was replaced with an alkyl chain (Table 2,
for synthetic details please see ESI†). Compounds 40 and 41
exhibited only modestly reduced activity when compared with
the parent compound possessing a rigid aryl linker. These
results suggest that the central aromatic ring is not essential for
inhibitory activity but rather serves as a spacer unit to appro-
priately position the two terminal functional groups with
respect to each other.
Having determined the inhibitory activity of 11–41 against
T. b. brucei in a preliminary screen, we were next interested in
selecting compounds for screening against a human infective
trypanosomal strain. To this end, 13 compounds (all with IC50
values <5 mM in the preliminary screen) were screened against
T. b. rhodesiense (strain STIB 900) (Table 3). In addition, these
compounds were assessed against three other parasitic
protozoa, namely, Trypanosoma cruzi (the causative agent of
American trypanosomiasis or Chagas disease), Leishmania
donovani (the cause of visceral leishmaniasis) and Plasmodium
falciparum (the most common causative agent of human
T. b. brucei
IC50 [mM]
Compound
Sel. indexa (SI)
7.35
5
1.66
2.03
50
41
a
SI ¼ (IC50 against HEK293)/(IC50 against T. b. brucei).
of activity with 37 and 38 exhibiting IC50 values of 1.73 and 3.51
mM, respectively.
Having identied the importance of both the ethoxy-derived
phenol moiety and either a piperidyl or N,N-diisopropylamide
Table 3 Results from T. b. rhodesiense screening (and other parasitic protozoa) of 13 active first generation compounds from preliminary
T. b. brucei testinga
T. b. rhodesiense
IC50 [mM]
T. cruzi
IC50 [mM]
L. donovani
IC50 [mM]
P. falciparum
IC50 [mM]
Cytotoxicity L6
Compound
R1
X
R2
IC50b [mM] (SI)
11
1
13
OMe
OEt
OEt
O
O
NH
2.64 ꢀ 0.243
0.985 ꢀ 0.076
4.94 ꢀ 1.96
65.4 ꢀ 22.7
107 ꢀ 34.5
15.3 ꢀ 6.73
35.1 ꢀ 4.27
35.7 ꢀ 6.22
37.8 ꢀ 2.70
35.9 ꢀ 2.27
22.3 ꢀ 1.06
24.9 ꢀ 1.04
79.3 ꢀ 44.6 (30)
186 ꢀ 94.2 (190)
26.9 ꢀ 1.24 (5.4)
16
17
18
OMe
OEt
OEt
O
O
NH
1.82 ꢀ 0.022
0.365 ꢀ 0.059
1.88 ꢀ 0.122
59.8 ꢀ 21.1
50.1 ꢀ 23.5
60.8 ꢀ 19.6
39.2 ꢀ 3.98
28.9 ꢀ 1.02
45.4 ꢀ 1.42
25.7 ꢀ 5.45
17.6 ꢀ 3.43
20.3 ꢀ 1.72
85.7 ꢀ 35.8 (47)
116 ꢀ 6.93 (320)
71.6 ꢀ 36.8 (38)
23
26
OEt
nPr
O
O
1.74 ꢀ 0.230
9.73 ꢀ 1.28
172 ꢀ 49.5
32.1 ꢀ 11.5
94.0 ꢀ 40.1
40.4 ꢀ 11.6
48.3 ꢀ 11.3
19.1 ꢀ 5.13
263 ꢀ 31.71 (150)
106 ꢀ 14.2 (11)
32
34
35
Et
OEt
OEt
O
O
NH
7.16 ꢀ 3.17
3.38 ꢀ 1.58
5.28 ꢀ 0.472
168 ꢀ 37.1
135 ꢀ 71.2
16.2 ꢀ 5.38
64.0 ꢀ 23.2
136.5 ꢀ 87.5
38.6 ꢀ 4.30
113 ꢀ 31.4
117 ꢀ 38.3
15.4 ꢀ 0.222
>275 (>38)
>275 (>81)
75.0 ꢀ 23.3 (14)
37
38
OEt
OEt
O
NH
0.464 ꢀ 0.162
3.61 ꢀ 1.12
229 ꢀ 26.3
50.5 ꢀ 15.1
>275
44.9 ꢀ 0.423
88.3 ꢀ 41.4
12.3 ꢀ 1.21
>275 (>590)
83.6 ꢀ 24.4 (23)
a
Control IC50 values as follows: IC50 (melarsoprol) ¼ 0.010 mM (T. b. rhodesiense); IC50 (benznidazole) ¼ 2.325 mM (T. cruzi); IC50 (miltefosine) ¼
b
0.572 mM (L. donovani); IC50 (chloroquine) ¼ 0.013 mM (P. falciparum); IC50 (podophyllotoxin) ¼ 0.022 mM (L6 cells). SI ¼ (IC50 against L6)/(IC50
against T. b. rhodesiense). Errors are standard errors of the mean of three independent experiments provided to three signicant gures.
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malaria) to gauge for the selectivity of the anti-trypanosomal
activity. The compounds were also screened against rat skeletal
myoblasts (L6 cells), as an additional measure of the selectivity
of the anti-trypanosomal activity over mammalian cells. Grati-
fyingly, all of the compounds proved to be more potent inhib-
itors of T. b. rhodesiense compared with the other species of
protozoa investigated (Table 2). Indeed, while compounds
exhibited high nanomolar-low micromolar inhibition of
T. b. rhodesiense (IC50 ¼ 0.37–9.7 mM), most compounds dis-
played moderate activity against T. cruzi, L. donovani and P.
falciparum (Table 3). For the most part, the compounds also
showed signicant selectivity for T. b. rhodesiense over the rat
skeletal myoblast (L6) cell line. The most potent inhibitors of
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and 37 (IC50 values of 0.37 mM and 0.46 mM) containing 2-
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