Synthesis and Evaluation of Indatraline-Based Inhibitors for Trypanothione Reductase

Article abstract of DOI:10.1002/cmdc.201000442

The search for novel compounds of relevance to the treatment of diseases caused by trypanosomatid protozoan parasites continues. Screening of a large library of known bioactive compounds has led to several drug-like starting points for further optimisation. In this study, novel analogues of the monoamine uptake inhibitor indatraline were prepared and assessed both as inhibitors of trypanothione reductase (TryR) and against the parasite Trypanosoma brucei. Although it proved difficult to significantly increase the potency of the original compound as an inhibitor of TryR, some insight into the preferred substituent on the amine group and in the two aromatic rings of the parent indatraline was deduced. In addition, detailed mode of action studies indicated that two of the inhibitors exhibit a mixed mode of inhibition. Give these inhibitors a TryR: Indatraline is a CNS-active inhibitor of trypanothione reductase (TryR) revealed in a previous high-throughput screen. For this study we prepared analogues of indatraline and tested their capacity to inhibit TryR and the proliferation of Trypanosoma brucei cells. Inhibitors of micromolar potency with a mixed mode of inhibition were identified.

Full text of DOI:10.1002/cmdc.201000442

DOI: 10.1002/cmdc.201000442
Synthesis and Evaluation of Indatraline-Based Inhibitors
for Trypanothione Reductase
Jeffrey G. A. Walton,[a] Deuan C. Jones,[b] Paula Kiuru,[a] Alastair J. Durie,[a]
Nicholas J. Westwood,*[a] and Alan H. Fairlamb*[b]
The search for novel compounds of relevance to the treatment
of diseases caused by trypanosomatid protozoan parasites
continues. Screening of a large library of known bioactive com-
pounds has led to several drug-like starting points for further
optimisation. In this study, novel analogues of the monoamine
uptake inhibitor indatraline were prepared and assessed both
as inhibitors of trypanothione reductase (TryR) and against the
parasite Trypanosoma brucei. Although it proved difficult to sig-
nificantly increase the potency of the original compound as an
inhibitor of TryR, some insight into the preferred substituent
on the amine group and in the two aromatic rings of the
parent indatraline was deduced. In addition, detailed mode of
action studies indicated that two of the inhibitors exhibit a
mixed mode of inhibition.
Introduction
Trypanosomatid protozoan parasites cause a variety of impor-
tant diseases, including human African sleeping sickness,
Chagas disease, and leishmaniasis. Sleeping sickness is caused
by Trypanosoma brucei ssp., and is endemic in certain regions
of sub-Saharan Africa, covering about 20 countries with an es-
timated 70–80000 people infected.[1] Chagas disease is present
in 19 countries on the American continent and is responsible
for disease in around 8–11 million people.[1] The causative
agent of this disease is the parasite Trypanosoma cruzi. Leish-
maniasis is caused by members of the genus Leishmania and is
found in 88 countries, threatening 350 million people.[1] In
combination, these diseases contribute to approximately
95000 deaths annually, but considering their socioeconomic
importance, the current scale of new drug discovery and vac-
cine development efforts is inadequate. In addition, the avail-
able therapies are often toxic, marginally effective, adminis-
tered by injection, and expensive. In particular, there is an
urgent need for new CNS-active drugs to treat late-stage
sleeping sickness to replace the current therapies that are
losing efficacy due to parasite resistance.[1]
Figure 1. Structures of trypanothione (1), glutathione (2), and indatraline (3).
As part of a concerted campaign to discover new treatments
for trypanosomatid-based diseases, we undertook a high-
throughput screen for inhibitors of TryR. The Sigma-LOPAC1280
The trypanosomatids use a polyamine–glutathione adduct,
trypanothione (1, Figure 1), as a key component of their de-
fence system. Compound 1 is prepared through a unique bio-
synthetic pathway in which glutathione (2) is conjugated to
spermidine.[2] In humans, glutathione and glutathione reduc-
tase (GR) are used to maintain the intracellular redox balance,
whereas the analogous chemistry in the parasite is carried out
by trypanothione reductase (TryR), which reduces trypano-
thione disulfide (T[S]2) to 1. Previous genetic knockout studies
have illustrated the essential role of TryR in parasite viability,[3]
validating it as a target for drug development in all three dis-
eases. Importantly, comparison of TryR and human GR crystal
structures reveal significant differences between their active
sites,[4] suggesting that these differences may be exploited to
gain selectivity for TryR over GR.
[a] Dr. J. G. A. Walton,+ Dr. P. Kiuru, Dr. A. J. Durie, Dr. N. J. Westwood
School of Chemistry and Biomedical Sciences Research Complex
University of St Andrews
North Haugh, St Andrews, Fife, KY16 9ST (UK)
Fax: (+44)1334-462595
E-mail: njw3@st-andrews.ac.uk
[b] Dr. D. C. Jones,+ Prof. A. H. Fairlamb
Division of Biological Chemistry and Drug Discovery
University of Dundee, Dundee, DD1 5EH (UK)
Fax: (+44)1382-385155
E-mail: a.h.fairlamb@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.201000442.
Re-use of this article is permitted in accordance with the Terms and Condi-
tions set out at http://onlinelibrary.wiley.com/journal/10.1002/(ISSN) 1860–
7179/homepage/2452_onlineopen.html
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321

A. H. Fairlamb, N. J. Westwood, et al.
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collection, a library of compounds with known pharmacologi-
cal activity, was screened against TryR.[5] The thinking behind
screening a library of known drugs is encapsulated in Sir
James Black’s famous quote: “The most fruitful basis for the
discovery of a new drug is to start with an old drug”.[6] It was
planned that hits derived from small molecules that already
have desirable drug-like properties could be modified to tune
their selectivity away from their original protein targets and to-
wards TryR without too much disruption of the desirable drug-
like properties.
As reported previously,[5] assessment of initial screening hits
against human GR and T. brucei cells together with in silico
analysis of chemical properties revealed three new classes of
TryR inhibitors that merited further development. Investigation
of one of these classes, based on 1-[1-(2-benzo[b]thienyl)cyclo-
hexyl)]piperidine (BTCP), was reported previously.[7] Herein we
describe a related investigation of a further class of com-
pounds based on indatraline (3), a nonselective monoamine re-
uptake inhibitor.[8] The molecule contains an indanamine core
structure with a second aromatic unit at the 3-position
(Figure 1). This CNS-active molecule was considered suitable
for extensive modification and was of sufficiently low molecu-
lar weight to allow adherence to Lipinski’s rules, even follow-
ing considerable alteration.[9] We identified four possible sites
(A–D, Figure 1) for modification of the core structure. Details of
the parallel synthetic routes that were developed along with
careful analysis of the activity of the analogues generated
against TryR in vitro and T. brucei in culture are reported.
Whilst it proved difficult in this chemical series to improve po-
tency against the desired target, a new important insight into
the mode of inhibition of TryR by these analogues was discov-
ered, progressing our thinking on how to inhibit effectively
this important enzyme.
