Synthesis and evaluation of analogs of the phenylpyridazinone NPD-001 as potent trypanosomal TbrPDEB1 phosphodiesterase inhibitors and in vitro trypanocidals

Article abstract of DOI:10.1016/j.bmc.2016.02.032

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. Knock down of both enzymes leads to cell cycle arrest and is lethal to the parasite. Recently, we reported the phenylpyridazinone, NPD-001, with low nanomolar IC₅₀ values on both TbrPDEB1 (IC₅₀: 4 nM) and TbrPDEB2 (IC₅₀: 3 nM) (J. Infect. Dis. 2012, 206, 229). In this study, we now report on the first structure activity relationships of a series of phenylpyridazinone analogs as TbrPDEB1 inhibitors. A selection of compounds was also shown to be anti-parasitic. Importantly, a good correlation between TbrPDEB1 IC₅₀ and EC₅₀ against the whole parasite was observed. Preliminary analysis of the SAR of selected compounds on TbrPDEB1 and human PDEs shows large differences which shows the potential for obtaining parasite selective PDE inhibitors. The results of these studies support the pharmacological validation of the Trypanosome PDEB family as novel therapeutic approach for HAT and provide as well valuable information for the design of potent TbrPDEB1 inhibitors that could be used for the treatment of this disease.

Full text of DOI:10.1016/j.bmc.2016.02.032

Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and evaluation of analogs of the phenylpyridazinone
NPD-001 as potent trypanosomal TbrPDEB1 phosphodiesterase
inhibitors and in vitro trypanocidals
Johan Veerman a, Toine van den Bergh a, Kristina M. Orrling b, Chimed Jansen b, Paul Cos c, Louis Maes c,
Eric Chatelain d, Jean-Robert Ioset d, Ewald E. Edink b, Hermann Tenor e, Thomas Seebeck f, Iwan de Esch b,
Rob Leurs b, Geert Jan Sterk a,b,
⇑
a Mercachem, PO Box 6747, 6503 GE Nijmegen, The Netherlands
b Division of Medicinal Chemistry, Faculty of Sciences, Amsterdam Institute of Molecules, Medicines & Systems (AIMMS), Vrije Universiteit Amsterdam, De Boelelaan 1083,
1081 HV Amsterdam, The Netherlands
c Laboratory for Microbiology, Parasitology and Hygiene (LMPH), University of Antwerp, Groenenborgerlaan 171, 2020 Wilrijk, Belgium
d DNDi (Drugs for Neglected Diseases initiative), 15 Chemin Louis Dunant, 1202 Geneva, Switzerland
e Takeda, Takeda Pharmaceuticals International GmbH, Thurgauerstrasse 130, 8152 Glattpark-Opfikon, Zurich, Switzerland
f Institute of Cell Biology, University of Bern, Baltzerstrasse 4, 3012 Bern, Switzerland
a r t i c l e i n f o
a b s t r a c t
Article history:
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. Knock down of both enzymes leads to cell cycle arrest and is lethal to the
parasite. Recently, we reported the phenylpyridazinone, NPD-001, with low nanomolar IC50 values on
both TbrPDEB1 (IC50: 4 nM) and TbrPDEB2 (IC50: 3 nM) (J. Infect. Dis. 2012, 206, 229). In this study, we
now report on the first structure activity relationships of a series of phenylpyridazinone analogs as
TbrPDEB1 inhibitors. A selection of compounds was also shown to be anti-parasitic. Importantly, a good
correlation between TbrPDEB1 IC50 and EC50 against the whole parasite was observed. Preliminary anal-
ysis of the SAR of selected compounds on TbrPDEB1 and human PDEs shows large differences which
shows the potential for obtaining parasite selective PDE inhibitors. The results of these studies support
the pharmacological validation of the Trypanosome PDEB family as novel therapeutic approach for
HAT and provide as well valuable information for the design of potent TbrPDEB1 inhibitors that could
be used for the treatment of this disease.
Received 18 December 2015
Revised 21 February 2016
Accepted 24 February 2016
Available online xxxx
Keywords:
Pyridazinone
Phosphodiesterase
Trypanosome brucei
African trypanosomiasis
TbrPDEB1
Human PDEs
Ó 2016 Elsevier Ltd. All rights reserved.
1. Introduction
list of Neglected Tropical Diseases for which there is an urgent
need for more effective and less toxic medications.2–4
Human African trypanosomiasis (HAT), or African sleeping sick-
ness, is a neglected tropical disease that affects tens of thousands
of patients annually.2 The causative agent of this disease is the par-
asite Trypanosoma brucei (T. brucei), which is transmitted by the
tsetse fly. The disease is fatal unless treated and current treatment
options are limited.2–4 Consequently, the disease ranks high on the
Spatiotemporal control of the cyclic nucleotide signaling path-
ways is essential for any cell and disruption of this control may
be detrimental to its survival.5,6 Cyclic nucleotide phosphodi-
esterases (PDEs) play a vital role in the control of the homeostasis
of the intracellular cyclic nucleotides cAMP and cGMP in any
eukaryotic cell. Inhibition of PDEs has therefore been recognized
as an effective therapeutic concept in man (e.g., erectile dysfunc-
tion, chronic obstructive pulmonary disease (COPD) and psoriasis),
and more recently also as a potential concept for the design of
novel antiparasitic drugs.7–9
Abbreviations: MOE, Molecular Operating Environment; MW, microwave;
TbrPDEB1, Trypanosoma brucei phosphodiesterase B1; TbrPDEB2, Trypanosoma
brucei phosphodiesterase B2.
