Optimisation of the Anti-Trypanosoma brucei Activity of the Opioid Agonist U50488

Article abstract of DOI:10.1002/cmdc.201100278

Screening of the Sigma-Aldrich Library of Pharmacologically Active Compounds (LOPAC) against cultured Trypanosoma brucei, the causative agent of African sleeping sickness, resulted in the identification of a number of compounds with selective antiproliferative activity over mammalian cells. These included (+)-(1R,2R)-U50488, a weak opioid agonist with an EC₅₀ value of 59nM as determined in our T.brucei invitro assay reported previously. This paper describes the modification of key structural elements of U50488 to investigate structure-activity relationships (SAR) and to optimise the antiproliferative activity and pharmacokinetic properties of this compound.

Full text of DOI:10.1002/cmdc.201100278

MED
DOI: 10.1002/cmdc.201100278
Optimisation of the Anti-Trypanosoma brucei Activity of
the Opioid Agonist U50488
Victoria C. Smith, Laura A. T. Cleghorn, Andrew Woodland, Daniel Spinks, Irene Hallyburton,
Iain T. Collie, N. Yi Mok, Suzanne Norval, Ruth Brenk, Alan H. Fairlamb, Julie A. Frearson,
Kevin D. Read, Ian H. Gilbert,* and Paul G. Wyatt*[a]
Screening of the Sigma–Aldrich Library of Pharmacologically
Active Compounds (LOPAC) against cultured Trypanosoma
brucei, the causative agent of African sleeping sickness, result-
ed in the identification of a number of compounds with selec-
tive antiproliferative activity over mammalian cells. These in-
cluded (+)-(1R,2R)-U50488, a weak opioid agonist with an EC50
value of 59 nm as determined in our T. brucei in vitro assay re-
ported previously. This paper describes the modification of key
structural elements of U50488 to investigate structure–activity
relationships (SAR) and to optimise the antiproliferative activity
and pharmacokinetic properties of this compound.
Introduction
Human African trypanosomiasis (HAT), or African sleeping sick-
ness, is a parasitic disease caused by protozoa parasites of the
species Trypanosoma brucei and is transmitted by the tsetse fly.
HAT is endemic in certain regions of sub-Saharan Africa, threat-
ening about 60 million people in 36 countries. The disease is a
major cause of morbidity and mortality in these developing re-
gions, resulting in an estimated 30000 deaths each year.[1]
Phase 1 of the disease, after a bite by an infected fly, results
in a systemic infection from the extracellular spread of para-
sites, causing episodes of fever, headache, sweating, and swel-
ling of the lymph nodes. Phase 2 of the disease results from
the spread of infection into the central nervous system. The
term sleeping sickness derives from the symptoms of this
second phase, in which the circadian rhythm is disturbed, re-
sulting in bouts of fatigue alternating with manic periods, pro-
gressing to daytime slumber and nighttime insomnia. Without
treatment, the disease is fatal; progressive mental deterioration
leads to coma and death. The current therapeutic product pro-
file for HAT requires new clinical candidates to show activity in
models of phase 2 of the disease, therefore requiring com-
pounds to cross the blood–brain barrier.[2]
(LOPAC) against T. brucei in culture, using MRC-5 cells as a
mammalian cell line counter-screen to exclude nonselective
compounds. We recently reported the output of this screen,[4]
and herein we discuss the medicinal chemistry programme
around one of the hits, (+)-(1R,2R)-U50488 (1).
Compound 1 (Table 1), is the enantiomer of (ꢀ)-(1S,2S)-
U50488 (2), which is a potent agonist of the k-opioid receptor.
In contrast, (+)-(1R,2R)-U50488 (1) is a weak agonist of the k-
opioid receptor.[5] Compound 1 represents an interesting start-
ing point for a drug discovery programme, as it has potent an-
tiproliferative activity against T. brucei (EC50 =59 nm) with sub-
stantial selectivity over MRC-5 cells. It is also able to cross the
blood–brain barrier (B/B ratio=8.2),[4] a key requirement to
treat phase 2 HAT.
Herein we describe our studies to identify the key pharma-
cophoric elements of 1, including the optimisation of T. brucei
antiproliferative activity and selectivity over the mammalian
MRC-5 cell line. Improvement in metabolic stability would also
be beneficial. Compound 1 was identified as a hit from the ini-
tial screen.[4] No other structurally related compounds were
present in the LOPAC. The alternative trans stereoisomer of
U50488 was purchased and tested to determine if antiprolifer-
Despite the burden of HAT and other neglected diseases,
there is a lack of validated drug discovery targets and lead
compounds for these diseases.[1,3] To address this gap, a
number of approaches to generate hits have been taken: the
exploitation of parasite-specific targets with little history of
drug discovery, the exploitation of target families with a history
of drug discovery for other indications, and hit identification
through phenotypic (in vitro whole-parasite) screening. The
Drug Discovery Unit (DDU) at the University of Dundee (http://
www.drugdiscovery.dundee.ac.uk/) is taking each of these ap-
proaches to develop a portfolio of projects to discover drugs
for HAT. Herein we report the optimisation of (+)-(1R,2R)-
U50488 (1), a hit compound discovered by screening the
Sigma–Aldrich Library of Pharmacologically Active Compounds
[a] V. C. Smith,+ Dr. L. A. T. Cleghorn,+ Dr. A. Woodland, D. Spinks,
I. Hallyburton, I. T. Collie, Dr. N. Yi Mok, S. Norval, Dr. R. Brenk,
Prof. A. H. Fairlamb, Prof. J. A. Frearson, Dr. K. D. Read, Prof. I. H. Gilbert,
Prof. P. G. Wyatt
Drug Discovery Unit, College of Life Sciences, James Black Centre
University of Dundee, Dundee, DD1 5EH, Scotland (UK)
E-mail: p.g.wyatt@dundee.ac.uk
i.h.gilbert@dundee.ac.uk
[+] These authors contributed equally to this work.
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201100278.
Re-use of this article is permitted in accordance with the Terms and Con-
ditions set out at http://onlinelibrary.wiley.com/journal/
10.1002/(ISSN)1860-7187/homepage/2452_onlineopen.html.
1832
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Optimisation of U50488
Table 1. Structure and EC50 values for (+)-(1R,2R)-U50488 and related
compounds.
Compd
Stereochemistry
EC50 [mm]
T. brucei
MRC-5
Scheme 1. Reagents and conditions: a) NR1R2, EtOH/iPrOH, 808C, 18 h;
b) MsCl/Et3N, 08C, 2 h; c) MeNH2, RT, 16 h; d) Bromotrispyrrolidinophosphoni-
um hexafluorophosphate (PyBrop), Et3N, CH2Cl2, 30 min.
