Catechol pyrazolinones as trypanocidals: Fragment-based design, synthesis, and pharmacological evaluation of nanomolar inhibitors of trypanosomal phosphodiesterase B1

Article abstract of DOI:10.1021/jm301059b

Trypanosomal phosphodiesterases B1 and B2 (TbrPDEB1 and TbrPDEB2) play an important role in the life cycle of Trypanosoma brucei, the causative parasite of human African trypanosomiasis (HAT), also known as African sleeping sickness. We used homology modeling and docking studies to guide fragment growing into the parasite-specific P-pocket in the enzyme binding site. The resulting catechol pyrazolinones act as potent TbrPDEB1 inhibitors with IC₅₀ values down to 49 nM. The compounds also block parasite proliferation (e.g., VUF13525 (20b): T. brucei rhodesiense IC₅₀ = 60 nM, T. brucei brucei IC₅₀ = 520 nM, T. cruzi = 7.6 μM), inducing a typical multiple nuclei and kinetoplast phenotype without being generally cytotoxic. The mode of action of 20b was investigated with recombinantly engineered trypanosomes expressing a cAMP-sensitive FRET sensor, confirming a dose-response related increase of intracellular cAMP levels in trypanosomes. Our findings further validate the TbrPDEB family as antitrypanosomal target.

Full text of DOI:10.1021/jm301059b

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Article
Catechol Pyrazolinones as Trypanocidals: Fragment-
Based Design, Synthesis and Pharmacological Evaluation of
Nanomolar Inhibitors of Trypanosomal Phosphodiesterase B1
Kristina Maria Orrling, Chimed Jansen, Xuan Lan Vu, Vreni Balmer, Patrick Bregy,
Anitha Shanmugham, Paul England, Paul Cos, Louis Maes, Emily Adams, Erika van
de Bogaart, Eric Chatelain, Jean-Robert Ioset, Andrea Stolpe, Stèphanie Zorg, Johan
Veerman, Thomas Seebeck, Geert Jan Sterk, Iwan J. P. de Esch, and Rob Leurs
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm301059b • Publication Date (Web): 10 Sep 2012
Downloaded from http://pubs.acs.org on October 7, 2012
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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
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Catechol Pyrazolinones as Trypanocidals: Fragment-
Based Design, Synthesis and Pharmacological
Evaluation of Nanomolar Inhibitors of
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Trypanosomal Phosphodiesterase B1
Kristina M Orrling,1 Chimed Jansen,1 Xuan Lan Vu,2 Vreni Balmer,3 Patrick Bregy,2† Anitha
Shanmugham,4 Paul England,4 Paul Cos,5 Louis Maes,5 Emily Adams,6 Erika van den Bogaart,6
Eric Chatelain,7 Jean-Robert Ioset,7 Andrea van de Stolpe,1 Stèphanie Zorg,1 Johan Veerman,8
Thomas Seebeck,2 Geert Jan Sterk,8 Iwan de Esch,1 Rob Leurs*,1
1Leiden/Amsterdam Centre of Drug Research (LACDR), Amsterdam Institute of Molecules,
Medicines & Systems (AIMMS), Division of Medicinal Chemistry, VU University Amsterdam,
De Boelelaan 1083,
1081 HV Amsterdam, The Netherlands,
2Institute of Cell Biology, University of Bern, Baltzerstrasse 4, 3012 Bern, Switzerland,
3Institute of Parasitology of the University of Bern, University of Bern, Baltzerstrasse 4, 3012
Bern, Switzerland,
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4IOTA Pharmaceuticals, St John’s Innovation Centre, Cowley Road, Cambridge CB4 0WS,
United Kingdom
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5Laboratory for Microbiology, Parasitology and Hygiene (LMPH), University of Antwerp,
Groenenborgerlaan 171, 2020 Wilrijk, Belgium
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6Department of Biomedical Research, Royal Tropical Institute Amsterdam, Meibergdreef 39,
1105 AZ Amsterdam, The Netherlands
7DNDi (Drugs for Neglected Diseases initiative), 15 Chemin Louis Dunant, 1202 Geneva,
Switzerland
8Mercachem, PO-Box 6747, 6503 GE Nijmegen, The Netherlands
*Corresponding Author E-mail: R.Leurs@vu.nl
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ABSTRACT
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Trypanosomal phosphodiesterases B1 and B2 (TbrPDEB1 and TbrPDEB2) play an important
role in the life cycle of Trypanosoma brucei, the causative parasite of human African
trypanosomiasis (HAT), also known as African sleeping sickness. We used homology modelling
and docking studies to guide fragment growing into the parasite-specific P-pocket in the enzyme
binding site. The resulting catechol pyrazolinones act as potent TbrPDEB1 inhibitors with IC50
values down to 49 nM. The compounds also block parasite proliferation (e.g. VUF13525 (20b):
T. brucei rhodesiense IC50 = 60 nM, T. brucei brucei IC50 = 520 nM, T. cruzi = 7.6 µM),
inducing a typical multiple nuclei- and kinetoplast phenotype without being generally cytotoxic.
The mode of action of 20b was investigated with recombinantly engineered trypanosomes
expressing a cAMP-sensitive FRET sensor, confirming a dose-response related increase of
intracellular cAMP levels in trypanosomes. Our findings further validate the TbrPDEB family as
antitrypanosomal target.