The T. brucei phosphodiesterases TbrPDEB1 and TbrPDEB2 are
essential for the survival of the bloodstream form of the parasite.
Concomitant RNAi-induced knock-down of both enzymes kills
⇑
Corresponding author.
E-mail address: g.j.sterk@vu.nl (G.J. Sterk).
http://dx.doi.org/10.1016/j.bmc.2016.02.032
0968-0896/Ó 2016 Elsevier Ltd. All rights reserved.
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2
J. Veerman et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
the parasite in vitro and also saves mice from parasitaemia in vivo.5
Next to this genetic evidence, pharmacological validation of the
TbrPDEB family as therapeutic target has been performed using
the potent tetrazole-containing phenylpyridazinone 1 (NPD-001,
previously reported as compd A)1 and its pyrazolone analog, 2
(VUF13525)10 and a series of piclamilast (3) analogs with low
micromolar activity against both TbrPDEB1 and -B211 (see Fig. 1).
Other chemical classes of PDE inhibitors have been investigated
as well for TbrPDEB1 inhibition but only with minor results.12–17
The phenylpyridazinone 1 shows low nanomolar inhibitory
activities against both TbrPDEB enzymes (TbrPDEB1: IC50 = 4 nM;
TbrPDEB2: IC50 = 3 nM), increases cAMP levels in the whole para-
sites and prevents cytokinesis, resulting in multinucleated, multi-
flagellated cells that eventually lyse, resulting in potent in vitro
trypanocidal activity against the bloodstream form of Trypanosoma
brucei.1 NPD-001 was originally described as a potent PDE4 inhibi-
tor with sub-nanomolar potency,18 limiting its use as anti-try-
panocidal.1 This limitation does not stem from the side effects of
O
R1
R2
R1
R2
a
X
X
O
+
O
CO2H
O
4a: R1=R2=MeO; X1
5a: R1=MeO; R2=H; X1
8a: R1=R2=MeO; X4
9a: R1=R2=MeO; X2
10a: R1=R2=MeO; X3
b
R1
R2
R1
R2
X
c
X
N
N
R3
O
N
N
H
O
5: R1=R2=MeO; X1; R3=c-Hept
6: R1=MeO; R2=H; X1; R3=c-Hept
8: R1=R2=MeO; X4; R3=c-Hept
9: R1=R2=MeO; X2; R3=c-Hept
10: R1=R2=MeO; X3; R3=c-Hept
11: R1=R2=MeO; X2; R3=2-Prop
4: R1=R2=MeO; X1
5b: R1=MeO; R2=H; X1
8b: R1=R2=MeO; X4
9b: R1=R2=MeO; X2
10b: R1=R2=MeO; X3
PDE4 inhibitors per se but mainly from its TNF
as this cytokine plays a crucial role in host defense during infec-
tions.19–21 TNF
suppression makes a patient more vulnerable to
a suppressive effect
H
H
a
H
H
infections which is a very undesirable condition in rural Africa.
However, as most potent TbrPDEB1 and -B2 inhibitor to date,
NPD-001 is an interesting scaffold for hit exploration and SAR
development. To evaluate the usefulness of the phenylpyridazi-
none scaffold for the design of a parasite-specific TbrPDEB1 inhibi-
tor, we report here on a series of phenylpyridazinone analogs and
their inhibitory activities on both TbrPDEB1 and on Trypanosoma
brucei growth inhibitory activity in the Alamar blue test.
X3
X1
X4
X2
Scheme 1. Synthetic route via Friedel–Craft acylation. Reagents: (a) AlCl3 DCM, rt,
16 h. (b) EtOH, N2H4, reflux, 2 h. (c) DMF/NaH, R3-Br, 0 °C, 20 min; rt, 16 h.
by potassium hydroxide to 17. The methyl-substituted tetrazoles
12 and 13 were prepared as mixture by alkylation of 1 and purified
by preparative HPLC. The regioisomer identity of the two methyl
isomers was established using NMR experiments; NOESY, HSQC
and HMBC.
2. Results
2.1. Synthesis
All compounds, except the achiral 8 and 10, were prepared as
racemic mixtures of the cis-diastereomers (4aS,8aR and 4aR,8aS).
The synthesis Schemes 1 and 2 depict the synthetic routes of all
compounds, some of which (1, 4, 5, 9, 11 and 14–18) have been
published before as PDE4 inhibitors,18,22 while the others (6, 7, 8,
10, 12, 13, 19–25) are new. For the compounds with the 3,4-
dimethoxy (4, 5, 8–11) and the 4-methoxy (6) substitution the syn-
thesis route (Scheme 1) started with a Friedel–Craft acylation. Sub-
sequent condensation with hydrazine gave the pyridazinones
which could be alkylated using sodium hydride in DMF. In all other
cases (1, 7, 12–25) the route started with a Grignard reaction
(Scheme 2) to obtain the gamma-ketopropionic acids. These car-
boxylic acids were then converted to the corresponding pyridazi-
nones using the same chemistry as already outlined in Scheme 1.