Scheme 1. Reagents and conditions: a) MeNH2, TiCl4, PhMe, À108C, 1 h;
b) NaBH4, MeOH, RT, 3 h (62%); c) NaBH4, MeOH, RT, 2 h (77%); d) SOCl2, Tol.,
RT, 3 h; e) NHR1R2, THF, 908C, 4 h; f) (PhO)2P(O)N3, DBU, THF, RT, o/n (93%);
g) PS–PPh3, H2O, THF, RT, 16 h (quant); h) R1CHO, NaBH(OAc)3 or CH3COCl or
TsCl, THF, RT, o/n. R1 and R2 are defined in Table 1 and Supporting Informa-
tion.[11]
rative HPLC, and the stereochemistry was assigned by
comparison with published work.[8a]
Having prepared analogues 5 and 8i–vi, we decided to eval-
uate the routes used for conversion into a parallel synthesis
protocol. This was viewed as challenging due to the required
separation of the isomeric mixtures of 8 on a small scale. We
therefore decided to adapt the original route by incorporating
a modified Mitsunobu protocol to convert indanol 6a to the
azide 9a (Scheme 1).[12] This reaction occurred with complete
inversion of the C1 stereochemistry in 6a and generated exclu-
sively the trans isomer of 9a, removing the requirement to
separate isomeric mixtures later in the route. A Staudinger re-
duction of azide 9a using polymer-supported triphenylphos-
phine generated the indanamine 10a in high yield and purity
following filtration to remove the reagent. Reductive amination
of 10a with a range of aldehydes in the presence of sodium
triacetoxyborohydride afforded the required trans-indanamines
8vii–xiii. Initially, purification of these compounds was neces-
sary, as over-alkylation of 10a was observed. However when
an excess of 10a (1.2 equivalents) was used in this reaction,
none of the dialkylated product was formed, and remaining
10a was removed using a polymer-supported benzaldehyde
scavenger resin. With this protocol it was possible to prepare
pure trans-indanamines 8vii–xiii in moderate to high yields. In-
danamine 10a was also treated with acetyl and tosyl chloride
to afford 8xiv and 8xv, respectively. The stereochemistry of 8
and 9 prepared by this route was assigned based on literature
precedent from Rice and co-workers.[12]
Results and Discussion
Synthesis of indatraline analogues
Initial studies focused on the amino substituent in 3 (site A,
Figure 1) starting from the common intermediate 3-phenylin-
danone (4a, Scheme 1). Compound 4a was prepared accord-
ing to published methods.[10] Treatment of 4a with methyl-
amine in the presence of titanium tetrachloride followed by re-
duction of the resulting imine with sodium borohydride afford-
ed indanamine 5 as the cis isomer, as reported by Bøgesø
et al.[8a] Access to the trans-indanamines was achieved as fol-
lows:[8a] Reduction of indanone 4a with sodium borohydride
gave 3-phenylindan-1-ol (6a) in high yield and high cis selec-
tivity (97:3). A single recrystallisation was required to afford the
pure cis isomer. Reaction of 6a with thionyl chloride resulted
in an isomeric mixture of cis- and trans-1-chloro-3-phenylin-
danes (7), with a cis/trans ratio of 7:3. Crude 7 was then react-
ed with a series of primary and secondary alkylamines to pro-
duce the corresponding 3-phenylindan-1-amines with, as ex-
pected, a reversal of the cis/trans ratio (3:7). The pure trans iso-
mers 8i–vi were isolated following purification by semi-prepa-
Having developed a robust route to amino-substituted ana-
logues, we decided to investigate modification of the two aro-
matic rings present in 3. Rice and colleagues reported a proto-
col to access the indanone core via an aldol condensation of
3-methoxyacetophenone (11 b, Scheme 2) with 3,4-dichloro-
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Indatraline-Based TryR Inhibitors
12b–e. The electronic nature of the substituent on the benzal-
dehydes did not influence the outcome of the Nazarov cyclisa-
tion, and all the chalcones afforded the desired indanones 4 f–
i. These were then converted into the indanamines 8xvi–xxi
via method B (Scheme 1).
Biological analysis against TryR, GR, and T. brucei
The analogues prepared according to the methods described
above were tested against T. cruzi TryR and in an assay for
T. brucei cell proliferation, as described below in the Experi-
mental Section.
The biological data for a selected group of these com-
pounds are summarised in Table 1. The cis isomer 5 (Table 1,
Entry 1) was less active against TryR than indatraline 3,
Entry 2). This resulted in the synthetic efforts being focused on
the generation of trans analogues. As the size and lipophilicity
of the amino substituent was increased (Entries 1 and 3–6),
there appeared to be a relatively flat SAR (see also analogues
S8xxii–S8xxvii in the Supporting Information[11]). This suggests
that this region of the molecule contributes little to the biolog-
ical activity and is probably incorporated in a large open
region of the active site. The slightly lower activity of the N-tBu
analogue 8iii may, however, indicate some steric constraint
close to the nitrogen atom. In addition, the piperidine (8v,
Entry 7) and morpholine (8vi, Entry 8) analogues were pre-
pared. Whilst the piperidine analogue 8v retained activity, the
insertion of an additional oxygen atom, in the case of the mor-
pholine analogue 8vi, caused a significant decrease in activity
(see also analogue S8xxx in the Supporting Information[11]). By
replacing the methyl substituent of indatraline with a benzyl
group (8vii, Entry 9), the activity was retained. This provided
the opportunity to synthesise a Topliss series of analogues.[14]
Therefore the 4-chloro (8viii, Entry 10), 3,4-dichloro (8ix,
Entry 11), 4-methyl (8x, Entry 12), and 4-methoxy (8xi,
Entry 13) analogues were prepared. The incorporation of elec-
tron-donating substituents led to the highest activity, whereas
activity diminished when electron-withdrawing groups were
introduced. This result led to the preparation of the remaining
two compounds in the electron-rich Topliss series: 4-dimethyl-
amino (8xii, Entry 14) and 4-amino (8xiii, Entry 15). These re-
sults further highlight the benefits associated with the inclu-
sion of an electron-donating substituent, although no further
improvement in activity was observed relative to the 4-me-
thoxy analogue 8xi. For data on additional substituted benzyl
analogues, see table S1, analogues S8xxxi–S8xxxv (Support-
ing Information). The acetyl (8xiv, Entry 16) and tosyl (8xv,
Entry 17) analogues were inactive against TryR (see also ana-
logue S8xxxvi in the Supporting Information[11]).