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INTRODUCTION
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Human African trypanosomiasis (HAT), commonly known as African sleeping sickness, is an
extremely deadly disease; without treatment, all patients entering second stage HAT following
the spread of the parasite to the CNS will eventually die.1 Millions of people are living in the
endemic areas in rural central Africa, where poverty and poor access to accurate healthcare
hamper the chances of survival of infected patients.2, 3 Even if the number of reported cases has
dropped to its lowest level in 50 years, epidemics, increased political turbulence, and resistance
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emergence can quickly alter this trend.3, HAT is caused by the kinetoplastid protozoa
Trypanosoma brucei and is transmitted by infected tsetse flies (Glossina ssp.). Two subspecies
of T. brucei are infectious to humans, T. b. gambiense and T. b. rhodesiense. The course of the
disease, symptoms and appropriate treatment depend on the etiological subspecies.2 Despite the
high medical need, current treatment options are limited and suffer from suboptimal dosage
regimens and/or severe toxicity.2 During the second stage of the disease, the only choices are the
arsenic-containing drug melarsoprol and the ornithine decarboxylase inhibitor eflornithine, used
as monotherapy or in combination with nifurtimox (NECT).5 However, eflornithine is not
effective against T. b. rhodesiense. Consequently, public-private initiatives such as the Drugs for
Neglected Disease initiative investigate new approaches to target HAT.6
The mapping of the T. brucei genome revealed the presence of five trypanosomal cyclic
nucleotide phosphodiesterases (PDEs).7 These enzymes play an important role in the regulation
of cyclic nucleotide levels by catalysing the hydrolysis of the phosphodiester bond of cAMP
and/or cGMP. In humans, 11 main families of PDEs can be distinguished, and several of the
human PDE enzymes have been extensively explored as molecular targets for a diverse set of
diseases conditions, e.g. COPD, erectile dysfunction, pulmonary arterial hypertension, asthma,
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psoriasis, type 2 diabetes, depression, cognitive disorders, and psychosis. Although the exact role
of cAMP in the lifecycle of the trypanosomes is not fully understood,8 two trypanosomal PDEs
have been found to play a pivotal role in T. brucei proliferation.9, 10 Knocking down of both
TbrPDEB1 and TbrPDEB2 simultaneously leads to an arrest of parasite cell division, lysis of the
parasites, and to elimination of the infection in vivo in the infected mouse model.11 Additional
studies on a hit from a high throughput screening campaign, PPS54019 (1), pharmacologically
validated TbrPDEB1 and TbrPDEB2 as anti-trypanosomal targets.12 In fact, 1 was once
synthesised as potent human PDE4 inhibitor.13 The benefits of extracting acquired knowledge
from very advanced drug discovery programs targeting human proteins for use in neglected
diseases drug discovery cannot be overestimated. Scarcity in the drug research pipeline can thus
be compensated with better pharmacological predictability and an early understanding of
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possible adverse effects. A recent investigation shows that the hPDE4 inhibitor piclamilast (2)
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and analogues also inhibit the TbrPDEB1 and TbrPDEB2 (Figure 1).12,
Inhibition of
TbrPDEB1 has furthermore been observed with analogues of tadalafil (3)15 and sildenafil (4),16
both hPDE5 inhibitors, however at high micromolar concentrations.
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Figure 1. Known TbrPDE inhibitors, 1,12 2,14 3,15 and 416.
These findings indicate that in the area of neglected infectious diseases, basing drug discovery
efforts on lead compounds originally developed for human PDE targets can be quite a successful
strategy. Such an approach proved not only successful in the discovery of the first TbrPDEB1
inhibitors 1–4, but also the recent discovery of antiplasmodial compounds based on the PDE5
inhibitor tadalafil.17
Following up on chemotypes related to HTS hit 1, catechol pyrazolinone 6a (TbrPDEB1
inhibition with IC50 = 12 µM) was discovered. This fragment contains several structural features
of known human PDE inhibitors and can be considered a merger of the known PDE4 inhibitor
rolipram (i.e., catechol moiety connected to a cyclopentyl group, also present in 2) and of
published cardiotonic hPDE3 inhibitors 518, 19 and hPDE4 inhibitors in the patent literature20 (i.e.,
the pyrazolinone moiety) as outlined in Scheme 1. Following these fragment-based drug design
considerations, we investigated the putative binding mode of these compounds by constructing a
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homology model of TbrPDEB1 based on the x-ray structure (PDB id 2R8Q)21 of the Leishmania
major orthologue LmjPDEB1 (66% sequence identity). Interestingly, the so-called P-pocket, a
unique sub-pocket that extends from the invariant glutamine (Gln874) through the protein to the
solvent in the ligand-binding site of the LmjPDEB1 proved to be conserved in our model of
TbrPDEB1.
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Docking studies of rolipram suggested good accommodation in the homology model of
TbrPDEB1. The docked binding poses of rolipram identified the catechol motif as the main
potential contributor of affinity to the protein, being well positioned in the hydrophobic clamp
between Phe877 and Val839 and hydrogen bonding with Gln874, while the lactam ring was less
well positioned, making no specific interactions (Figure 2a). However, biochemical
measurements indicate rolipram to be inactive at >100 µM on the TbrPDEB1 enzyme.
A pyrazolinone is a heterocycle related to the lactam ring of rolipram, but encompasses a
conjugated π-system and additional possibility to interact with aromatic residues in the binding
pocket (Figure 2b).
Figure 2. Proposed binding modes of (a) rolipram and (b) pyrazolinone 6a in the TbrPDEB1
homology model. Both compounds interact with the highly conserved Gln874 and Phe877 and
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the cyclopentyl moiety is placed at the entrance of the P-pocket. (b) The pyrazolinone ring is
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involved in a π– π interaction with Phe843.
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In this binding pose, the cyclopentyl ring of 6a is placed at the entrance of the P-pocket,
making this an interesting region to optimize, leading to compounds with the general structure 7
(Scheme 1). A second vector to be decorated was placed at the pyrazolinone nitrogen (R2), in
line with for example phthalazinone-based hPDE4 inhibitors.22
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Scheme 1. The design idea behind the original TbrPDEB1 inhibitor 6a, where rolipram
contributed the catechol moiety and 518, 19 the pyrazolinone scaffold. The general structure of the
herein reported TbrPDEB1 inhibitors is represented by 7.
RESULTS AND DISCUSSION
Homology model of TbrPDEB1. The homology model of TbrPDEB1 was constructed based
on the TbrPDEB1 sequence published by Zoraghi and Seebeck (Uniprot: Q8WQX9)9 and the
structure of LmjPDB1 (2R8Q),21 the only orthologue of TbrPDEB1 crystallized to date. The B
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chain of the LmjPDEB1 structure was used to construct the homology model, since the B-factors
in the region of the catalytic site is lower in the B chain. The residues on the surface of the
catalytic site show a very high sequence homology with only one V836I substitution. The P-
pocket region has two substitutions N881M and R885K, both on the far side of the P-pocket
from the invariant glutamine (Gln874). The overall sequence identity of the catalytic domains is
66%.