1
2
3
R,R (+)
S,S (ꢀ)
0.028[a]
9.9
44
48
40
R,R/S,S (ꢁ)
0.052
[a] Data acquired with our 384-well format assay (see Experimental Sec-
tion), which is similar to data acquired with the original 96-well format
assay.[4]
Biological assays
Initial investigations focussed on modification of the phenyla-
cetamide moiety (Table 2). Removal of both chlorines to give
the unsubstituted phenylacetamide 10 resulted in a dramatic
loss of activity relative to 3. Removal of either chlorine resulted
in a six- to eightfold decrease in activity, as observed with
compounds 11 and 12, as did replacement with 3,4-difluoro-
phenyl (compound 15). Although this decrease could have
been driven by the decrease in lipophilicity upon removal or
replacement of the chlorines, the 2,4- and 2,6-dichloro substi-
ative activity is stereospecific. It was found that the (+)-R,R ste-
reoisomer 1 is ~350-fold more potent than the (ꢀ)-S,S stereo-
isomer 2, but roughly equipotent with the (ꢁ)-R,R/S,S racemate
3 (Table 1), suggesting that the less active stereoisomer does
not antagonise the effects of the active stereoisomer. In light
of this result, and to simplify our chemistry programme, we
chose to synthesise racemic mixtures for our initial explora-
tions of SAR. Enantiospecific syntheses of these compounds
were reported previously and could be adapted for the synthe-
sis of any hit compound, should it be developed further.[6,7]
Table 2. Phenyl substitution variation in compounds 10–32.
Results and Discussion
[b]
Compd
R
EC50 [mm]
CLi [mLminꢀ1 gꢀ1
]
Chemistry
T. b.
MRC-5 Human
Mouse
brucei[a]
To probe the SAR of starting compound 1, a number of modifi-
cations to the core were planned (Figure 1). Analogues of
U50488 were prepared by using the chemistry outlined in
Scheme 1. Cyclohexene oxide 4 was treated with an amine to
effect epoxide ring opening. The resulting aminocyclohexanol
5 was treated with methanesulfonyl chloride to form mesylate
6, which was displaced by the amine to form an aziridine ring
7, which ring-opened upon reaction with methylamine to give
diamine 8.[6] The remaining free NH group was then allowed to
react with a range of acids or acid chlorides to give the desired
acetamides 9.
3
3,4-dichlorophenyl
0.052
11
0.41
0.33
11
0.33
0.32
1.3
1.0
7.2
0.49
3.0
0.70
0.74
0.40
0.12
0.47
0.34
0.034
0.099
0.16
40
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
46
3.3
0.4
0.6
ND
3.7
1.9
3.6
8.0
14
ND
11
4.6
1.8
9.9
11
9.8
5.9
9.1
20
10 phenyl
11 3-chlorophenyl
12 4-chlorophenyl
13 2,6-dichlorophenyl
14 2,4-dichlorophenyl
15 3,4-difluorophenyl
16 3-fluorophenyl
17 4-fluorophenyl
18 3-methoxyphenyl
19 4-methoxyphenyl
20 2-tolyl
21 3-tolyl
22 4-tolyl
23 4-(isopropyl)phenyl
24 3-(trifluoromethyl)phenyl
25 4-(trifluoromethyl)phenyl
26 1-naphthyl
1.2
<0.5
<0.5
1.2
1.9
0.6
2.8
ND
ND
ND
ND
2.3
13
14
3.8
<0.5
22
27 2-naphthyl
28 (1,1’-biphenyl)-4-yl
29 3-bromophenyl
30 3-bromo-4-methoxyphenyl 0.090
31 3-pyridyl
4.7
20
19
ND
ND
ND
ND
ND
2.7
9.5
6.5
ND
5.2
>50
>50
>50
>50
9.8
2.3
32 cyclohexyl
[a] Hill slopes in the range of 0.3–2.2. [b] Microsomal intrinsic clearance;
ND: not determined.
Figure 1. Planned alterations to U50488.
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I. H. Gilbert, P. G. Wyatt, et al.
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tution patterns in 14 and 13, showing a respective six- and
210-fold loss in potency, indicate a structural element in the
SAR. The ortho-methyl substituent in 20 was also poorly toler-
ated, perhaps suggesting a twisted conformation is not favour-
able. The 1-naphthyl group in 26 conferred some loss in activi-
ty relative to 3, but the 2-naphthyl compound 27 is equipo-
tent. The 2-naphthyl compound was, in fact, the only analogue
in this subset to exhibit activity against the trypanosomes simi-
lar to that of the original 3,4-dichlorophenyl compound 3, pre-
sumably with the second benzene ring occupying a similar
space as the 3,4-dichloro substituents of the original molecule.
On the whole, single substitutions at the 3- or 4-positions are
roughly equipotent, as observed with the chlorophenyls (11
and 12), fluorophenyls (16 and 17), tolyls (21 and 22), and (tri-
fluoromethyl)phenyls (24 and 25). The much larger biphenyl
substituent in 28 was also tolerated; however, this compound
showed the highest level of MRC-5 cell toxicity observed in
this subset. Lastly, the aliphatic carbocycle 32 and the pyridyl
31 both suffered significantly lower activity than compound 3.
Encouragingly, the potency of these compounds against the
human MRC-5 cell line remained low in general, with the ma-
jority exhibiting EC50 >50 mm. Intrinsic clearance rates in
mouse (the standard efficacy model species for HAT) are gener-
ally high, with nearly all compounds having higher metabolic
instability than 3, at 3.6 mLminꢀ1 gꢀ1. However, with human
liver microsomes, the clearance rates are encouragingly much
lower; the fluorophenyl compounds 16 and 17 are quite
stable, showing rates of <0.5 mLminꢀ1 gꢀ1.
Table 3. Amine variation in compounds 33–37.
[b]
Compd
R
EC50 [mm]
T. b. brucei[a]
CLi [mLminꢀ1 gꢀ1
]
Human
MRC-5
40
Mouse
3
0.052
0.076
3.3
3.6
33
>50
>50
>50
27
11
15
29
3.4
ND
--
34
35
36
0.18
0.73
3.3
ND
ND
37
3.2
>50
ND
ND
[a] Hill slopes in the range of 1.2–3.9. [b] Microsomal intrinsic clearance;
ND: not determined.
Table 4. Modifications to the amide N-alkyl substituent in compounds
38–41.
A small series of amines were synthesised to explore the
SAR around the pyrrolidine in 3 (Table 3). Enlarging the ring
size to 6 and 7 atoms (33 and 34) did not change the potency
significantly. A more significant loss in potency was observed
with the introduction of a second heteroatom to the ring in
compounds 35 and 36, as with the presence of gem-difluoro
substituents on the pyrrolidine ring (compound 37). In these
compounds, the decreased basicity of the nitrogen atom could
be the cause of this trend. Activity against MRC-5 cells re-
mained low for these compounds (EC50 >50 mm), except for
the N-methylpiperazine 36, with an EC50 value of 27 mm.
Human intrinsic clearance measurements indicate that the
larger alkylamine rings and the morpholine exhibit much lower
metabolic stability than 3, whereas the N-methylpiperazine
shows a similar rate.
[b]
Compd
R
EC50 [mm]
T. b. brucei[a]
CLi [mLminꢀ1 gꢀ1
]
Human
MRC-5
Mouse
3
methyl
H
ethyl
tert-butyl
benzyl
0.052
0.27
8.6
11
2.4
40
28
42
50
8.0
3.3
2.6
ND
ND
ND
3.6
1.8
ND
ND
1.1
38
39
40
41
Our next investigation focussed on the amide N-alkyl sub-
stituent (Table 4). This work demonstrated that the original N-
methyl group is optimal, as its removal (compound 38) led to
a 10-fold loss in potency; its homologation (in 39 and 40) re-
sulted in a significant decrease in activity (80–400-fold). Both
38 and 41 showed an improvement in microsomal stability,
suggesting that demethylation may be an issue in this regard.