Scheme 2. Reagents and conditions: a) R4CHO, KOH, EtOH, 08C, 2 h; b) TFA,
1208C, microwave, 10 min; c) NaBH4, MeOH, RT, 2 h; d) (PhO)2P(O)N3, DBU,
THF, RT, o/n; e) PS–PPh3, H2O, THF, RT, 16 h; f) R1CHO, NaBH(OAc)3, THF, RT, o/
n. R1–R4 are defined in Table 1 and Supporting Information.[11]
benzaldehyde (12a) to generate chalcone (13b).[12,13] In their
hands, a subsequent Nazarov cyclisation in the presence of tri-
fluoroacetic acid afforded 6-methoxyindanone (4b) in high
yield. This route was particularly appealing, as it allowed rapid
access to the indanone core in two steps. Furthermore, the
product from the aldol reaction crystallised out of the reaction
solution and required no further purification. These factors
made this approach potentially adaptable to a high-through-
put synthesis format. To investigate this further, a series of
chalcones (compounds 13a–e) were prepared from the aldol
reaction between acetophenones 11 a–e and 3,4-dichloroben-
zaldehyde (12a). The chalcones 13a–e were then submitted to
cyclisation reactions using trifluoroacetic acid. The Nazarov re-
action only occurred when a 3-methoxy substituent was pres-
ent in the chalcone (for substrate 13b). In this case, it was pos-
sible to decrease the reaction time considerably by carrying
out the reactions in a microwave reactor as opposed to con-
ventional heating (10 min versus 4 h, respectively). Having de-
termined the requirements for a successful Nazarov reaction, a
collection of indanones (4b,f–i) was prepared by using 3-me-
thoxyacetophenone (11 b) and a variety of benzaldehydes
We decided to use the isoamyl group (see 8ii, Entry 4) as
the substituent for site A, whilst an exploration of the B and C
rings was carried out. The inclusion of a 6-methoxy substituent
in the B ring was investigated, as this was a synthetic require-
ment for the Nazarov reaction. Analogue 8xvi (Entry 18) was
prepared as a direct comparison with 8ii (Entry 4). The in-
creased activity of 8xvi relative to 8ii demonstrated that the
presence of an electron-donating substituent in the B ring is
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A. H. Fairlamb, N. J. Westwood, et al.
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However, compounds 8ii (Fig-
ure 2b) and 8iii were confirmed
by an F-test to show a mode of
mixed inhibition. The mixed in-
hibition mode indicates that
these inhibitors are able to bind
to the substrate–enzyme com-
plex as well as to the free
enzyme, whilst the competitive
mode indicates binding only to
the free enzyme. However, even
for the mixed mode inhibitors,
the Ki’ values are approximately
fivefold greater than the Ki
values, suggesting that these
compounds bind much more fa-
vourably to the free enzyme.
This subtle shift in mode of in-
hibition may represent a useful
direction for further develop-
ment, because mixed inhibitors
are less susceptible to the effects
of substrate accumulation than
competitive inhibitors. For these
series, driving down the Ki’ value
may be more effective than fo-
cussing solely on the potency
(IC50) of the inhibitors.
Table 1. Biological data for selected indatraline analogues.
Entry
Compound
R1
R2
R3
R4
TryR
T. brucei
EC50 [mm]
IC50 [mm][a]
Pentamidine[b]
3
0.0037Æ0.0001
1.06Æ0.05
1.50Æ0.23
2.12Æ0.06
1.08Æ0.07
0.48Æ0.04
0.66Æ0.04
1.31Æ0.11
6.62Æ0.77
3.53Æ0.35
3.22Æ0.26
8.87Æ0.60
1.29Æ0.04
2.36Æ0.22
1.98Æ0.23
2.68Æ1.04
3.64Æ0.28
4.33Æ0.33
1.26Æ0.13
2.47Æ0.25
2.51Æ0.33
1.49Æ0.08
6.99Æ1.55
2.30Æ0.22
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
Me
Me
Et
Isoamyl
tBu
Octyl
–CH2(CH2)3CH2–
–(CH2)2O(CH2)2–
Benzyl
4-Cl-Bn
3,4-Cl2-Bn
4-Me-Bn
4-OMe-Bn
4-Me2N-Bn
4-NH2-Bn
Acetyl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
3,4-Cl2
3,4-Cl2
3,4-Cl2
3,4-Cl2
3,4-Cl2
3,4-Cl2
3,4-Cl2
3,4-Cl2
3,4-Cl2
3,4-Cl2
3,4-Cl2
3,4-Cl2
3,4-Cl2
3,4-Cl2
3,4-Cl2
3,4-Cl2
3,4-Cl2
3,4-Cl2
3,4-Br2
4-Cl
8.84Æ0.24
13.5Æ0.7
7.19Æ0.40
4.05Æ0.39
15.3Æ1.4
7.93Æ0.78
5.47Æ0.32
173Æ56
5[c]
8i[b]
8ii
8iii[b]
8iv
8v[b]
8vi
8vii[b]
8viii
8ix
H
8.04Æ0.91
17.6Æ3.3
45.9Æ4.2
13.0Æ1.6
4.14Æ0.41
7.38Æ1.21
4.94Æ0.43
>200
H
H
H
H
H
H
H
H
H
H
H
H
H
H
8x
8xi
8xii
8xiii
8xiv
8xv
8xvi
8xvii
8xviii
8xix
8xx
Tosyl
>200
Isoamyl
Isoamyl
Isoamyl
Isoamyl
Isoamyl
Benzyl
6-OMe
6-OMe
6-OMe
6-OMe
6-OMe
6-OMe
2.23Æ0.66
3.15Æ0.23
15.8Æ1.6
32.9Æ4.3
125Æ17
4-Me
3-OMe
3,4-Cl2
8xxi
3.07Æ0.45
[a] Inhibition of trypanothione reductase (TryR). [b] Pentamidine was included as a reference drug in biological
testing.[16a] [c] See Ref. [11].
favourable. Therefore a collection of analogues was synthes-
ised containing the 6-methoxy substituent but with variations
Conclusions
in substituents in the C ring. The 3,4-dibromo (8xvii, Entry 19),
We previously reported the screening of the Sigma-LOPAC1280
collection of bioactive compounds for inhibitors of TryR and
have described follow-up studies on one of the hits we ob-
tained from this collection.[5,7] Herein we report our attempts
to progress a second hit obtained in this screen. Indatraline 3,
a CNS-active, nonselective monoamine reuptake inhibitor, pro-
vided the drug-like starting point for our studies. Using a
range of synthetic routes, novel indatraline analogues were
prepared in order to explore structure–activity relationships.
Whilst it proved difficult to significantly increase the potency
of the original compound as an inhibitor of TryR, some insight
into the preferred substituent on the amine and in the two ar-
omatic rings was deduced. In addition, detailed mode of
action studies indicated that two of the inhibitors exhibit a
mixed mode of inhibition. This interesting observation has led
us to further refine the criteria that need to be considered
when developing inhibitors of this important enzyme.
4-chloro (8xviii, Entry 20), 4-methyl (8xix, Entry 21), and 3-me-
thoxy (8xx, entry 22) substituted analogues were prepared
and tested. A clear preference for electron-withdrawing sub-
stituents in the C ring was observed (see also 8xxi and addi-
tional analogues in table S1, Supporting Information).
All the analogues were also tested against T. brucei cultured
in vitro. These data predominantly followed a trend similar to
that of the IC50 data, with decreases in the IC50 values being
mirrored by corresponding decreases in the EC50 values. How-
ever, there were a few notable exceptions. Compounds 8iii
(EC50 =0.48 mm, IC50 =15.29 mm) and 8iv (EC50 =0.66 mm, IC50 =
7.93 mm) showed much improved EC50 values relative to their
corresponding IC50 values. Furthermore, although 8xiv (EC50 =
3.64 mm, IC50 >200 mm) and 8xv (EC50 =4.33 mm, IC50 >200 mm)
were inactive against TryR, they showed reasonable activity
against T. brucei. These results could suggest that these ana-
logues have additional off-target effects, or are selectively con-
centrated/metabolically activated, or a combination of these.