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Synthetic Chemistry. The pyrazolinones 6,8–10a were formed in good yields (67 – 99%)
from a heterocyclisation reaction with aqueous hydrazine and the corresponding β-ketoesters
11a–d.18, 19 In contrast to previously reported synthetic routes, the β-ketoesters 11a–d were
synthesised from the corresponding benzoic acids via the benzoyl chloride and the enolate of
methyl isobutyrate (Scheme 2). The ring-nitrogen was alkylated using NaH and
isopropylbromide or cycloheptylbromide and heated either by microwave (MW) irradiation or
refluxing overnight, depending on the reaction scale, to give 8c,d and 9–10,b,c. The methyl
group in pyrazolinone 8b was introduced in a similar fashion, but using K2CO3 as base. In an
attempt to reduce the number of reaction steps cycloheptylhydrazine hydrogen chloride salt was
used in the pyrazolinone-formation reaction, but this resulted in a complex reaction mixture and
poor yield (17%) of required product 9c. Reductive debenzylation of 8c,d afforded the phenols
12a,b required for further derivatization.
Scheme 2. Synthetic route to pyrazolinonesa.
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a
Reagents: (a) (COCl)2, DMF, CH2Cl2, 1-18 h or SOCl2, rt, 1-2 h; (b) methyl isobutanoate,
LDA, THF, –45 °C, 30 min, then added to acyl chloride, –55 °C to rt, 1–18 h; (c) N2H4, EtOH,
reflux, 18 h; (d) 8b: K2CO3, DMF, MW (145 °C, 20 min), then MeI, MW (145 °C, 20 min);
8c,d, 9–10b,c: R2Br, NaH, DMF/MeCN, MW (120 °C, 40 min); (e) Pd/C, NH4CHO2,
MeOH:CH2Cl2 (2:1), 40 °C, 18 h.
A diverse set of benzyl derivatives was introduced on the phenols 12a,b with either of two
MW-assisted nucleophilic substitution protocols to afford inhibitors 13–20a,b (Scheme 3, Table
2) using substituted bromides or chlorides, where chlorides normally needed longer reaction
times for full conversion. For the transformation of nitriles 19a,b into the corresponding
tetrazoles 20a,b a novel expedient MW-assisted protocol was developed (Scheme 3). Compared
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to conventional refluxing overnight, the yield was improved from 19% to 62–75% and reaction
time was reduced to one hour.
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Scheme 3. Synthetic route to catechol pyrazolinones.a
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aReagents: a) Method A: R1Br, K2CO3, DMF/MeCN, MW (120 °C, 30 min), then R1Br or
R1Cl, MW (120 °C, 20–60 min); method B: NaH, DMF, R1Br, MW (120 °C, 15–30 min; b)
NaN3, NH4Cl, DMF, MW (160 °C, 1 h).
Pharmacological measurements. All compounds were tested in at least two independent
3
experiments for inhibition of TbrPDEB1-mediated H-cAMP hydrolysis, using scintillation
proximity assay and recombinant TbrPDEB1 expressed in Sf21 insect cells, as described
previously.12 The compounds were further tested for inhibition of T. brucei bloodstream form
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trypomastigote proliferation and cytotoxicity on MRC5 fibroblasts using established
techniques.23, 24
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To insure a fast testing–design iteration, assessment of TbrPDEB2 inhibition was omitted at
the initial screening stage, as suppression of parasite proliferation would imply concomitant
inhibition of TbrPDEB1 and TbrPDEB2. In addition, two previous studies on TbrPDE inhibitors
suggest high degree of equipotency against the paralogues,12, 14 as expected from the high
sequence homology. Similarly, the antiparasitic effect of the compounds was constrained to the
non-infective disease model subspecies T. brucei brucei. The most advanced inhibitor was tested
against an extended panel of PDEs, including TbrPDEB2, and trypanosome species. The
inhibition of the isolated catalytic domain of TbrPDEB1 was determined using the PDELight™
HTS cAMP phosphodiesterase kit.25, 26
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Table 1. Inhibitory potency against TbrPDEB1, T. b. brucei proliferation and cytotoxicity of
pyrazolinones.
%
IC50 (µM) CC50 (µM) LEc
TbrPDE T. b.
Yielda
compd
Rolipram
6a
R1
R2
H
B1
brucei
n.t.b
>64
>64
8.6
MRC5
n.t.b
>64
>64
55
>100
12
–
Ǝ
Ǝ
OcPen
OcPen
OBn
15
89
97
98
0.30
0.30
0.30
0.27
cHep
H
0.41
6.3
10
6b
8a
OBn
Me
32
>64
8b
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OBn
OBn
OH
OH
-Cl
iPr
73
1.0
4.5
52
0.30
0.28
–
8c
cHep
iPr
75
0.50
>10
>10
>10
>10
2.0
6.3
>64
>64
>64
>64
>64
32
8d
96
>64
8.6
12a
12b
9a
cHep
H
quant
67
–
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>64
>64
10.0
>64
>64
34
–
-Cl
iPr
71
–
9b
-Cl
cHep
H
17
0.32
–
9c
-Br
-Br
-Br
87
>10
>10
2.5
>64
>64
30
10a
10b
iPr
73
–
cHep
88
0.32
10c
a
b
c
Isolated yield of the last step. not tested. ligand efficiency = (ꢀG)/N, where ꢀG = –
RTln(IC50(TbrPDEB1)) and N is the number of non-hydrogen atoms.27
Fragment growing.
Compared to hit structure 6a, the benzyl analogue 8a was slightly more potent (IC50 = 6.3 µM,
Table 1), possibly attributed to a π – π interaction between the phenyl ring of 8a and Phe843 in
the binding pocket. By introducing aliphatic substituents (R2) on the nitrogen atom in the
pyrazolinone ring the IC50 values could be further improved in a size-dependent manner (R2 =
cHep < iPr < Me ≈ H) and submicromolar activities were observed for the cycloheptyl
derivatives (e.g. 8d IC50 = 0.50 µM). According to our docking studies, this gain appears to
result from the hydrophobic interaction with Phe843 and slightly shifted positioning of the
pyrazolinone ring strengthening the π – π interactions with Phe843 (Figure 3a). Removal of the
catechol ether (12a,b) or replacing it with a chloride or bromide (9,10a–c) was not tolerated,
although measurable potencies were observed for the two cycloheptyl derivatives 9,10c. An
analysis of the ligand efficiencies (LEs)27 of this series indicates that with the 24-fold
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improvement in IC50 values the LE values remain constant. Whereas the cyclopentyl derivatives
6a,b were moderately active in the TbrPDEB1 enzyme assay, no anti-proliferative effect was
observed in the parasite assay (Table 1). In contrast, the benzyloxy derivatives 8a,c,d, all
inhibited parasite proliferation with IC50 values < 10 µM, without being cytotoxic in MRC5 cells
in this concentration range. The difference in antiproliferative efficacy might be attributed to
differential parasite cell membrane permeability.