We decided to investigate changes to the cyclohexyl ring to
determine whether an increase in potency is possible here. In
addition, one possible mechanism of metabolism is through
hydroxylation of this ring. The corresponding tetrahydrofuranyl
42 and cyclopentyl 43 derivatives (Table 5) were synthesised
by using the same chemistry as described in Scheme 1, start-
ing with cyclopentyl and tetrahydrofuran oxide instead of cy-
[a] Hill slopes in the range of 1.3–8.9. [b] Microsomal intrinsic clearance;
ND: not determined.
clohexyl oxide. These modifications resulted in a significant
loss in activity. Similarly, removal and replacement with an eth-
ylene linker (to give 45) resulted in a >60-fold decrease in po-
tency, whilst replacement of the cyclohexyl ring with a planar
phenyl ring in 44 also resulted in a >66-fold loss of potency
relative to 38. Interestingly, the introduction of a non-fused
phenyl ring adjacent to the amide resulted in a stereochemi-
cally sensitive increase in activity, with the R isomer 46 demon-
strating fivefold more potency that the S isomer 47. Another
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Optimisation of U50488
to compound 11. Inclusion of
the best elements from the SAR
studies into one compound
gave the 3,4-dichlorophenyl cy-
clopropyl analogue 57, which
demonstrated single-digit nano-
molar inhibition of parasite
growth in culture, with low
levels of toxicity towards MRC-5
cells. Both this compound and
the dimethyl analogue 56
showed similar or decreased
clearance rates in human liver
microsomes relative to parent
compound 3. Unfortunately,
these compounds had higher
mouse liver microsome clear-
ance rates, which may make effi-
cacy testing in a mouse model
of infection problematic.
Table 5. Core ring variation in compounds 42–48.
[b]
Compd
Ring A
R
EC50 [mm]
T. b. brucei[a]
CLi [mLminꢀ1 gꢀ1
]
Human
MRC-5
40
Mouse
3
methyl
methyl
methyl
H
0.052
3.3
3.6
42
43
44
45
16.6
50
50
50
50
ND
ND
ND
0.6
ND
ND
ND
ND
50
17.7
16.3
H
46
47
methyl
methyl
0.88
9.8
8.3
2.2
1.4
6.8
7.4
Modelling
5.4
We were interested to investi-
gate if there is a direct correla-
tion between the activity of
these molecules and their three-
dimensional shape and physico-
chemical properties. Such a cor-
relation could indicate a specific
molecular target. Based on the
similarity principle, which de-
scribes that similar molecules ex-
hibit similar biological effects,[8]
48
–
9.9
50
ND
ND
[a] Hill slopes in the range of 1.1–8.8. [b] Microsomal intrinsic clearance; ND: not determined.
strategy was to fuse the cyclohexyl ring to the amide nitrogen
to form a piperidine ring and to extend the pyrrolidine with a
methylene linker to produce 48; this compound also showed
poor activity.
we first attempted to deduce the bioactive conformation of hit
compound 1 by overlaying its multiple 3D conformations with
multiple conformations of the most potent analogue 57. Using
ROCS software,[9] the conformation of 1 derived from the best-
fit overlay of these two molecules was chosen as the bioactive
conformation of the hit, and was subsequently used as the
template conformation for further overlay experiments with
other structural analogues within the compound series. Multi-
ple conformations generated for the remaining 48 compounds
were then overlaid with the proposed bioactive conformation
of 1, and ranked according to similarity in molecular shape
and functional group complementarity relative to 1.
Figure 2a shows the plot of the potency values observed in
T. b. brucei cells (pEC50) against the ShapeTanimoto score,[9]
which describes the molecular shape resemblance of the struc-
tural analogues compared with hit compound 1. Similarly, Fig-
ure 2b shows the plot of pEC50 against the ScaledColor score,[9]
which represents functional group complementarity to hit
compound 1. The higher the value of these scores, the more
similar the compound is to hit compound 1. It was apparent
from these graphs that there is no correlation between either
of these properties and the observed potency. This could sug-
gest that the mechanism of inhibition of parasitic growth ob-
All changes to the cyclohexyl moiety led to a significant de-
crease in activity, suggesting that the stereochemistry and re-
sulting conformation is very important for activity. This is con-
sistent with the results obtained with (ꢀ)-(1S,2S)-U50488 (2),
which shows a significant decrease in activity relative to the
(+)-(1R,2R) enantiomer 1.
To investigate the role of the linker between the amide car-
bonyl group and the phenyl ring, the linker was extended and
also rigidified (Table 6). Removal of the methylene unit in the
3- and 4-biphenyls (49 and 50) demonstrated a significant loss
in potency (compare with 28). Addition of an oxygen atom (in
51) was reasonably tolerated (sevenfold loss in activity). Chain
extension to either two or three methylene units showed an
increasing loss of potency (52: 11-fold; 53: 27-fold). Rigidifica-
tion through the introduction of cyclopropyl (in 54) and cyclo-
pentyl (in 55) in place of the methylene linker showed a great-
er than fivefold improvement in potency over compound 10,
with the three-membered ring being better tolerated than the
five. gem-Dimethyl 56 gave a sixfold increase in activity relative
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I. H. Gilbert, P. G. Wyatt, et al.
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Table 6. Linker variation in compounds 49–57.
[b]
Compd
R
EC50 [mm]
CLi [mLminꢀ1 gꢀ1
]
T. b. brucei[a] MRC-5 Human
Mouse
3
0.052
1.8
40
3.3
3.6
49
50
51
50
50
33
ND
ND
3.3
12
11
ND
5.4
0.35
0.56
52
50
1.3
3
53
54
1.4
50
50
ND
ND
5.3
25
0.34
55
56
0.58
50
50
2.8
1.8
14
0.053
9.1
57
0.007
46
2.9
17
[a] Hill slopes in the range of 1.3–12.0. [b] Microsomal intrinsic clearance;
ND: not determined.
served for these compounds might involve polypharmacologi-
cal effects, which could also explain the very “tight” SAR. Alter-
natively, the active conformation of compound 1 might be sig-
nificantly different from the predicted conformation.
Conclusions
(+)-(1R,2R)-U50488 (1) is an exciting starting point for a medici-
nal chemistry programme for HAT, as it has good in vitro po-
tency against T. brucei, acceptable in vitro and in vivo drug me-
tabolism and pharmacokinetics (DMPK) properties, and is
blood–brain barrier penetrant (B/B ratio=8.2).[4] Our previous
studies[4] indicated that the compound has good oral exposure,
a half-life in mice of 2.6 h and a large unbound fraction (0.69).