In addition, all compounds were tested for inhibition of human
GR as described in the Experimental Section below. Com-
pounds were tested in duplicate at a single final concentration
of 25 mm. All compounds exhibited <12% inhibition.
Experimental Section
Procedures for the synthesis of analogues of indatraline (3)
Unless otherwise stated, starting materials and reagents were ob-
tained from commercial suppliers and were used without further
purification. 1H and 13C NMR spectra were measured on a Bruker
Advance 300/400 instrument. Chemical shifts are calibrated with
reference to the residual proton and carbon resonances of the sol-
Three of the analogues were chosen for assessment of the
mode of inhibition with respect to T[S]2, as described in the Ex-
perimental Section. Indatraline (3), the original indatraline start-
ing point (Figure 2a), is a linear competitive inhibitor of TryR.
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Indatraline-Based TryR Inhibitors
m, CHsyn), 2.53–2.48 (1H, m, CHanti), 1.76–1.61 (3H, m, CH and CH2),
0.99 (3H, d, J=6.7 Hz, CH3), 0.98 (3H, d, J=6.7 Hz, CH3); 13C NMR
(100 MHz, MeOD): d=148.8 (C), 145.9 (C), 138.4 (C), 133.7 (C),
132.0 (CH ꢁ2), 131.9 (C), 131.2 (CH), 129.3 (CH), 129.1 (CH), 127.3
(CH), 127.0 (CH), 63.3 (CH), 49.4 (CH), 45.8 (CH2), 40.3 (CH2), 36.1
(CH2), 27.3 (CH), 22.7 (CH3), 22.6 (CH3); LRMS (CI+) m/z: 348.1
[M+H]+; HRMS (CI+) [M+H]+ m/z expected for C20H24N35Cl37Cl
350.1256, obtained 350.1246.
Trans-[3-(3,4-dichlorophenyl)indan-1-yl]octylamine (8iv): Yield
60%, colourless oil; 1H NMR (300 MHz, CDCl3): d=7.33–7.26 (2H,
m, CHar), 7.20–7.15 (3H, m, CHar), 6.91–6.88 (2H, m, CHar), 4.44 (1H,
t, J=7.6 Hz, CH-N), 4.28 (1H, dd, J=6.8, 3.4 Hz, CH-Ar), 2.65–2.60
(2H, m, CH2), 2.41–2.32 (1H, m, CHsyn), 2.21–2.12 (1H, m, CHanti),
1.52–1.39 (2H, m, CH2), 1.28–1.12 (10H, m, CH2 ꢁ5), 0.83–0.79 (3H,
m, CH3); 13C NMR (100 MHz, CDCl3): d=145.7 (C), 145.4 (C), 145.4
(C), 132.5 (C), 130.4 (CH), 130.3 (C), 129.9 (CH), 128.5 (CH), 127.4
(CH), 127.3 (CH), 125.3 (CH), 124.8 (CH), 124.8 (CH), 61.9 (CH), 48.5
(CH), 47.5 (CH2), 43.3 (CH2), 31.8 (CH2), 29.8 (CH2), 29.5 (CH2), 27.4
(CH2), 22.7 (CH2), 14.1 (CH3); LRMS (CI+) m/z: 390.2 [M+H]+; HRMS
(CI+) [M+H]+ m/z expected for C23H30NCl2 390.1755, obtained
390.1755.
Trans-4-[3-(3,4-dichlorophenyl)indan-1-yl]morpholine (8vi): Yield
1
61%, colourless oil; H NMR (300 MHz, MeOD): d=7.80–7.77 (1H,
m, CHar), 7.52–7.43 (3H, m, CHar), 7.37 (1H, d, J=1.8 Hz, CHar), 7.12
(1H, dd, J=8.3, 1.8 Hz, CHar), 7.07–7.04 (1H, m, CHar), 5.10 (1H, d,
J=8.0 Hz, CH-N), 4.80 (1H, t, J=8.0 Hz, CH-Ar), 4.09–4.04 (2H, m,
CH2), 3.94–3.82 (2H, m, CH2), 3.50–3.45 (1H, m, CH), 3.37–3.35 (2H,
m, CH2), 3.26–3.06 (2H, m, CH2, CHsyn), 2.56–2.45 (1H, m, CHanti);
13C NMR (75 MHz, MeOD): d=150.3 (C), 145.8 (C), 135.6 (C), 133.7
(C), 132.6 (CH), 132.0 (CH), 131.4 (CH), 129.3 (CH), 129.2 (CH), 128.7
(CH), 127.1 (CH), 72.0 (CH), 65.2 (CH2), 65.0 (CH2), 50.8 (CH2), 50.3
(CH2), 50.1 (CH), 38.3 (CH2); LRMS (CI+) m/z: 348.1 [M+H]+; HRMS
(CI+) [M+H]+ m/z expected for C19H20NOCl2 348.0922, obtained
348.0900.
Figure 2. Mode of inhibition by indatraline and compound 8ii. a) Indatraline:
linear competitive inhibition with respect to trypanothione disulfide,
Ki =5.0Æ0.2 mm. b) 8ii: linear mixed inhibition, Ki =3.5Æ0.4 mm;
Ki’=16.8Æ3.3 mm.
General procedure for chalcone 13a–i formation: A solution of
KOH (6 mmol) in H2O (3 mL) was slowly added to a solution of
ketone 11 (2 mmol) and aldehyde 12 (2 mmol) in EtOH (6 mL) at
08C. The reaction mixture was stirred for 2 h, and the resulting pre-
cipitate was collected by filtration, washed with EtOH (3 mL), and
dried in vacuo. See Supporting Information for analytical data for
13a–i.
vent (CDCl3: dH =7.26, dC =77.0 ppm). Low- and high-resolution
mass spectrometric analyses were recorded using chemical ionisa-
tion operating in positive or negative ion mode. For assessment of
purity of novel compounds that were tested, see table S2 (Sup-
porting Information).[11]
General procedure for the Nazarov reaction (4b,f–i): A solution
of 13 (1 mmol) in TFA (4 mL) was heated in a sealed 10 mL tube in
a multimode CEM DiscoverTM microwave at 1208C for 10 min. The
powermax mode was enabled, and the microwave was used at its
maximum power of 300 W until the set temperature was reached.
The solvent was removed in vacuo, and the residue was poured
onto ice water and extracted with EtOAc (3ꢁ10 mL). The com-
bined extracts were washed with saturated NaHCO3 solution
(20 mL) and brine (20 mL), dried (Na2SO4) and reduced in vacuo.
Purification through a plug of silica (Hex/EtOAc 20:1) afforded the
desired indanone. See Supporting Information for analytical data
for 4b,f–i.
General procedure for the formation of amines 8 (Method A):
The corresponding amine (0.5 mmol) was added to a solution of
7[11] (100 mg, 0.34 mmol) in THF (3 mL), and the solution was
stirred at 908C in a sealed tube for 4 h. The solvent was removed
in vacuo, and the residue partitioned between H2O (10 mL) and
Et2O (10 mL). The organic layer was extracted with 10% citric acid
(3ꢁ10 mL), and the extract was basified with 10% aqueous NH4OH
solution then extracted with Et2O (3ꢁ20 mL). The combined organ-
ics were dried (Na2SO4) and concentrated in vacuo. Purification by
semi-preparative HPLC (Phenomenex Luna silica column, EtOAc/
Hex 6:4 (0.1% Et2NH)) afforded the desired amines.