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Figure 3. Proposed binding modes of a) 8d and b) 20b in the TbrPDEB1 homology model. The
cycloheptyl rings are folded over the benzene ring of Phe843. The entrance of the P-pocket is
occupied by a) the benzyl moiety of 8d and b) the aliphatic tether of 20b of the tetrazole, which
interacts with Tyr844 further into the P-pocket.
The observed antitrypanosomal efficacy in combination with expedient synthetic access to
analogues led us to proceed with the benzyl-catechol chemotype. In the docking poses, the
benzene ring of these compounds is accommodated at the entrance of the P-pocket suggesting
that it should be possible to grow the inhibitors further into this pocket from the 4-position,
which is directed towards the P-pocket channel (Figure 3a). It was for example hypothesized that
Thr854 in the P-pocket could be targeted by H-bond acceptors. We therefore synthesized the
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nitriles 13a,b and anisoles 14a,b providing opposite electronic influences on the aromatic ring.
In addition, methoxy derivatives with a two-carbon spacer (15a,b), as well as napthyl-substituted
analogues 16a,b were synthesized to monitor the size of the P-pocket. As the pyrazolinones with
R2 = isopropyl provided better LEs (0.30) and ligand–lipophilicity efficiencies (LLEs, Table 2,
Supporting Information),28 while good potencies were observed for R2 = cycloheptyl in the first
series, both analogues were synthesized and evaluated. As can be seen in Table 2, all analogues
were active TbrPDEB1 inhibitors with IC50 values in the range of the parent compounds 8c,d. In
fact, naphthyl 16b provided a further 2-fold increase in IC50 value.
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The phenyl of 8c,d was also replaced with ortho-pyridine (17a,b) and 5-methylisoxazol
(18a,b) rings in order to potentially pick up an additional hydrogen bond with the second
hydrogen of the Gln874 nitrogen, uniquely accessed from the P-pocket. However, this approach
did not result in an increase in activity (Table 2). Interestingly, as in the previously identified
TbrPDEB1 inhibitor 1, a butyloxy spacer could be incorporated to tether the benzonitriles
(19a,b) without major loss of activity; in fact, the cycloheptyl derivative 19b was even more
potent than 13b with an IC50 value of 0.16 µM (Table 2).
Table 2. Inhibitory potency against TbrPDEB1, T. b. brucei proliferation and cytotoxicity of
catechol pyrazolinones.
R2
iPr
cHep
IC50
CC50
(µM)
LE
IC50
CC50
(µM)
LEa
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(µM)
TbrP
(µM)
TbrP
T. b.
compd DEB1 brucei MRC5
T. b.
MRC
5
R1
compd DEB1 brucei
7
8
9
4-CN-benzyl
4-MeO-benzyl
4-MeO-phenethyl
1-naphthylmethyl
2.0
>64
32
>64
>64
48.1
>64
0.27
0.27
0.28
0.27
1.0
9.5
10
>64
>64
>64
>64
0.25
0.25
0.25
0.26
13a
14a
15a
16a
13b
14b
15b
16b
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2.0
0.79
0.63
0.25
0.83
0.79
32
8.1
9.6
9.6
2-pyridinmethyl
2.5
5.0
35
33
>64
>64
0.28
0.27
0.50
0.63
0.16
2.5
8.4
2.3
>64
33
0.28
0.27
17a
18a
17b
18b
3-(5-
methylisoxazolyl)-
methyl
4-(4-cyano-
phenyloxy)butyl
5.0
9.1
2.0
>64
>64
0.22
0.24
>64
>64
0.25
0.25
19a
20a
19b
20b
4-(4-tetrazole-
phenyloxy)butyl
0.40
0.049 0.52
a
ligand efficiency = (ꢀG)/N, where ꢀG = –RTln(IC50(TbrPDEB1)) and N is the number of
non-hydrogen atoms.27
Inspired by the structure of the very potent tetrazole-encompassing tetrahydrophthalazinone
1,12 nitriles 19a,b were converted to the corresponding tetrazoles 20a,b. This transformation
afforded 20b (VUF13525), which rewardingly proved to be an efficacious inhibitor of
TbrPDEB1 (IC50 = 49 nM) (Table 2). Docking studies of 20b indicated that the increase in
affinity could be attributed to interactions with residues in the P-pocket (Figure 3b). The
proposed conformation of the catechol group is similar to the one of rolipram in its hPDE4
cocrystals29, 30 and shows that the extended alkyl chain is able to reach and enter the P-pocket.
This potentially displaces water from the length of the P-pocket and places the tetrazole at the
solvent-exposed exit of the pore. In addition, the tetrazole may also form a hydrogen bond with
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Tyr844, further stabilizing the conformation. Both suggestions may explain the high activity of
20b and indicate that targeting the P-pocket may be an effective strategy for the design of potent
TbrPDEB1 inhibitors.
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The antitrypanosomal potency did not strictly follow the same trend as the TbrPDEB1
inhibitory activities in this series of analogues (Table 2 and Chart 1, Supporting Information).