It is possible to obtain concentrations above EC99 in mice for
4 h following oral dosing of 150 mgkgꢀ1. Unfortunately it was
not curative in mice under a dosing regimen that maintained
levels above EC99 for 32 h. The reasons for this are not entirely
clear, but are probably due to the compound only being cyto-
cidal at high concentrations. Furthermore, 1 displays a relative-
ly low Hill slope (s=1.3), therefore requiring >10 times the
Figure 2. Plots of: a) molecular shape similarity (ShapeTanimoto score) and
b) functional group complementarity (ScaledColor score) to hit compound 1
against potency values in T. b. brucei cells represented as pEC50. c) The Com-
boScore value plot represents the sum of the ShapeTanimoto and ScaledCol-
or scores.[9]
EC50 to obtain EC99. This contrasts with eflornithine, a cytostatic
HAT drug (s=4.0) which requires only three times its EC50 to
obtain EC99.[4] Therefore, (+)-(1R,2R)-U50488 (1) requires further
optimisation to achieve efficacy.
Herein we report studies to optimise potency and to system-
atically generate structure–activity relationships. Further im-
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Optimisation of U50488
adhere as monolayers. A working stock (2 mL of 2 nm to 50 mm
final concentration) of each test compound was transferred to an
intermediate 384-well plate using a Matrix Platemate Plus (Thermo)
and pre-diluted with 38 mL minimum essential media (MEM). The
pre-diluted stocks (9 mL) were then transferred onto the cell mono-
layers using a Beckman FX instrument, and the plates were incu-
bated for 68 h as described above. After 68 h, Resazurin (10 mL of
500 mm stock to give a final concentration of 50 mm) was added to
each well, plates were incubated and measured for fluorescence as
described for the T. brucei assay.
provement in metabolic stability would also be beneficial. Our
investigations across all moieties of the molecule showed that
it displays very tight SAR, but there are some possibilities for
increasing the potency. Alternative aromatic groups to the 3,4-
dichlorophenyl in the original hit are reasonably well tolerated,
although the activity was not significantly improved. The con-
formation of the central cyclohexyl ring appears to be vital for
activity, as modification of the core resulted in significant loss
of potency. Other changes to the pyrrolidine ring and amide N
substituent resulted in loss of activity. The greatest gain in po-
tency was achieved by rigidifying the methylene linker of the
amide moiety, which gave compound 57 (EC50 =0.007 mm),
with a sevenfold increase in potency relative to the parent
compound 3 (EC50 =0.052 mm). Compound 57 has similar met-
abolic stability in human microsomes to that of the parent.
However, there is a decrease in mouse microsomal stability,
which could make efficacy testing problematic. This compound
has a similar Hill slope to that of 3 (3: s=1.5; 57: s=1.4), indi-
cating that a large concentration of compound would be re-
quired to obtain the EC99 in vivo.
EC50 curve fitting for both cell types employed a four-parameter lo-
gistic dose–response curve using IDBS XLfit 4.2 Model 205 [Eq. (1)]:
BꢀA
y ¼ A þ
ð1Þ
D
1 þ ðC=xÞ
For which A is the minimum y value, B is the maximum y value, C
is the EC50 and D is the Hill slope. All test compounds had floating
maximum and minimum, and pre-fit was used for all four parame-
ters. Pentamidine was used as a standard comparator drug for
T. brucei in all experiments (pEC50 =8.45ꢁ0.38, n=63), and doxoru-
bicin as a standard comparator drug for MRC-5 (pEC50 =7.15ꢁ0.55,
n=47).
Molecular modelling experiments were employed to com-
pare the molecular shape and functional group complementar-
ity of structural analogues with hit compound 1. Assessments
of these calculations revealed little correlation between the ob-
served potency and either of these properties. This could sug-
gest that the compounds do not inhibit just a single molecular
target, but show more complex polypharmacological behav-
iour. Alternatively, the inhibitors may bind in an unexpected
conformation.
Drug metabolism and pharmacokinetics (DMPK)
Each test compound (0.5 mm) was incubated with female CD1
mouse or pooled human liver microsomes (Tebu-Bio, UK;
0.5 mgmLꢀ1 in 50 mm potassium phosphate buffer, pH 7.4), and
the reaction was started with the addition of excess NADPH
(8 mgmLꢀ1 in 50 mm potassium phosphate buffer, pH 7.4). Immedi-
ately at time zero, then at 3, 6, 9, 15, and 30 min, aliquots (50 mL)
of the incubation mixture were removed and mixed with MeCN
(100 mL) to stop the reaction. Internal standard was added to all
samples, the samples were centrifuged (1800 g, 48C, 10 min) to
sediment precipitated protein, and the plates were sealed prior to
UPLC–MS–MS analysis using a Quattro Premier XE instrument
(Waters).
We have established that it is possible to increase the poten-
cy of the lead compound, although there are relatively limited
options for modification of the parent structure. Further work
to determine the molecular target(s) of the compound series
may indicate further strategies to increase potency and allow
scope for improving the DMPK properties of the molecules.
XLfit (IDBS, UK) was used to calculate the exponential decay and
consequently the rate constant (k) from the ratio of peak area of
test compound to internal standard at each time point. The rate of
intrinsic clearance (CLi, [mLminꢀ1 (g liver)ꢀ1]) of each compound
was then calculated [Eq. 2]:
Experimental Section
Biology
Growth inhibition was determined using T. brucei (S427 single
marker line). T. brucei culture (50 mL at a density of 1ꢁ104 cellsmLꢀ1
in HMI9-T media)[4] was added, using a Wellmate (ThermoMatrix),
to 384-well plates containing test compounds (250 nL at 2 nm to
50 mm final concentration), yielding a final DMSO concentration of
0.5%. Columns 11 and 12 of the plate were used as full signal con-
trol wells (0% inhibition) and columns 23 and 24 as low signal con-
trol (100% inhibition). Plates were then incubated at 378C in a hu-
midified incubator with an atmosphere of 5% CO2. After 68 h, Re-
sazurin (5 mL of 500 mm stock to give a final concentration of
45 mm) was added to each well, and the plates were incubated for
a further 4 h before being measured for fluorescence (lex =528 nm,
lem =590 nm). The fluorescence signal for each well was back-
ground subtracted and expressed as a percentage of inhibition rel-
ative to the full signal control wells.
CLi ¼ k ꢂ V ꢂ ðmicrosomal protein yieldÞ
ð2Þ
For which V [mL(mg protein)ꢀ1] is the incubation volume per mg
protein added, and microsomal protein yield is taken as 52.5 mg
protein per g liver. Verapamil was used as a positive control to con-
firm acceptable assay performance.
Molecular modelling
Multiple 3D conformations of all compounds were generated using
OMEGA2 (OpenEye Scientific Software Inc.), with a threshold RMS
distance of 0.5 per conformer, and a maximum of 200 conformers
for each molecule; 92 conformations were selected for hit com-
pound 1, and 32 conformations for analogue 57 in the initial ex-
periment to determine the bioactive conformation of compound 1.
The number of conformations for all other analogues ranged from
one single conformer (compound 55) to the maximum number al-
lowed of 200 (compounds 37 and 45–47).
A counter-screen against normal diploid human fibroblasts (MRC-5
cell line) was carried out to exclude nonselective compounds. Cells
(80 mL) were plated at a density of 1.25ꢁ104 cellsmLꢀ1 into wells
1–22 of a 384-well plate and incubated overnight to allow them to
ChemMedChem 2011, 6, 1832 – 1840
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
1837

I. H. Gilbert, P. G. Wyatt, et al.
MED
Structural overlay was performed using ROCS[9] (OpenEye Scientific
Software Inc.) by applying the ImplicitMillsDean force field as the
Color force field for functional group complementarity compari-
sons. Compounds were ranked by ComboScore, which represented
a combination of the ShapeTanimoto and the ScaledColor scores.
chromatography to give 13.5 g of the title compound (75%).