General procedure for the formation of alcohols (6b,f–i): NaBH4
(0.6 mmol) was added to a solution of 4 (0.6 mmol) in MeOH
(3 mL), and the reaction was stirred at room temperature for 2 h. A
2m solution of aqueous KOH (3 mL) was added, and the reaction
mixture was extracted with CH2Cl2 (3ꢁ7 mL). The combined organ-
ic layers were dried (Na2SO4) and reduced in vacuo. Purification of
the residue through a plug of silica (Hex/EtOAc 10:1) afforded the
Trans-[3-(3,4-dichlorophenyl)indan-1-yl]-(3-methylbutyl)amine
hydrochloride (8ii): Yield 72%, white solid; mp: 190–1918C;
1H NMR (400 MHz, MeOD): d=7.71 (1H, d, J=6.2 Hz, CHar), 7.49
(1H, d, J=8.3 Hz, CHar) 7.45–7.43 (2H, m, CHar), 7.34 (1H, d, J=
1.8 Hz, CHar), 7.12–7.06 (2H, m, CHar), 4.97–4.95 (1H, m, CH-N), 4.76
(1H, t, J=7.6 Hz, CH-Ar), 3.17–3.13 (2H, m, CH2N), 2.86–2.81 (1H,
ChemMedChem 2011, 6, 321 – 328
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www.chemmedchem.org
325

A. H. Fairlamb, N. J. Westwood, et al.
MED
desired alcohol. See Supporting Information for analytical data for
6b,f–i.
CH2-Ar), 2.45–2.37 (1H, m, CHsyn), 2.26 (3H, s, CH3), 2.22–2.13 (1H,
m, CHanti); 13C NMR (75 MHz, CDCl3): d=145.6 (C), 145.5 (C), 145.5
(C), 137.2 (C), 136.6 (C), 132.4 (C), 130.4 (CH), 130.2 (C), 129.9 (CH),
129.1 (CH ꢁ2), 128.2 (CH), 128.1 (CH ꢁ2), 127.4 (CH), 127.2 (CH),
125.2 (CH), 124.6 (CH), 61.4 (CH), 51.5 (CH2), 48.6 (CH), 43.8 (CH2),
21.1 (CH3); LRMS (CI+) m/z: 382.1 [M+H]+; HRMS (CI+) [M+H]+
m/z expected for C23H22NCl2 382.1129, obtained 382.1134.
General procedure for the formation of azides 9b,f–i: Diphenyl-
phosphoryl azide (0.48 mmol) was added to a solution of 6
(0.4 mmol) in anhydrous THF (2 mL) at 08C, and the reaction was
stirred for 10 min. DBU (0.48 mmol) was slowly added, and the re-
action mixture was stirred overnight. H2O (3 mL) was added, and
the reaction mixture was extracted with CH2Cl2 (3ꢁ7 mL). The
combined organic layers were dried (Na2SO4) and reduced in va-
cuo. Purification of the residue through a plug of silica (Hex/EtOAc
40:1) afforded the desired azide. See Supporting Information for
analytical data for 9b,f–i.
Trans-[3-(3,4-dichlorophenyl)indan-1-yl]-(4-methoxybenzyl)a-
mine (8xi): Yield 83%, colourless oil; 1H NMR (300 MHz, CDCl3):
d=7.42–7.39 (1H, m, CHar), 7.35 (1H, d, J=8.3 Hz, CHar), 7.30–7.22
(5H, m, CHar), 6.99–6.94 (2H, m, CHar), 6.91–6.85 (2H, m, CHar), 4.54
(1H, t, J=7.7 Hz, CH-N), 4.41 (1H, dd, J=6.8, 3.5 Hz, CH-Ar), 3.84
(2H, s, CH2-Ar), 3.80 (3H, s, CH3), 2.49 (1H, ddd, J=13.1, 7.7, 3.5 Hz,
CHsyn), 2.25 (1H, dt, J=13.1, 6.8 Hz, CHanti), 1.69 (1H, brs, NH);
13C NMR (75 MHz, CDCl3): d=158.7 (C), 145.6 (C), 145.5 (C), 145.5
(C), 145.3 (C), 132.4 (C), 130.4 (CH), 130.2 (C), 129.9 (CH), 129.3 (CH
ꢁ2), 128.2 (CH), 127.4 (CH), 127.2 (CH), 125.2 (CH), 124.6 (CH), 113.8
(CH ꢁ2), 61.5 (CH), 55.3 (CH3), 51.2 (CH2), 48.6 (CH), 43.6 (CH2);
LRMS (CI+) m/z: 398.1 [M+H]+; HRMS (CI+) [M+H]+ m/z expected
for C23H22NOCl2 398.1078, obtained 398.1082.
General procedure for Staudinger reduction to form 10b,f–i:
PS–PPh3 (0.6 mmol) was added to a solution of 9 (0.3 mmol) in an-
hydrous THF (5 mL), and the reaction was stirred for 16 h. H2O
(1 mL) was added, and the reaction was stirred for a further 4 h.
The reaction mixture was filtered and extracted with CH2Cl2 (3ꢁ
5 mL). The combined organic layers were dried (Na2SO4) and con-
centrated in vacuo to give the desired amine 10. See Supporting
Information for analytical data for 10b,f–i.
Trans-[3-(3,4-dichlorophenyl)indan-1-yl]-(4-dimethylaminoben-
zyl)amine (8xii): Yield 68%, colourless oil; 1H NMR (300 MHz,
CDCl3): d=7.46–7.43 (1H, m, CHar), 7.38 (1H, d, J=8.3 Hz, CHar),
7.32–7.24 (5H, m, CHar), 7.02–6.98 (2H, m, CHar), 6.76 (2H, d, J=
8.5 Hz, CHar), 4.58 (1H, t, J=7.7 Hz, CH-N), 4.46 (1H, dd, J=6.9,
3.3 Hz, CH-Ar), 3.84 (2H, s, CH2-Ar), 2.97 (6H, s, CH3 ꢁ2), 2.53 (1H,
ddd, J=13.0, 7.7, 3.3 Hz, CHsyn), 2.28 (1H, dt, J=13.0, 6.9 Hz, CHanti),
2.15 (1H, brs, NH); 13C NMR (75 MHz, CDCl3): d=149.8 (C), 145.6
(C), 145.5 (C), 145.3 (C), 132.4 (C), 130.4 (CH), 130.1 (C), 129.9 (CH),
129.1 (CH ꢁ2), 128.1 (CH), 128.0 (C), 127.4 (CH), 127.2 (CH), 125.1
(CH), 124.6 (CH), 112.7 (CH ꢁ2), 61.3 (CH), 51.3 (CH2), 48.5 (CH), 43.7
(CH2), 40.9 (CH3 ꢁ2); LRMS (CI+) m/z: 411.1 [M+H]+; HRMS (CI+)
[M+H]+ m/z expected for C24H25N2Cl2 411.1395, obtained 411.1385.