The addition of lipophilic substituents on the benzyl group R1 often led to low IC50 values in the
enzymatic assay, but not in the parasitic assay (c.f. 13a and 16b, Table 2). Moreover, we are also
aware of the high cLogP31 values (Table 1, Supporting information) of many of the cycloheptyl
derivatives, accompanied with reduced aqueous solubility (visually observation), which may
have been troubling the whole cell parasite assays to some extent. Yet, the most potent
TbrPDEB1 inhibitor 20b, carrying a solubilising charged tetrazole group, concentration-
dependently reduced proliferation of T. b. brucei in the cellular assay at submicromolar
concentrations (IC50 = 520 nM, Table 2). Compound 20b was therefore analysed in a broader set
of assays (Table 3). As could be expected on the basis of the high homology (88% sequence
identity) between the catalytic domains of the TbrPDEB1 and B2 isoenzymes, 20b also acted as
a potent TbrPDEB2 inhibitor (IC50 = 72 nM). Similarly, the inhibition of the isolated TbrPDEB1
catalytic domain using the PDE light assay25, 26 remained invariant (IC50 = 55 nM), suggesting
that 20b is not binding to any allosteric sites such as the GAF domains. To evaluate the
selectivity across the PDE families, 20b was also tested against a panel of other PDE enzymes
(Table 3). The L. major orthologue LmjPDEB1 was inhibited with a slightly lower potency
(IC50 = 0.13 µM). On the other hand 20b was more potent to inhibit representative isoenzymes of
PDE4A–D families (IC50 at 0.0012 – 0.010 µM). The relatively low bias for hPDE4 inhibition (4
– 35-fold), compared to, for example, piclamilast 2 (>1000-fold), might be attributed to the
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proposed interactions with the parasite-specific P-pocket. Nevertheless, the data indicates that
the binding mode of the compounds in TbrPDEB1 and hPDE4 still needs careful considerations.
Evidently, the flexibility of the ligands and/or the hPDE4 protein allows accommodation in the
binding site. Considering that the clinical development of hPDE4 inhibitors has been
accompanied by emesis and adverse gastrointestinal and pro-inflammatory side effects,32, 33 the
level of hPDE4 inhibition must be considered. The selectivity ratio needed for a safe PDE-
targeting trypanocidal is difficult to predict at this stage. The narrow therapeutic window of
hPDE4 inhibitors that have advanced to clinical trials indicates that at least equipotency is
preferred and most likely future lead optimization efforts should focus on a 10–100-fold
selectivity for the parasite PDE.
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Interestingly, besides being a good inhibitor of T. b. brucei proliferation, 20b was also an
excellent inhibitor of T. b. rhodesiense (IC50 = 60 nM, Table 3), the causative agent of acute
East-African HAT, for which the only treatment is the arsenic-containing drug melarsoprol,
associated with fatal encephalopathy in 5-10% patients.34 Moreover, 20b also showed some
activity against T. cruzi, the causative agent of Chagas disease.35 Pyrazolinone 20b was well-
tolerated by the human fibroblast cell line (CC50 > 64 µM) and murine macrophages, providing a
sensitivity index of at least 250-fold for T. b. rhodesiense, i.e. well within the conventional
thresholds.36
Table 3. Pharmacological profiling of pyrazolinone 20b.
IC50
(µM)
Cellular assay
IC50 (µM)
Enzymatic assay
TbrPDEB1
0.049
0.072
T. b. brucei
0.52
0.06
T. b.
rhodesiense
TbrPDEB2
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TbrPDEB1 catalytic
domain
0.055
T. cruzi
7.6
6
7
8
9
LmjPDEB1
hPDE4A1
hPDE4B1
hPDE4C1
hPDE4D3
0.13
MRC5
>64a
0.0021
0.0046
0.010
0.0012
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a Mouse macrophage cytotoxicity was observed at 64 µM.
To confirm that the trypanocidal effect of 20b acts via PDE inhibition, the accumulation of
intracellular trypanosomal cAMP was monitored using a FRET-based cAMP sensor that was
recombinantly expressed in procyclic trypanosomes. As can be seen in Figure 4a, administration
of 20b to the parasites induced a rapid and dose-dependent increase in intracellular cAMP levels,
implying fast penetration over the cellular membrane. When wild-type blood-form T. b. brucei
were incubated with 20b for 12 hours they display the typical phenotype, associated with
duplicate or multiple nuclei and kinetoplasts (Figure 4b–d), which has previously been observed
in genetic11 and chemical12 interference of TbrPDE function. The occurrence of aberrant forms
of parasites increased with the doses of 20b administrated. After 48 hours incubation with 10 µM
20b, parasite cells had lysed confirming the cidal effect of prolonged exposure of TbrPDEB
family inhibitors.
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Figure 4. (a) Time-response curves after administration of TbrPDEB1 inhibitor 20b to
engineered T. brucei expressing cAMP-FRET sensors. (b) Occurrence of different T. b. brucei
phenotypes after 12 h incubation with 20b; 1N1K (1 nucleus + 1 kinetoplast); 1N2K (1 nucleus
+ 2 kinetoplasts); 2N1K (2 nuclei + 1 kinetoplast) and >2N2K (aberrant forms with more than 2
nuclei and/or 2 kinetoplasts). Microscope images of (c) control T. b. brucei 1N1K parasites and
(d) T. b. brucei parasites after 12 h incubation with 10 µM pyrazolinone 20b displaying (left)
duplicate nuclei and (right) multiple nuclei and kinetoplasts.
CONCLUSIONS
With this study it has been shown that P-pocket directed design of TbrPDEB1 inhibitors is a
successful strategy to find highly potent antitrypanosomal compounds. Structure-based and
ligand efficiency-led design resulted in the cathecol pyrazolinone (20b) with nanomolar
potencies against both TbrPDEB1 and TBrPDEB2 (49 nM and 72 nM, respectively). It also
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shows excellent in vitro activity against T. b. brucei and, clinically more relevant, T. b.
rhodesiense (60 nM), which is the causative agent of the acute East-African form of HAT. At
prolonged exposure, the inhibitor also has a cidal effect on the parasites. On the contrary,
mammalian cell lines seem to tolerate the catechol pyrazolinone chemotype very well; fibroblast
toxicity was only observed for a handful of compounds.
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For the first time, the increase in cAMP levels in the parasites upon administration of PDE
inhibitor could be followed in real-time using a FRET-based cAMP sensor expressed in
engineered trypanosomes. Being aware of the many challenges that remain, such as selectivity
over hPDE4, blood-brain barrier penetration, acceptable pharmacokinetic profile and metabolic
stability, we still believe that the promising pharmacological profile of 20b brings new hope to
the prospect of developing a less toxic alternative to melarsoprol.
EXPERIMENTAL SECTION
Homology model of TbrPDEB1.
The homology model was constructed using the TbrPDEB1 sequence published by Zoraghi
and Seebeck (Uniprot: Q8WQX9).9 The closest homologue of TbrPDEB1 crystallized to date is
Leishmania major PDEB1, the structure is published as 2R8Q on the RCSB Protein Data Bank.