1H NMR (500 MHz, CDCl3): d=2.54–2.60 (m, 2H), 2.49–2.54 (m,
2H), 2.41–2.47 (m, 1H), 2.38 (s, 3H, CH3), 2.13–2.19 (m, 1H), 2.05–
2.11 (m, 1H), 1.74–1.79 (m, 2H), 1.64–1.73 (m, 5H), 1.13–1.26 (m,
3H), 0.93–1.02 ppm (m, 1H); LCMS m/z: 183 [M+H]+, tR =0.6–
0.8 min.
Chemistry
1H NMR spectra were recorded on a Bruker Avance DPX 500 instru-
ment. Chemical shifts (d) are expressed in ppm. Signal splitting
patterns are described as singlet (s), broad singlet (bs), doublet (d),
triplet (t), quartet (q), multiplet (m), or combination thereof. Low-
resolution electrospray (ES) mass spectra were recorded on a
Bruker MicroTof mass spectrometer, run in positive ion mode,
using either MeOH, MeOH/H2O (95:5) or H2O/MeCN (1:1) and 0.1%
formic acid as the mobile phase. High-resolution electrospray MS
measurements were performed on a Bruker MicroTof mass spec-
trometer. LC–MS analyses were performed with an Agilent HPLC
1100 instrument (Phenomenex Gemini Column 5 mm C18 110A 50ꢁ
3.0 mm, eluted with (0–3 min 20% MeOH/H2O) and a diode array
detector in series with a Bruker MicroTof mass spectrometer. All
synthesised compounds were determined to be of >95% purity
by LC–MS. Thin-layer chromatography (TLC) was carried out on
Merck silica gel 60 F254 plates using UV light and/or KMnO4 for visu-
alisation. Column chromatography was performed using RediSep 4
or 12 g silica pre-packed columns. All reactions were carried out
under dry and inert conditions unless otherwise stated.
General procedure for amide formation from 8 with an acid
or acid chloride 9
Method A: A flask was charged with 8 (1 molequiv), acid (2 mole-
quiv), and HOBt (2 molequiv) in DMF (anhydrous, 2 mLmmolꢀ1).
DIPEA (2 molequiv) and EDCI (2 molequiv) were added, and the re-
action mixture was stirred at room temperature for 1.5–4 h. After
concentration in vacuo, the resulting residue was partitioned be-
tween EtOAc and a saturated aqueous NaHCO3 solution, the organ-
ics were dried over MgSO4, concentrated, and purified by column
chromatography. The resulting gum was taken up in EtOAc/Et2O
(1:2), 2m HCl in Et2O (1–2 mL) was added dropwise, and the HCl
salt of 9 was collected by filtration.
Method B: Acid chloride (1.1 molequiv) was added to an ice-cold
solution of 8 (1 molequiv) in CH2Cl2 (anhydrous, 2 mLmmolꢀ1). The
reaction mixture was allowed to warm to room temperature and
stirred for a total of 2 h, then diluted with further CH2Cl2, washed
with a saturated aqueous NaHCO3 solution, and the organics were
dried over MgSO4 and concentrated. An HCl salt of 3 was obtained
as in Method A.
Experimental details for key compounds and some intermediates
are included herein; specifics for other compounds are included in
the Supporting Information.
Method C: Compound 8 (1 molequiv), acid (1 molequiv), Et3N
(0.4 mLmmolꢀ1),
and
PyBrop
(1.2 molequiv)
in
CH2Cl2
General procedure for diamine formation 8: Cyclohexene oxide
(1 mL, 10 mmol) and amine (12 mmol) in EtOH (8 mL) were heated
by microwave at 1408C for 40 min, concentrated to dryness, and
used without further purification. Methanesulfonyl chloride (MsCl;
1 mL, 12 mmol) was added dropwise to an ice-cold solution of the
resulting aminocyclohexanol and Et3N (4.39 mL, 30 mmol) in anhy-
drous Et2O (10 mL). The reaction mixture was stirred for 10 min,
methylamine (41% aqueous solution, 5 mL) was added, and the re-
action mixture was allowed to warm to room temperature with
vigorous stirring for 18 h. The layers were separated, the aqueous
layer further extracted with Et2O (150 mL), and the combined or-
ganics were dried over MgSO4 and concentrated in vacuo. The re-
sulting gum was purified by column chromatography to give 8.
(2 mLmmolꢀ1) were stirred under argon for 40 min at 48C. The re-
action was diluted with CH2Cl2 and washed with H2O (3ꢁ10 mL).
Purification by column chromatography eluting with CH2Cl2/MeOH
95:5 afforded the desired products.
2-(3,4-Dichlorophenyl)-N-methyl-N-(trans-(ꢁ))-[2-(pyrrolidin-1-yl)-
cyclohexyl]acetamide·HCl (3): Prepared according to Method A
from
trans-(ꢁ)-N-methyl-2-(pyrrolidin-1-yl)cyclohexanamine
(100 mg, 0.55 mmol), 3,4-dichlorophenylacetic acid (226 mg,
1.1 mmol), HOBt (149 mg, 1.1 mmol), DIPEA (192 mL, 1.1 mmol),
and EDCI (211 mg, 1.1 mmol) in DMF (anhydrous, 5 mL) in 56%
1
yield. H NMR (500 MHz, [D6]DMSO): d=9.58 (bs, HCl), 7.55 (d, 1H,
ArH, J=8.3 Hz), 7.52 (d, 1H, J=1.8 Hz, ArH), 7.24 (dd, 1H, ArH, J=
8.3 and 1.9 Hz), 4.52 (bs, 1H), 3.93 (d, 1H, J=16.2 Hz), 3.72 (d, 1H,
J=16.4 Hz), 3.53–3.62 (m, 1H), 3.41–3.48 (m, 1H), 3.10–3.28 (m,
3H), 2.94 (s, 3H, CH3), 2.02–2.10 (m, 1H), 1.80–1.96 (m, 4H), 1.73–
1.80 (m, 1H), 1.65–1.72 (m, 1H), 1.46–1.63 (m, 3H), 1.21–1.36 ppm
(m, 2H); 13C NMR (125 MHz, [D6]DMSO): d=171.4, 137.6, 131.9,
130.5, 130.2, 129.9, 128.7, 60.1, 51.4, 47.7, 39.2, 28.6, 24.2, 23.9,
23.4, 23.0 ppm; LC–MS m/z: 369 [M+H]+, tR =3.9 min; HRMS (ESI)
calcd for C19H27N2OCl2 369.1500 [M+H]+, found 369.1508.
trans-(ꢁ)-2-(Pyrrolidin-1-yl)cyclohexanol
(5):
Pyrrolidine
(13.37 mL, 162 mmol) was added to a solution of cyclohexene
oxide (10 mL, 99 mmol) in EtOH (100 mL). The reaction mixture
was stirred at reflux for 18 h, then concentrated to dryness in va-
cuo to give the title compound in quantitative yields. The resulting
1
material was used without further purification. H NMR (300 MHz,
CDCl3): d=4.00 (bs, 1H), 3.28–3.37 (m, 1H), 2.61–2.72 (m, 2H),
2.50–2.60 (m, 2H), 2.39–2.50 (m, 1H), 2.04–2.14 (m, 1H), 1.63–1.83
(m, 7H), 1.10–1.33 ppm (m, 4H).