General procedure for reductive amination of 10: The corre-
sponding aldehyde (0.15 mmol) was added to a solution of 10
(0.18 mmol) in anhydrous THF (1 mL), and the solution was stirred
for 1 h. Sodium triacetoxyborohydride (0.36 mmol) was added, and
the mixture was stirred overnight. A 2m solution of aqueous KOH
(1 mL) was added, and the reaction was extracted with CH2Cl2 (3ꢁ
1 mL). The combined organics were dried (Na2SO4) and filtered.
PS–benzaldehyde resin (0.1 mmol) was added, and the mixture
was agitated for 2 h. The resin was removed by filtration, and the
solvent was concentrated in vacuo to afford the desired amines.
Trans-[3-(3,4-dichlorophenyl)indan-1-yl]-(4-chlorobenzyl)amine
(8viii): Yield 54%, colourless oil; 1H NMR (300 MHz, CDCl3): d=
7.35–7.14 (9H, m, CHar), 6.93–6.86 (2H, m, CHar), 4.46 (1H, t, J=
7.5 Hz, CH-N), 4.32 (1H, dd, J=6.8, 3.5 Hz, CH-Ar), 3.79 (2H, s,
CH2Ar), 2.44–2.36 (1H, m, CHsyn), 2.23–2.14 (1H, m, CHanti); 13C NMR
(100 MHz, CDCl3): d=145.5 (C), 145.5 (C), 145.1 (C), 138.8 (C), 132.7
(C), 132.5 (C), 130.5 (CH), 130.3 (C), 129.9 (CH), 129.4 (CH ꢁ2), 128.5
(CH ꢁ2), 128.4 (CH), 127.4 (CH), 127.3 (CH), 125.3 (CH), 124.6 (CH),
61.6 (CH), 51.0 (CH2), 48.5 (CH), 43.8 (CH2); LRMS (CI+) m/z: 402.1
[M+H]+; HRMS (CI+) [M+H]+ m/z expected for C22H19NCl3
402.0583, obtained 402.0573.
Trans-[3-(3,4-dichlorophenyl)indan-1-yl]-(4-aminobenzyl)amine
(8xiii): Yield 76%, colourless oil; 1H NMR (400 MHz, CDCl3): d=
7.41–7.39 (1H, m, CHar), 7.34 (1H, d, J=8.3 Hz, CHar), 7.26–7.21 (3H,
m, CHar), 7.15 (2H, d, J=8.4 Hz, CHar), 6.98–6.95 (2H, m, CHar), 6.66
(2H, d, J=8.4 Hz, CHar), 4.54 (1H, t, J=7.8 Hz, CH-N), 4.41 (1H, dd,
J=6.9, 3.3 Hz, CH-Ar), 3.78 (2H, d, J=3.0 Hz, CH2-Ar), 2.49 (1H,
ddd, J=13.2, 7.8, 3.3 Hz, CHsyn), 2.24 (1H, dt, J=13.2, 6.9 Hz, CHanti),
1.75 (3H, brs, NH ꢁ3); 13C NMR (100 MHz, CDCl3): d=149.6 (C),
145.6 (C), 145.5 (C), 145.3 (C), 132.4 (C), 130.4 (CH), 130.1 (C), 129.9
(CH), 129.3 (CH ꢁ2), 128.2 (CH), 128.0 (C), 127.4 (CH), 127.2 (CH),
125.2 (CH), 124.6 (CH), 115.2 (CH ꢁ2), 61.3 (CH), 51.3 (CH2), 48.6
(CH), 43.7 (CH2); LRMS (CI+) m/z: 383.1 [M+H]+; HRMS (CI+)
[M+H]+ m/z expected for C22H21N2Cl2 383.1082, obtained 383.1084.
Trans-[3-(3,4-dichlorophenyl)indan-1-yl]-(3,4-dichlorobenzyl)a-
mine (8ix): Yield 73%, colourless oil; 1H NMR (300 MHz, CDCl3):
d=7.42 (1H, d, J=2.0 Hz, CHar), 7.34 (1H, dd, J=6.4, 2.6 Hz, CHar),
7.31 (1H, d, J=8.2 Hz, CHar), 7.28 (1H, d, J=8.2 Hz, CHar), 7.22–7.18
(2H, m, CHar), 7.16–7.12 (2H, m, CHar), 6.94–6.91 (1H, m, CHar), 6.88
(1H, dd, J=8.2, 2.0 Hz, CHar), 4.46 (1H, t, J=7.5 Hz, CH-N), 4.32
(1H, dd, J=6.7, 3.6 Hz, CH-Ar), 3.78 (2H, s, CH2-Ar), 2.43–2.35 (1H,
m, CHsyn), 2.24–2.15 (1H, m, CHanti); 13C NMR (100 MHz, CDCl3): d=
145.4 (C), 145.4 (C), 145.0 (C), 140.8 (C), 132.5 (C), 132.4 (C), 130.8
(C), 130.5 (CH), 130.3 (CH), 129.9 (CH), 129.8 (CH), 129.5 (C), 128.5
(CH), 127.4 (CH), 127.4 (CH), 127.3 (CH), 125.3 (CH), 124.6 (CH), 61.6
(CH), 50.5 (CH2), 48.5 (CH), 43.8 (CH2); LRMS (CI+) m/z: 436.0
[M+H]+; HRMS (CI+) [M+H]+ m/z expected for C22H18NCl4
436.0193, obtained 436.0194.
Trans-N-[3-(3,4-dichlorophenyl)indan-1-yl]acetamide
(8xiv):
Acetyl chloride (15.6 mL, 0.22 mmol) and DIPEA (115 mL, 0.66 mmol)
were added to a solution of 10a (50 mg, 0.18 mmol) in anhydrous
THF (1 mL), and the solution was stirred overnight. A saturated
aqueous solution of NaHCO3 (1 mL) was added, and the reaction
was extracted with CH2Cl2 (3ꢁ1 mL). The combined organics were
dried (Na2SO4) and concentrated in vacuo. Purification through a
plug of silica (Hex/EtOAc 5:1) afforded 8xiv as a white solid (84%);
mp: 127–1288C; 1H NMR (300 MHz, CDCl3): d=7.42–7.39 (1H, m,
CHar), 7.35 (1H, d, J=8.2 Hz, CHar), 7.32–7.28 (2H, m, CHar), 7.19
(1H, d, J=2.1 Hz, CHar), 7.05–7.02 (1H, m, CHar), 6.92 (1H, dd, J=
8.3, 2.1 Hz, CHar), 5.67–5.56 (2H, m, NH, CH-N), 4.46 (1H, t, J=
7.2 Hz, CH-Ar), 2.52–2.42 (2H, m, CH2), 2.03 (3H, s, CH3); LRMS (CI+)
m/z: 320.1 [M+H]+; HRMS (CI+) [M+H]+ m/z expected for
C17H16Cl2NO 320.0609, obtained 320.0616.