Aligning the sequences using ClustalW 2.137 gave a sequence identity of 66%. The residues on
the surface of the catalytic site show an even higher homology with only one substitution
(V836I). The P-pocket region has two substitutions, N881M and R885K, both on the far side of
the P-pocket from the invariant glutamine (G874).
The homology model was built using the Molecular Operating Environment (MOE) 2010.10
software package.38 The B chain of the LmjPDEB1 structure was used to construct the homology
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model, since the B chain showed lower B factors in the region of the catalytic site. The
LmjPDEB1 structure was opened in MOE and all solvent and metal atoms were removed. The
TbrPDEB1 sequence was added and the sequence alignment from ClustalW 2.1 applied. Those
residues which were identical in both sequences had their atoms tethered at 200 in the 2R8Q.B
model. The Amber99 forcefield and born solvation model were used during the homology model
construction. There were 10 main-chain and 1 side-chain models formed, the intermediate
models were refined to an RMS of 0.1 and the final model was refined once more to an RMS of
0.5. Models were protonated using the Protonate 3D function prior to refinement and scored
using the GB/VI scoring function. The final model had the metal ions and the six metal-
coordinating water molecules included directly from the LmjPDEB1 model, with the hydrogen
atoms minimized following inclusion.
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The full-length amino acid sequences of the TbrPDEB1 and TbrPDEB2 (Uniprot: Q38F42)
catalytic domains were aligned using ClustalW 2.1 giving a sequence identity of 75%.
Restricting the sequence alignment to the 334 residues of the catalytic domain increased the
sequence identity to 88%.
Inhibitor docking.
The structures of the ligands were prepared in MOE and exported as an SDF database. A
single low energy conformation was generated for each structure using Omega 2.4.3, OpenEye
Scientific Software.39 The mol2 file generated by Omega was used as input for GOLD.40 The
compounds were docked into the homology model of TbrPDEB1 described above, which was
used as prepared. GOLD uses a genetic algorithm to efficiently explore the conformational space
of the ligand in the protein pocket. The parameters used in GOLD include, autoscaling of
operations per ligand (dependent on the number of rotatable bonds), PLP scoring function, water
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included with spin, metal ions included, and the generation of 30 solutions per ligand with an
RMS tolerance of 1.5 Å. Results were analyzed using MOE and binding poses were rendered in
PyMOL.41
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Synthetic Chemistry.
General Information.
Chemicals and reagents were purchased from commercial suppliers and were used without
further purification. Microwave reactions were performed with Biotage Initiator single mode
cavity, producing controlled irradiation at 2450 MHz in sealed reaction vials (capable of
withholding elevated pressure) and with magnetic stirring. For column chromatography
commercially available Silica Gel 60 (particle size 0.040–0.063 mm) was used. Gradient flash
column chromatography purification was performed on Biotage Isolera with prepacked silica
(KP-sil) cartridges supplied by Biotage and ethylacetate in n-heptane as eluent, unless otherwise
stated. Analytical thin layer chromatography was performed using glass sheets precoated with
Silica Gel 60 F254 and visualization of components was made by UV (254 nm), I2 and/or
Pancaldi’s solution followed by heating. Analytical HPLC–MS was performed on a Shimadzu
LC-20AD liquid chromatography pump system with a Shimadzu LCMS-2010 liquid
chromatography mass spectrometer equipped with Xbridge (C18) 5 ꢁm column (50 mm × 4.6
mm), using acetonitrile in 0.10% aqueous formic acid and a 5% – 90% gradient over 4.5 min
1
13
followed by 2 min isocratic 90% mobile phase. H NMR and C NMR spectra were measured
on a Bruker 200, 400 or 500 at 250 MHz, 400 MHz or 500 MHz, respectively. The chemical
1
shifts for H NMR and 13C NMR were referenced to TMS via the CDCl3 solvent signal (1H
NMR at 7.26 ppm and 13C NMR at 77.16 ppm). Exact molecular masses (HRMS) were
determined on Bruker micrOTOF-Q mass spectrometer equipped with an electrospray ion source
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using caffeine as reference. The purity for all compounds was established to be ≥95% by HP-LC,
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1H NMR and C NMR, unless otherwise stated. Acyl chloride precursors of β-keto esters 11a–
7
8
9
d42-45 are known compounds. The synthetic preparation and spectroscopic data of catechol
pyrazolinone 8a has previously been published in a patent.20
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Experimental details and spectroscopic data for precursors 11a,b.
Methyl 3-(3-(cyclopentyloxy)-4-methoxyphenyl)-2,2-dimethyl-3-oxopropanoate (11a).
3-(cyclopentyloxy)-4-methoxybenzoic acid (5.0 g, 21.1 mmol) was suspended in 25 mL dry
DCM and DMF (3 drops) was added. Oxalyl chloride (2.78 ml, 31.7 mmol) was added dropwise
under vigorous stirring and N2 atmosphere. The reaction mixture was stirred at room temperature
for 1.5 h. The reaction was monitored with TLC of small aliquots of the reaction mixture, which
were diluted with MeOH and heated with a heat-gun. When full conversion was observed, the
volatiles were evaporated in vacuo to give 3-(cyclopentyloxy)-4-methoxybenzoyl chloride as a
yellow crystalline solid, which was used as such in the subsequent step without any further
purification.
Lithium diisopropyl amide (13 mL, 1.8 M in THF/Hex/ether, 1.1 equiv) was added to a dry 3-
neck flask with 25 mL freshly distilled THF and cooled to -45 °C and methyl isobutyrate (3.64
ml, 31.7 mmol, 1.5 equiv) was added dropwise. The mixture was stirred for 30 min at -40 °C.