N-Methyl-2-(naphthalen-2-yl)-N-(trans-(ꢁ))-[2-(pyrrolidin-1-yl)cy-
clohexyl]acetamide·HCl (27): Prepared according to Method A
trans-(ꢁ)-N-Methyl-2-(pyrrolidin-1-yl)cyclohexanamine (8a): MsCl
(9.14 mL, 117 mmol) was added dropwise to an ice-cold solution of
trans-(ꢁ)-2-(pyrrolidin-1-yl)cyclohexanol (99 mmol) and Et3N
(41.4 mL, 297 mmol) in anhydrous Et2O (150 mL). The reaction mix-
ture was stirred for 30 min, methylamine (41% aqueous solution,
48 mL) was added, and the reaction mixture was allowed to warm
to room temperature with vigorous stirring for 18 h. The layers
were separated, the aqueous layer further extracted with Et2O
(150 mL), and the combined organics were dried over MgSO4 and
concentrated in vacuo. The resulting gum was purified by column
from
trans-(ꢁ)-N-methyl-2-(pyrrolidin-1-yl)cyclohexanamine
(100 mg, 0.55 mmol), 2-(naphthalen-2-yl)acetic acid (205 mg,
1.1 mmol), HOBt (149 mg, 1.1 mmol), DIPEA (192 mL, 1.1 mmol),
and EDCI (211 mg, 1.1 mmol) in DMF (anhydrous, 5 mL) in 35%
1
yield. H NMR (500 MHz, CDCl3): d=11.39 (bs, 1H, HCl), 7.72–7.81
(m, 4H, ArH), 7.40–7.48 (m, 3H, ArH), 4.66–4.87 (m, 1H), 4.25 (d,
1H, J=15.9 Hz), 4.05 (d, 1H, J=15.9 Hz), 3.88–3.98 (m, 1H), 3.60–
3.68 (m, 1H), 3.25–3.39 (m, 1H), 3.10 (s, 3H, CH3), 2.96–3.07 (m,
2H), 2.08–2.30 (m, 3H), 1.90–1.98 (m, 1H), 1.74–1.88 (m, 4H), 1.54–
1.64 (m, 1H), 1.44–1.54 (m, 1H), 1.33–1.43 (m, 1H), 1.24–1.33 ppm
1838
www.chemmedchem.org
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2011, 6, 1832 – 1840

Optimisation of U50488
(m, 1H); 13C NMR (125 MHz, [D6]DMSO): d=172.1, 133.8, 132.9,
131.7, 128.4, 127.6, 127.4, 127.3, 127.2, 125.9, 125.4, 60.0, 51.2, 47.8,
40.7, 39.7, 28.7, 24.1, 24.0, 23.8, 23.4, 23.3 ppm; LC–MS m/z: 351
[M+H]+, tR =3.8 min; HRMS (ESI) calcd for C23H31N2O 351.2436
[M+H]+, found 351.2434.
over MgSO4, and concentrated to give an oil in near quantitative
yield. H NMR (500 MHz, CDCl3): d=6.98–7.01 (m, 1H, ArH), 6.87–
6.91 (m, ArH, 1H), 6.72–6.76 (m, 2H, ArH), 3.89 (bs, 2H, NH2), 3.02–
3.09 (m, 4H), 1.89–1.96 ppm (m, 4H); LC–MS m/z: 163 [M+H]+,
tR =0.7 min.
1
2-(3,4-Dichlorophenyl)-N-methyl-N-(trans-(ꢁ))-(2-morpholinocy-
clohexyl)acetamide·HCl (35): Prepared according to Method A
from trans-(ꢁ)-N-methyl-2-morpholinocyclohexanamine (100 mg,
0.5 mmol), 3,4-dichlorophenylacetic acid (205 mg, 1.0 mmol), HOBt
(135 mg, 1.0 mmol), DIPEA (174 mL, 1.0 mmol), and EDCI (192 mg,
1.0 mmol) in DMF (anhydrous, 5 mL) in 75% yield. 1H NMR
(500 MHz, CDCl3): d=11.58 (bs, 1H, HCl), 7.40 (d, 1H, ArH, J=
2.1 Hz), 7.37 (d, 1H, ArH, J=8.3 Hz), 7.20 (dd, 1H, ArH, J=8.3 and
2.1 Hz), 5.02–5.09 (m, 1H), 4.80–4.88 (m, 1H), 4.28–4.34 (m, 1H),
4.25 (d, 1H, J=16.4 Hz), 3.94–3.99 (m, 1H), 3.83–3.91 (m, 2H), 3.65
(d, 1H, J=16.4 Hz), 3.21–3.30 (m, 1H), 3.12–3.19 (m, 2H), 3.12 (s,
3H, CH3), 2.86–2.94 (m, 1H), 2.21–2.26 (m, 1H), 1.97–2.02 (m, 1H),
1.82–1.90 (m, 2H), 1.59–1.69 (m, 1H), 1.48–1.56 (m, 2H), 1.25–
1.42 ppm (m, 2H); 13C NMR (125 MHz, [D6]DMSO): d=171.9, 137.4,
131.8, 130.3, 130.3, 130.0, 128.8, 63.4, 62.6, 50.5, 48.8, 47.2, 30.3,
29.2, 23.9, 23.6 ppm; LC–MS m/z: 385 [M+H]+, tR =4.2 min; HRMS
(ESI) calcd for C19H27N2O2Cl2 385.1450 [M+H]+, found 385.1448.
(R)-2-(3,4-Dichlorophenyl)-N-methyl-N-[1-phenyl-2-(pyrrolidin-1-
yl)ethyl]acetamide·HCl (46): 2-(3,4-Dichlorophenyl)acetic acid
(402 mg, 1.96 mmol) was stirred in excess thionyl chloride at room
temperature for 10 min, and then concentrated in vacuo to form
the corresponding acid chloride. (R)-N-Methyl-1-phenyl-2-(pyrroli-
din-1-yl)ethanamine (200 mg, 0.98 mmol) in CH2Cl2 (3 mL) was
added slowly to a stirring solution of the acid chloride in CH2Cl2
(30 mL) at 08C. DIPEA (325 mL, 1.96 mmol) was added, and the re-
action mixture was heated at 408C for 18 h. After concentrating in
vacuo, the resulting residue was partitioned between CH2Cl2 and
2n NaOH, the organics were dried over MgSO4, concentrated, and
purified by column chromatography. The resulting gum was taken
up in Et2O, 2m HCl in Et2O was added dropwise, and the HCl salt
was collected by filtration in 20% yield. 1H NMR (500 MHz,
[D6]DMSO): d=9.99 (bs, 1H, HCl), 7.57 (d, 1H, ArH, J=8.2 Hz), 7.56
(d, 1H, ArH, J=1.9 Hz), 7.41 (t, 2H, ArH, J=7.4 Hz), 7.35 (t, 1H, ArH,
J=7.4 Hz), 7.29 (dd, 1H, ArH, J=8.2 and 1.9 Hz), 7.26 (d, 2H, ArH,
J=7.4 Hz), 6.13 (dd, 1H, J=12.1 and 2.6 Hz), 4.05–4.12 (m, 1H),
3.94 (d, 1H, J=16.3 Hz), 8.34 (d, 1H, 16.3 Hz), 3.61–3.70 (m, 2H),
3.50–3.57 (m, 1H), 3.10–3.24 (m, 2H), 2.78 (s, 3H, CH3), 1.88–
2.06 ppm (m, 4H); 13C NMR (125 MHz, [D6]DMSO): d=171.9, 137.5,
136.5, 132.0, 130.6, 130.2, 129.9, 128.8, 128.7, 128.0, 127.2, 55.1,
52.5, 51.1, 38.9, 29.8, 23.0, 22.6 ppm; LC–MS m/z: 391 [M+H]+,
tR =4.1 min; HRMS (ESI) calcd for C21H25N2OCl2 391.1344 [M+H]+,
found 391.1329.