Trans-[3-(3,4-dichlorophenyl)indan-1-yl]-(4-methylbenzyl)amine
1
(8x): Yield 77%, colourless oil; H NMR (300 MHz, CDCl3): d=7.35–
7.34 (1H, m, CHar), 7.27 (1H, d, J=8.2 Hz, CHar), 7.19–7.14 (5H, m,
CHar), 7.08–7.06 (2H, m, CHar), 6.92–6.86 (2H, m, CHar), 4.46 (1H, t,
J=7.5 Hz, CH-N), 4.33 (1H, dd, J=6.8, 3.5 Hz, CH-Ar), 3.78 (2H, s,
326
www.chemmedchem.org
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2011, 6, 321 – 328

Indatraline-Based TryR Inhibitors
Trans-N-[3-(3,4-dichlorophenyl)indan-1-yl]-4-methylbenzenesul-
fonamide (8xv): p-Toluenesulfonyl chloride (42 mg, 0.22 mmol)
and DIPEA (115 mL, 0.66 mmol) were added to a solution of 10a
(50 mg, 0.18 mmol) in anhydrous THF (1 mL), and the solution was
stirred overnight. A saturated aqueous solution of NaHCO3 was
added, and the reaction was extracted with CH2Cl2 (3ꢁ1 mL). The
combined organics were dried (Na2SO4) and concentrated in vacuo.
Purification through a plug of silica (Hex/EtOAc 5:1) afforded 8xv
1.39 (2H, m, CH2), 0.91 (3H, d, J=6.6 Hz, CH3), 0.90 (3H, d, J=
6.6 Hz, CH3); LRMS (CI+) m/z: 324.2 [M+H]+; HRMS (CI+) [M+H]+
m/z expected for C22H30NO 324.2327, obtained 324.2332.
Trans-[3-(4-methoxyphenyl)-6-methoxyindan-1-yl]-(3-methylbu-
tyl)amine (8xx): Yield (76%), colourless oil; 1H NMR (300 MHz,
CDCl3): d=7.22–7.16 (1H, m, CHar), 6.94–6.91 (2H, m, CHar), 6.78–
6.61 (4H, m, CHar), 4.45 (1H, t, J=7.4 Hz, CH-N), 4.33 (1H, dd, J=
6.7, 3.7 Hz, CH-Ar), 3.82 (3H, s, CH3), 3.75 (3H, s, CH3), 2.74–2.70
(2H, m, CH2), 2.46–2.39 (1H, m, CHsyn), 2.31–2.26 (1H, m, CHanti),
1.64 (1H, sept, J=6.6 Hz, CH), 1.44–1.39 (2H, m, CH2), 0.91 (3H, d,
J=6.6 Hz, CH3), 0.90 (3H, d, J=6.6 Hz, CH3); LRMS (CI+) m/z: 340.2
[M+H]+; HRMS (CI+) [M+H]+ m/z expected for C22H30NO2
340.2277, obtained 340.2276.
1
as a white solid (77%); mp: 149–1508C; H NMR (300 MHz, CDCl3):
d=7.82 (2H, d, J=8.3 Hz, CHar), 7.36–7.31 (3H, m, CHar), 7.26–7.23
(2H, m, CHar), 7.13–7.10 (2H, m, CHar), 7.00–6.97 (1H, m, CHar), 6.85
(1H, d, J=8.3, 2.1 Hz, CHar), 4.97–4.91 (1H, m, CH-N), 4.65 (1H, d,
J=8.0 Hz, NH), 4.44–4.39 (1H, m, CH-Ar), 2.46 (3H, s, CH3), 2.44–
2.38 (1H, m, CHsyn), 2.27–2.18 (1H, m, CHanti); 13C NMR (75 MHz,
CDCl3): d=144.9 (C), 144.4 (C), 143.7 (C), 141.9 (C), 137.8 (C), 134.2
(C), 132.6 (C), 130.6 (CH), 129.9 (CH ꢁ2), 129.6 (CH), 129.5 (CH),
129.3 (CH), 128.1 (CH), 127.1 (CH ꢁ2), 125.3 (CH), 124.7 (CH), 57.7
(CH), 47.9 (CH), 44.1 (CH2), 21.6 (CH3); LRMS (CI+) m/z: 432.1
[M+H]+; HRMS (CI+) [M+H]+ m/z expected for C22H20NO2Cl2S
432.0592, obtained 432.0590.
Trans-benzyl-[3-(3,4-dichlorophenyl)-6-methoxyindan-1-yl]amine
(8xxi): Yield (59%), colourless oil; 1H NMR (300 MHz, CDCl3): d=
7.32–7.12 (7H, m, CHar), 6.89–6.81 (3H, m, CHar), 6.72 (1H, dd, J=
8.3, 2.5 Hz, CHar), 4.39 (1H, t, J=7.3 Hz, CH-N), 4.31 (1H, dd, J=6.8,
4.0 Hz, CH-Ar), 3.82 (2H, s, CH2-Ph), 3.75 (3H, s, CH3), 2.46–2.37 (1H,
m, CHsyn), 2.24–2.15 (1H, m, CHanti); 13C NMR (75 MHz, CDCl3): d=
159.5 (C), 144.4 (C), 144.4 (C), 139.2 (C), 137.5 (C), 132.6 (C), 130.6
(CH), 130.5 (CH ꢁ2), 130.1 (C), 129.9 (CH), 129.1 (CH ꢁ2), 127.4
(CH), 126.3 (CH), 117.9 (CH), 110.8 (CH), 59.7 (CH), 55.7 (CH3), 47.9
(CH), 47.7 (CH2), 39.1 (CH2); LRMS (CI+) m/z: 398.1 [M+H]+; HRMS
(CI+) [M+H]+ m/z expected for C23H22NOCl2 398.1078, obtained
398.1090.
Trans-[3-(3,4-dichlorophenyl)-6-methoxyindan-1-yl]-(3-methylbu-
tyl)amine (8xvi): Yield (52%), colourless oil; 1H NMR (300 MHz,
CDCl3): d=7.28 (1H, d, J=8.3 Hz, CHar), 7.13 (1H, d, J=2.2 Hz,
CHar), 6.90–6.80 (3H, m, CHar), 6.71 (1H, dd, J=8.3, 2.2 Hz, CHar),
4.36 (1H, t, J=7.3 Hz, CH-N), 4.25 (1H, dd, J=6.8, 4.0 Hz, CH-Ar),
3.75 (3H, s, CH3), 2.67–2.62 (2H, m, CH2), 2.40–2.32 (1H, m, CHsyn),
2.23–2.14 (1H, m, CHanti), 1.58 (1H, sept, J=6.6 Hz, CH), 1.38–1.31
(2H, m, CH2), 0.84 (3H, d, J=6.6 Hz, CH3), 0.83 (3H, d, J=6.6 Hz,
CH3); 13C NMR (75 MHz, CDCl3): d=159.5 (C), 144.3 (C), 139.1 (C),
137.4 (C), 132.7 (C), 130.9 (C), 130.6 (CH), 129.9 (CH), 127.3 (CH),
126.3 (CH), 117.9 (CH), 110.9 (CH), 60.9 (CH), 55.7 (CH2), 48.0 (CH),
42.8 (CH2), 39.4 (CH2), 34.4 (CH2), 26.2 (CH), 22.3 (CH3), 22.1 (CH3);
LRMS (CI+) m/z: 378.1 [M+H]+; HRMS (CI+) [M+H]+ m/z expected
for C21H26Cl2NO 378.1391, obtained 378.1399.