Freshly prepared 3-(cyclopentyloxy)-4-methoxybenzoyl chloride (approx. 21 mmol) was
dissolved in dry THF (25 mL) and added dropwise during 30 min at -50 – -40 °C. The reaction
mixture was stirred for another 1 h before the cooling source was removed and the stirring
continued at rt overnight. The reaction mixture was acidified with 3 M HCl (aq), diluted with
EtOAc (10 mL) and the aqueous layer was extracted with EtOAc (2 x 10 mL). The combined
organic layers were washed with sat NaHCO3 and brine, dried over MgSO4 and reduced in
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vacuo. The crude product was purified with flash chromatography to give the title compound as a
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pale syrup (1.1 g, 15%): H NMR (500 MHz, CDCl3) δ (ppm) 7.50 – 7.41 (m, 2H), 6.83 (d, J =
7
8
9
9.08 Hz, 1H), 4.82 – 4.73 (m, 1H), 3.83 (s, 3H), 3.64 (s, 3H), 2.07 – 1.94 (m, 2H), 1.94 – 1.75
(m, 4H), 1.72 – 1.59 (m, 2H), 1.54 (s, 6H); 13C NMR (126 MHz, CDCl3) δ (ppm) 196.14,
176.13, 153.94, 147.59, 127.85, 122.77, 114.49, 110.64, 80.55, 56.18, 53.09, 52.66, 32.93, 24.33,
24.31; LC–MS–ESI+ found 321 [M+H]+.
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Methyl 3-(3-(benzyloxy)-4-methoxyphenyl)-2,2-dimethyl-3-oxopropanoate (11b).
Following the procedure to 11b using 3-(benzyloxy)-4-methoxybenzoic acid on a 148 mmol
scale first gave 3-(benzyloxy)-4-methoxybenzoyl chloride as a yellow semi-solid (41.5 g, quant.,
>95% purity according to 1H NMR); 1H NMR (250 MHz, CDCl3) δ (ppm) 7.78 (dd, J = 8.45,
1.94 Hz, 1H), 7.66 (d, J = 1.90 Hz, 1H), 7.48 (d, J = 7.26 Hz, 2H), 7.43 – 7.28 (m, 3H), 6.94 (d,
J = 8.52 Hz, 1H), 5.30 (s, 1H), 5.19 (s, 3H), 3.95 (s, 3H)) and the title compound as a yellow
syrup (59 g, quant., 90% purity): 1H NMR (250 MHz, CDCl3) δ (ppm) 7.55 – 7.44 (m, 4H), 7.43
– 7.26 (m, 3H), 6.88 (d, J = 8.4 Hz, 1H), 5.18 (s, 2H), 3.94 (s, 3H), 3.61 (s, 3H), 1.51 (s, 6H); 13C
NMR (63 MHz, CDCl3) δ (ppm) 195.81, 175.85, 153.58, 147.94, 136.54, 128.63, 128.05,
127.85, 127.49, 123.30, 113.78, 110.59, 70.85, 56.06, 52.97, 52.47, 24.13; LC–MS–ESI+ found
343 [M+H]+.
Experimental details and spectroscopic data for pyrazolinones.
3-(3-(Cyclopentyloxy)-4-methoxyphenyl)-4,4-dimethyl-1H-pyrazol-5(4H)-one (6a). To a
reaction tube with 11a (0.851 g, 2.66 mmol), dissolved in ethanol (5.5 mL, 0.5 M reaction
molarity), aqueous hydrazine hydrate (80%, 1.4 mL, 8 equiv) was added. The reaction mixture
was stirred at 50 °C overnight. The reaction mixture was cooled on ice and the precipitate was
filtered off over a glass filter, washed with cold ethanol and dried at 40 °C under vacuum to give
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the title compound as a solid foam (0.72 g, 90%): 1H NMR (400 MHz, CDCl3) δ (ppm) 9.43 (s,
1H), 7.42 (d, J = 1.93 Hz, 1H), 7.27 (dd, J = 8.35, 2.06 Hz, 1H), 6.86 (d, J = 8.46 Hz, 1H), 4.92 –
4.69 (m, 1H), 3.87 (s, 3H), 2.04 – 1.74 (m, 6H), 1.71 – 1.55 (m, 2H), 1.51 (s, 6H); 13C NMR
(101 MHz, CDCl3) δ (ppm) 181.71, 163.35, 151.93, 148.11, 123.93, 119.36, 112.34, 111.41,
80.68, 56.13, 47.40, 32.93, 24.21, 22.85; LC–MS–ESI+ found 303, [M+H]+; HRMS–ESI+:
[M+H]+ calcd for C17H23N2O3: 303.1703 found 303.1698.
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1-Cycloheptyl-3-(3-(cyclopentyloxy)-4-methoxyphenyl)-4,4-dimethyl-1H-pyrazol-5(4H)-
one (6b). Sodium hydride (52 mg, 1.3 mmol, 2 equiv.) was added to a chilled suspension of 6a
(195 mg, 0.65 mmol) in DMF/MeCN (2:1) under stirring and N2 atmosphere. The reaction
mixture was stirred for 15 min before bromocycloheptane (0.31 ml, 2.3 mmol, 3.5 equiv.) was
added. The reaction mixture was heated at 120 °C overnight. The volatiles were evaporated in
vacuo. The residue was diluted with EtOAc (50 mL) and washed with brined (3 x 15 mL), dried
over Na2SO4, filtered and reduced in vacuo. The residue was purified via flash chromatography
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to give title compound as a pale solid (230 mg, 89 % yield): H NMR (400 MHz, CDCl3) δ
(ppm) 7.39 (d, J = 1.90 Hz, 1H), 7.28 (dd, J = 8.47, 1.90 Hz, 1H), 6.85 (d, J = 8.47 Hz, 1H), 4.91
– 4.72 (m, 1H), 4.37 – 4.18 (m, 1H), 3.86 (s, 3H), 2.03 – 1.72 (m, 12H), 1.72 – 1.47 (m, 8H),
1.43 (s, 6H); 13C NMR (126 MHz, CDCl3) δ (ppm) 177.50, 161.48, 151.59, 147.82, 124.10,
119.35, 112.55, 111.35, 80.69, 56.07, 54.57, 48.58, 33.43, 32.95, 28.33, 24.81, 24.27, 22.86;
LC–MS–ESI+: m/z 399, [M+H]+; HRMS–ESI+: [M+H]+ calcd for C24H35N2O3: 399.2642 found
399.2624.