2-(3,4-Dichlorophenyl)-N-(trans-(ꢁ))-[2-(pyrrolidin-1-yl)cyclohexy-
l]acetamide (38): Prepared according to Method A from trans-(ꢁ)-
2-(pyrrolidin-1-yl)cyclohexanamine (100 mg, 0.59 mmol), 3,4-di-
chlorophenylacetic acid (242 mg, 1.18 mmol), HOBt (159 mg,
1.18 mmol), DIPEA (205 mL, 1.18 mmol), and EDCI (226 mg,
1.18 mmol) in DMF (anhydrous, 5 mL) in 14% yield. 1H NMR
(500 MHz, [D6]DMSO): d=7.81 (bd, 1H, NH, J=8.2 Hz), 7.54 (d, 1H,
ArH, J=8.2 Hz), 7.51 (d, 1H, ArH, J=2.1 Hz), 7.26 (dd, 1H, ArH, J=
8.2 and 2.1 Hz), 3.61–3.68 (m, 1H), 3.46 (d, 1H, J=14.0 Hz), 3.37 (d,
1H, J=14.0 Hz), 2.50–2.55 (m, 2H), 2.35–2.46 (m, 3H), 1.79–1.85
(m, 1H), 1.72–1.78 (m, 1H), 1.63–1.70 (m, 1H), 1.46–1.63 (m, 5H),
1.13–1.30 ppm (m, 4H); 13C NMR (125 MHz, [D6]DMSO): d=168.3,
138.1, 130.8, 130.6, 130.1, 129.3, 128.8, 61.4, 49.7, 47.5, 41.5, 31.4,
23.7, 23.7, 23.3 ppm; LC–MS m/z: 355 [M+H]+, tR =3.8 min; HRMS
(ESI) calcd for C18H25N2OCl2 355.1344 [M+H]+, found 355.1348.
1-Phenyl-N-[trans-(ꢁ)-2-(pyrrolidin-1-yl)cyclohexyl]cyclopropane-
carboxamide (54): Prepared according to Method C from trans-
(ꢁ)-N-methyl-2-(pyrrolidin-1-yl)cyclohexanamine (91 mg, 0.5 mmol),
1-phenylcyclopropane carboxylic acid (81 mg, 0.5 mmol), Et3N
(0.2 mL), and PyBrop (312 mg, 0.6 mmol) in CH2Cl2 (anhydrous,
1 mL). The crude residue was purified by column chromatography
eluting with CH2Cl2/MeOH/NH3 95:5:0.1 to afford 54 (86 mg, 53%).
1H NMR (500 MHz, [D6]DMSO): d=8.62 (bs, 1H), 7.15 (t, 2H, J=
7.6 Hz, PhH), 7.04 (t, 1H, J=7.3 Hz, PhH), 6.98 (d, 2H, J=7.3 Hz,
PhH), 4.35 (t, 1H, J=10.5 Hz), 3.42 (t, 1H, J=11.5), 3.08–3.05 (m,
2H), 2.98–2.89 (m, 2H), 2.45 (s, 3H, CH3), 1.91 (d, 1H), 1.67–1.64 (m,
3H), 1.58–1.55 (m, 2H), 1.49 (d, 1H, J=12.3 Hz), 1.35–1.27 (m, 5H),
1.24 (s, 2H), 1.16–1.09 (m, 1H), 1.04–0.97 ppm (m, 1H); 13C NMR
(125 MHz, [D6]DMSO): d=172.8, 139.9, 128.8, 126.3, 125.4, 62.0,
59.7, 51.0, 49.3, 30.3, 28.4, 24.7, 23.9, 23.5, 23.1, 22.9, 16.1,
13.1 ppm; LC–MS: m/z 327 [M+H]+, tR =5.1–5.2 min; HRMS (ESI)
calcd for C21H31N2O 327.2431 [M+H]+, found 327.2425.
2-(3,4-Dichlorophenyl)-N-[2-(pyrrolidin-1-yl)phenyl]acetamide
(44): Prepared according to Method A from 2-(pyrrolidin-1-yl)ani-
line 8b (200 mg, 1.23 mmol), 3,4-dichlorophenylacetic acid
(380 mg, 1.85 mmol), HOBt (250 mg, 1.85 mmol), DIPEA (322 mL,
1.85 mmol), and EDCI (355 mg, 1.85 mmol) in DMF (anhydrous,
1
5 mL) in 8% yield. H NMR (500 MHz, CDCl3): d=8.27–8.33 (m, 2H,
ArH, NH), 7.44–7.50 (m, 2H, ArH), 7.18–7.23 (m, 1H, ArH), 7.06–7.11
(m, 2H, ArH), 7.00–7.05 (m, 1H, ArH), 3.73 (s, 2H), 2.71–2.79 (m,
4H), 1.72–1.79 ppm (m, 4H); 13C NMR (125 MHz, [D6]DMSO): d=
168.2, 143.8, 137.1, 131.3, 130.7, 130.4, 129.7, 129.3, 127.0, 126.6,
125.8, 119.3, 116.2, 50.1, 41.7, 24.5 ppm; LC–MS m/z: 349 [M+H]+,
tR =5.1 min; HRMS (ESI) calcd for C18H19N2OCl2 349.0874 [M+H]+,
found 349.0861.
trans-(ꢁ)-1-Phenyl-N-[trans-(ꢁ)-2-(pyrrolidin-1-yl)cyclohexyl]cy-
clopentanecarboxamide (55): Prepared according to Method C
from trans-(ꢁ)-N-methyl-2-(pyrrolidin-1-yl)cyclohexanamine (91 mg,
0.5 mmol), 1-phenylcyclopentanecarboxylic acid (95 mg, 0.5 mmol),
Et3N (0.2 mL), and PyBrop (312 mg, 0.6 mmol) in CH2Cl2 (anhydrous,
1 mL). The crude residue was purified by column chromatography
eluting with CH2Cl2/MeOH/NH3 95:5:0.1 to afford 55 (74 mg, 42%).