Inhibition of trypanothione reductase: Compounds were tested
for inhibition of T. cruzi TryR by using a high-throughput micro-
plate assay.[15] Briefly, assays were set up in 96-well plates using a
Biotek Precision 2000 automated liquid handler and initiated with
NADPH. The final assay mixtures (0.18 mL) contained TryR
(20 mUmLÀ1), 40 mm HEPES (pH 7.5), 1 mm EDTA, 0.15 mm NADPH,
50 mm DTNB, 6 mm T[S]2, and inhibitor (100 mm–5 nm in threefold
serial dilutions). The rate of TNBÀ formation was monitored over
5 min in a Spectramax 340PC plate reader (Molecular Devices) at
l=412 nm. Raw data were processed with Microsoft Excel.
GraFit 5.0 (Erithacus software) was used to fit the data to a three-
parameter equation and output the concentration resulting in
50% inhibition (IC50 value). Compounds were tested against TryR
on three separate occasions, and the IC50 values were used to cal-
culate a mean weighted to the standard error. These values are
given in Table 1. The mean Z’ value throughout the IC50 testing
was 0.88, indicating suitable assay performance.
Trans-[3-(3,4-dibromophenyl)-6-methoxyindan-1-yl]-(3-methyl-
1
butyl)amine (8xvii): Yield (71%), colourless oil; H NMR (300 MHz,
CDCl3): d=7.50 (1H, d, J=8.2 Hz, CHar), 7.38 (1H, d, J=2.0 Hz,
CHar), 6.93–6.86 (3H, m, CHar), 6.79 (1H, dd, J=8.4, 2.5 Hz, CHar),
4.42 (1H, t, J=7.5 Hz, CH-N), 4.31 (1H, dd, J=6.8, 3.6 Hz, CH-Ar),
3.82 (3H, s, CH3), 2.73–2.68 (2H, m, CH2), 2.47–2.39 (1H, m, CHsyn),
2.29–2.21 (1H, m, CHanti), 1.64 (1H, sept, J=6.6 Hz, CH), 1.45–1.37
(2H, m, CH2), 0.91 (3H, d, J=6.6 Hz, CH3), 0.90 (3H, d, J=6.6 Hz,
CH3); LRMS (CI+) m/z: 468.0 [M+H]+; HRMS (CI+) [M+H]+ m/z ex-
pected for C21H26NO79Br81Br 468.0361, obtained 468.0356.
Inhibition of glutathione reductase: Compounds were tested for
inhibition of human GR by using a modification of the TryR assay
method and processed as before. The final conditions in the assay
were 7 mm glutathione disulfide, 150 mm NADPH, 50 mm DTNB, and
16 nm human GR.
Trans-[3-(4-chlorophenyl)-6-methoxyindan-1-yl]-(3-methylbutyl)-
amine (8xviii): Yield (56%), colourless oil; 1H NMR (300 MHz,
CDCl3): d=7.24–7.22 (2H, m, CHar), 7.05–6.86 (4H, m, CHar), 6.77
(1H, dd, J=8.3, 2.6 Hz, CHar), 4.45 (1H, t, J=7.3 Hz, CH-N), 4.31
(1H, dd, J=7.1, 3.8 Hz, CH-Ar), 3.82 (3H, s, CH3), 2.72–2.69 (2H, m,
CH2), 2.47–2.38 (1H, m, CHsyn), 2.31–2.22 (1H, m, CHanti), 1.62 (1H,
sept, J=6.6 Hz, CH), 1.45–1.38 (2H, m, CH2), 0.91 (3H, d, J=6.6 Hz,
CH3), 0.90 (3H, d, J=6.6 Hz, CH3); LRMS (CI+) m/z: 344.2 [M+H]+;
HRMS (CI+) [M+H]+ m/z expected for C21H27NOCl 344.1781, ob-
tained 344.1784.
Assessment of mode of inhibition: Three of the compounds (3,
8ii, and 8iii) were tested for mode of inhibition with respect to try-
panothione. The standard TryR assay was used as before. Aliquots
of the assay mixture (180 mL) containing three different concentra-
tions of test compound were added to three rows of a microtitre
plate, a fourth row contained only the assay mixture. T[S]2 was seri-
ally diluted across a fifth row of the plate to produce a 12-point
range from 500–5.8 mm. The assay was initiated by transferring
20 mL of T[S]2 row to each of the assay rows. The final 200 mL assay
contained 150 mm NADPH, 50 mm DTNB, and 20 mUmLÀ1 TryR and
50–0.58 mm T[S]2. The final inhibitor concentration in the three
rows of the plate ranged from 0.5ꢁ to 3ꢁ IC50. The rate of reaction
was measured as before. Each data set was fitted by nonlinear re-
gression to the Michaelis–Menten equation using GraFit 5.0 (Eritha-
Trans-[3-(4-methylphenyl)-6-methoxyindan-1-yl]-(3-methylbutyl)-
amine (8xix): Yield (73%), colourless oil; 1H NMR (300 MHz,
CDCl3): d=7.05 (2H, d, J=8.0 Hz, CHar), 7.01 (2H, d, J=8.0 Hz,
CHar), 6.97–6.89 (2H, m, CHar), 6.76 (1H, dd, J=8.2, 2.4 Hz, CHar),
4.44 (1H, t, J=7.4 Hz, CH-N), 4.32 (1H, dd, J=6.7, 3.7 Hz, CH-Ar),
3.82 (3H, s, CH3), 2.74–2.70 (2H, m, CH2), 2.45–2.39 (1H, m, CHsyn),
2.31–2.24 (4H, m, CH3, CHanti), 1.64 (1H, sept, J=6.6 Hz, CH), 1.44–
ChemMedChem 2011, 6, 321 – 328
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
327

A. H. Fairlamb, N. J. Westwood, et al.
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from 50 to 0.2 mm in a total volume of 200 mL. Pentamidine (250–
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Plates were incubated at 378C in a 5% CO2 humidified atmosphere
for 72 h, then 45 mm resazurin (Sigma) was added. After incubation
for 4–5 h, fluorescence due to formation of resorufin was measured
at lex =528 nm, lem =590 nm. Compounds were tested against
T. brucei cells on three separate occasions, and the EC50 values
were used to calculate a mean weighted to the standard error.
These values are given in Table 1. The mean Z’ value throughout
the T. brucei testing was 0.67, indicating suitable assay perfor-
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Acknowledgements
A.H.F. is a Wellcome Principal Research Fellow, and N.J.W. a Royal
Society University Research Fellow. This work was funded by
grants from the Wellcome Trust (WT079838 and WT083481) and
the Scottish Funding Council.
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Keywords: antiprotozoal agents · drug discovery · indatraline ·
Nazarov reaction · trypanothione reductase
Received: October 12, 2010
Revised: November 11, 2010
[1] K. Stuart, R. Brun, S. Croft, A. H. Fairlamb, R. E. Gurtler, J. McKerrow, S.
Reed, R. Tarleton, J. Clin. Invest. 2008, 118, 1301–1310.
Published online on December 15, 2010
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ChemMedChem 2011, 6, 321 – 328