3-(3-(Benzyloxy)-4-methoxyphenyl)-4,4-dimethyl-1H-pyrazol-5(4H)-one (8a). Following
the procedure to 6a using 11b on a 0.15 mol scale gave the title compound as a yellow solid
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(47.36 g. 97%): H NMR (400 MHz, CDCl3) δ (ppm) 9.67 (br s, 1H), 7.44 (appar. d, J = 7.36
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Hz, 2H), 7.41 (d, J = 1.93 Hz, 1H), 7.39 – 7.26 (m, 4H), 6.89 (d, J = 8.48 Hz, 1H), 5.18 (s, 2H),
3.91 (s, 3H), 1.41 (s, 6H); 13C NMR (101 MHz, CDCl3) δ (ppm) 181.72, 163.11, 151.49, 148.35,
136.85, 128.68, 128.05, 127.39, 123.88, 119.99, 111.78, 111.38, 71.16, 56.07, 47.32, 22.62; LC–
MS–ESI+: m/z 325, [M+H]+; HRMS–ESI+: [M+H]+ calcd for C19H21N2O3: 325.1547 found
325.1531.
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3-(3-(Benzyloxy)-4-methoxyphenyl)-1-isopropyl-4,4-dimethyl-1H-pyrazol-5(4H)-one (8c).
Following the procedure to 6b using isopropylbromide and 8a on a 2.46 mmol scale gave the
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title compound as a white solid (706 mg, 78%): H NMR (400 MHz, CDCl3) δ (ppm) 7.49 (d, J
= 7.3 Hz, 2H), 7.44 (d, J = 1.9 Hz, 1H), 7.34 (m, 4H), 6.91 (d, J = 8.5 Hz, 1H), 5.22 (s, 2H), 4.50
(hept, J = 6.7 Hz, 1H), 3.93 (s, 3H), 1.38 (s, 6H), 1.37 (d, J = 7.0 Hz, 6H); 13C NMR (101 MHz,
CDCl3) δ (ppm) 177.85, 161.28, 151.30, 148.28, 137.00, 128.66, 128.05, 127.47, 124.11, 119.92,
112.17, 111.38, 71.36, 56.06, 48.73, 45.26, 22.67, 20.82; LC–MS–ESI+: m/z 367, [M+H]+;
HRMS–ESI+: [M+H]+ calcd for C22H27N2O3: 367.2016 found 367.1998.
3-(3-(Benzyloxy)-4-methoxyphenyl)-1-cycloheptyl-4,4-dimethyl-1H-pyrazol-5(4H)-one
(8d). Following the procedure to 6b on a 1.59 mmol scale using 8a gave the title compound as a
white solid (544 mg, 81% yield): 1H NMR (400 MHz, CDCl3) δ (ppm) 7.49 – 7.43 (m, 2H), 7.41
(d, J = 2.00 Hz, 1H), 7.38 – 7.33 (m, 2H), 7.32 – 7.26 (m, 2H), 6.87 (d, J = 8.49 Hz, 1H), 5.20 (s,
2H), 4.36 – 4.16 (m, 1H), 3.90 (s, 3H), 1.98 – 1.74 (m, 6H), 1.67 – 1.47 (m, 6H), 1.34 (s, 6H);
13C NMR (101 MHz, CDCl3) δ (ppm) 177.41, 161.16, 151.21, 148.23, 136.98, 128.61, 127.99,
127.38, 124.09, 119.83, 112.10, 111.32, 71.31, 56.02, 54.47, 48.39, 33.39, 28.34, 24.73, 22.62;
LC–MS–ESI+: m/z 421, [M+H]+; HRMS–ESI+: [M+H]+ calcd for C26H33N2O3: 421.2486 found
421.2467.
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3-(3-Hydroxy-4-methoxyphenyl)-1-isopropyl-4,4-dimethyl-1H-pyrazol-5(4H)-one (12a).
Ammonium formate (10 g, 159 mmol) and 10% Pd/C (50 mg, 0.047 mmol) were added to
benzylether 8c (12 g, 32.7 mmol) dissolved in MeOH/CH2Cl2 (2:1, 200 mL). The reaction
mixture was stirred for 5 h at 40 °C. The reaction mixture was filtered through a cake of celite
and the volatiles were evaporated. The residue was diluted with EtOAc, washed with brine (2 x
200 mL), dried over Na2SO4, filtered and reduced in vacuo with CH2Cl2 as co-solvent to give the
title compound as an off-white solid (8.73 g, 96% yield): 1H NMR (400 MHz, CDCl3) δ 7.44 (d,
J = 2.0 Hz, 1H), 7.29 (dd, J = 8.4, 2.0 Hz, 1H), 6.86 (d, J = 8.5 Hz, 1H), 5.97 (s, 1H), 4.49 (hept,
J = 6.6 Hz, 1H), 3.92 (s, 3H), 1.44 (s, 6H), 1.34 (d, J = 6.7 Hz, 6H); 13C NMR (101 MHz,
CDCl3) δ (ppm) 178.06, 161.60, 148.22, 145.99, 124.91, 118.78, 112.61, 110.62, 56.10, 49.00,
45.37, 22.74, 20.85; LC–MS–ESI+: m/z 277, [M+H]+; HRMS–ESI+: [M+H]+ calcd for
C15H21N2O3: 277.1547 found 277.1543.
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1-Cycloheptyl-3-(3-hydroxy-4-methoxyphenyl)-4,4-dimethyl-1H-pyrazol-5(4H)-one (12b).
Following the procedure of 12a using 8d on a 1.32 mmol scale afforded the title compound as a
pale oil (1.35g, quantitative yield, 99.8% purity): 1H NMR (400 MHz, CDCl3) δ (ppm) 7.46 (d, J
= 2.05 Hz, 1H), 7.29 (dd, J = 8.43, 2.08 Hz, 1H), 6.87 (d, J = 8.49 Hz, 1H), 6.30 (br s, 1H), 4.36
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– 4.22 (m, 1H), 3.92 (s, 3H), 2.03 – 1.74 (m, 6H), 1.70 – 1.47 (m, 6H), 1.45 (s, 6H); C NMR
(101 MHz, CDCl3) δ 177.62, 161.58, 148.30, 146.00, 124.72, 118.64, 112.63, 110.63, 56.00,
54.58, 48.66, 33.37, 28.25, 24.72, 22.61; LC–MS–ESI+: m/z 331, [M+H]+; HRMS–ESI+: [M+H]+
calcd for C19H27N2O3: 331.2016 found 331.1997.
General procedure to O-alkylations A. Corresponding phenol 12a or 12b was added to a
MW reaction tube with K2CO3 (3.0 equiv) and MeCN/DMF (1:1, 0.2 M final reaction molarity).
It was MW irradiated for 30 min at 120 °C. After releasing the CO2-gas from the MW tube, the
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