1H NMR (500 MHz, [D6]DMSO): d=8.79 (bs, 1H), 7.28 (t, 2H, J=
7.7 Hz, PhH), 7.17 (t, 1H, J=7.4 Hz, PhH), 7.12 (d, 2H, J=7.45 Hz,
PhH), 4.54 (bs, 1H), 3.50 (bs, 1H), 3.09–2.90 (m, 2H), 2.39–2.33 (m,
1H), 2.28 (s, 3H, CH3), 2.25–2.21 (m, 1H), 2.18–2.13 (m, 1H), 2.04–
2.02 (m, 1H), 1.84–1.78 (m, 3H), 1.70–1.57 (m, 6H), 1.54–1.45 (m,
5H), 1.34–1.32 (m, 2H), 1.24–1.16 (m, 1H), 1.11–1.05 ppm (m, 1H);
13C NMR (125 MHz, [D6]DMSO): d=172.1, 153.9, 133.7, 129.9, 129.3,
112.3, 110.1, 60.1, 56.1, 51.4, 48.5, 38.1, 28.6, 27.4, 24.3, 24.1, 23.9,
2-(Pyrrolidin-1-yl)aniline (8b): 1-Chloro-2-nitrobenzene (1.25 g,
7.9 mmol) was stirred in neat pyrrolidine (20 mL) at room tempera-
ture for 6 h, then concentrated in vacuo. The residue was parti-
tioned between EtOAc and saturated aqueous NaHCO3 solution,
the organics were dried over MgSO4, concentrated, and the result-
ing bright-yellow oil was used without further purification. The
formed 1-(2-nitrophenyl)pyrrolidine was subjected to standard hy-
drogenation conditions in EtOH (50 mL) with 10% Pd/C (800 mg).
The reaction mixture was stirred at room temperature under hy-
drogen balloon for 72 h, filtered through Celite, and the filtrate
was concentrated. The resulting oil was taken up in CH2Cl2, dried
ChemMedChem 2011, 6, 1832 – 1840
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
1839

I. H. Gilbert, P. G. Wyatt, et al.
MED
23.6, 23.5, 23.4, 23.3, 23.0 ppm; LC–MS: m/z 355 [M+H]+, tR =3.3–
3.4 min; HRMS (ESI) calcd for C23H35N2O 355.2744 [M+H]+, found
355.2733.
Acknowledgements
We thank the Wellcome Trust for financial support for these stud-
ies (WT077705 and WT083481), OpenEye for free software licens-
es, and Daniel James for data management.
2-(3,4-Dichlorophenyl)-2-methyl-N-[trans-(ꢁ)-2-(pyrrolidin-1-yl)-
cyclohexyl]propanamide (56): Prepared according to Method C
from trans-(ꢁ)-N-methyl-2-(pyrrolidin-1-yl)cyclohexanamine (91 mg,
0.5 mmol), 4-chloro-a,a-dimethylacetic acid (99 mg, 0.5 mmol),
Et3N (0.2 mL), and PyBrop (312 mg, 0.6 mmol) in CH2Cl2 (anhydrous,
1 mL). The crude residue was purified by column chromatography
eluting with CH2Cl2/MeOH/NH3 95:5:0.1 to afford 56 (121 mg,
Keywords: antiprotozoal
trypanosomiasis · medicinal chemistry · U50488
agents
·
human
African
1
67%). H NMR (500 MHz, [D6]DMSO): d=8.91 (bs, 1H0, 7.43 (d, 2H,
J=8.4 Hz, ArH), 7.21 (d, 2H, J=8.4 Hz, ArH), 4.59 (bs, 1H), 3.89 (bs,
1H), 3.31 (bs, 1H), 3.16–3.08 (m, 2H), 2.28 (s, 3H, CH3), 2.11 (d, 1H,
J=10.2 Hz), 1.87–1.82 (m, 3H), 1.78–1.76 (m, 1H), 1.72 (d, 1H, J=
12.2 Hz), 1.66 (d, 1H, J=12.2 Hz), 1.55 (s, 3H, CH3), 1.50–1.43 (m,
3H), 1.37 (s, 3H, CH3), 1.28–1.26 (m, 1H), 1.20–1.14 ppm (m, 1H);
13C NMR (125 MHz, [D6]DMSO): d=175.8, 144.3, 131.0, 128.8, 126.8,
59.8, 50.8, 50.0, 46.8, 31.2, 30.5, 28.0, 26.0, 25.2, 23.9, 23.5, 23.0,
22.5 ppm; LC–MS: m/z 363 and 365 35Cl and 37Cl, [M+H]+, tR =3.2–
3.4 min; HRMS (ESI) calcd for C21H32ClN2O 363.2198 35Cl [M+H]+,
found 363.2180.
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1-(3,4-Dichlorophenyl)-N-[trans-(ꢁ)-2-(pyrrolidin-1-yl)cyclohexyl]-
cyclopropanecarboxamide (57): Prepared according to Method C
from trans-(ꢁ)-N-methyl-2-(pyrrolidin-1-yl)cyclohexanamine (91 mg,
0.5 mmol),
1-(3,4-dichlorophenyl)cyclopropanecarboxylic
acid
(115 mg, 0.5 mmol), Et3N (0.2 mL), and PyBrop (312 mg, 0.6 mmol)
in CH2Cl2 (anhydrous, 1 mL). The crude residue was purified by
column chromatography eluting with CH2Cl2/MeOH/NH3 95:5:0.1
to afford 57 (33 mg, 17%). 1H NMR (500 MHz, CDCl3): d=7.41 (d,
1H, ArH, J=8.3 Hz), 7.29 (d, 1H, ArH, J=2.1 Hz), 7.03 (dd, 1H, ArH,
J=8.3 and 2.1 Hz), 4.51 (bs, 1H), 3.91 (bs, 1H), 3.75 (bs, 1H), 3.54
(bs, 2H), 3.25 (bs, 1H), 3.15 (bs, 1H), 2.75 (s, 3H, CH3), 2.26–2.18 (m,
2H), 2.13–2.01 (m, 4H), 1.72–1.69 (m, 4H), 1.63–1.58 (m, 3H), 1.45–
1.42 (m, 2H), 1.40–1.39 (m, 1H), 0.85 ppm (bs, 1H); 13C NMR
(125 MHz, [D6]DMSO): d=171.4, 141.8, 131.9, 130.8, 130.7, 119.0,
109.2, 60.1, 51.5, 48.4, 40.2, 28.6, 27.5, 24.1, 23.9, 23.8, 23.6, 23.4,
23.1 ppm; LC–MS: m/z 395 and 397 35Cl and 37Cl [M+H]+, tR =3.1–
3.3 min; HRMS (ESI) calcd for C21H29Cl2N2O 395.1651 35Cl [M+H]+,
found 395.1634.
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D. C. Rees, D. Schofield, J. Med. Chem. 1988, 31, 831–836.
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74–82; b) A. Nicholls, G. B. McGaughey, R. P. Sheridan, A. C. Good, G.
Warren, M. Mathieu, S. W. Muchmore, S. P. Brown, J. A. Grant, J. A. Haigh,
N. Nevins, A. N. Jain, B. Kelley, J. Med. Chem. 2010, 53, 3862–3886.
Received: June 2, 2011
Revised: July 18, 2011
Published online on August 10, 2011
1840
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ChemMedChem 2011, 6, 1832 – 1840