For the preparation of 1 and 12–25 the 3-cyclopentyloxy group
of intermediate 1c was hydrolyzed by 4-toluenesulfonic acid in
toluene in a Dean Stark apparatus.18 The obtained phenol was then
substituted to obtain the desired compounds using different condi-
tions. The bromoalkyl intermediate 1e was further derivatized by
reacting with a phenol (1f, 14, 17a) or 5H-pyrrolo[3,2-d]pyrimidine
(15). The nitrile 1f was converted to the tetrazole 1 using sodium
azide and ammonium chloride while the ester 17e was hydrolyzed
2.2. Pharmacological testing
All analogs were tested in at least two independent experi-
ments for inhibition of TbrPDEB1-mediated [3H]-cAMP hydrolysis,
using a scintillation proximity assay as described previously.1
Selected compounds were tested for inhibition of T. brucei blood-
stream form trypomastigote proliferation and for cytotoxicity on
MRC-5 fibroblasts using established techniques.23,24 The antipara-
sitic effect of the compounds was determined for the subspecies T.
brucei brucei, which is not infective to humans.
At this early stage of research, assessment of TbrPDEB2 inhibi-
tion was omitted, as suppression of parasite proliferation would
imply concomitant inhibition of TbrPDEB1 and TbrPDEB2. In addi-
tion, two previous studies on TbrPDE inhibitors suggest high
degree of equipotency against the paralogues,1,10 as expected from
the high sequence homology. The binding sites of these two
enzymes are completely identical and the sequence identity of
the catalytic domain is 88%, of which the major discrepancies occur
O
Cl
O
O
H
N
O
O
O
O
O
O
O
N
Cl
N
N
N
N
N
N
N
O
N
NH
Piclamilast
3
VUF13525
2
TbrPDEB1 IC50: 0.049µM
TbrPDEB2 IC50: 0.072µM
T.b. brucei IC50: 0.52µM
N
NH
NPD-001
1
TbrPDEB1 IC50: 0.004
TbrPDEB2 IC50: 0.003
T.b. brucei IC50: 0.08µM
N
TbrPDEB1 IC50: 7.6µM
µ
µ
M
M
TbrPDEB2 IC50: not tested
T.b. brucei IC50: 9.8µM
Figure 1. Known TbrPDE inhibitors with antitrypanosomal activity; 1,1 2,10 and 3.11
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J. Veerman et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
3
R1
R2
O
R1
R2
a
b
R1
R2
+
O
Br
N
O
CO2H
N
H
O
O
1b: R1=MeO; R2=c-PentO
7b: R1=H; R2=MeO
1a: R1=MeO; R2=c-PentO
7a: R1=H; R2=MeO
O
O
R1
R2
c
d
e
HO
R4
O
N
N
N
N
O
N
O
N
O
1e: R4=Br(CH2)4-
1c: R1=MeO; R2=c-PentO
7: R1=H; R2=MeO
1d
16: R4=5-Ph(CH2)5-
18: R4=HOCH2CH2-
19: R4=MeOCH2CH2-
20: R4=2,3-diMeOBn
21: R4=3,5-diMeOBn
22: R4=2-PyrCH2-
23: R4=4-PyrCH2-
24: R4=4-(Me)2N-PhCH2CH2-
25: R4=4-MeOPhCH2CH2-
O
O
O
h
R5
O
f
R6
N
N
N
O
N
O
i
1f: R5=4-CNPhO(CH2)4-
1: R6=4-TetrazolePhO(CH 2)4-
14: R4=4-Imidazol-Ph-O(CH 2)4-
15: R5=4-(Purin-7-yl)(CH 2)4-
17a: R5=4-(CO2Et)PhO(CH2)4-
17: R5=4-(CO2H)PhO(CH2)4-
12: R6=4-(2-MeTetrazole)PhO(CH2)4-
13: R6=4-(1-MeTetrazole)PhO(CH2)4-
g
Scheme 2. Synthetic route via Grignard reaction. Reagents: (a) THF/Mg, 2 h, reflux, rt, anhydride, 18 h, 1 h reflux. (b) EtOH, N2H4, reflux, 2 h. (c) DMF/NaH,
bromocycloheptane. (d) Toluene, 4-TsOH, 4 h Dean–Stark. (e) DMF/K2CO3, R-Br, 60 °C, 2 h or PPh3-DIAD, THF, 0 °C. (f) DMF/K2CO3, ROH or RxNH, 60 °C, 2 h. (g) KOH/H2O/THF.
(h) DMF/NaN3/NH4Cl, 8 h, 120 °C. (i) DMF/K2CO3, MeI, MW, 145 °C, 20 min.
in a single stretch of 33 amino acids in a mostly solvent-exposed
loop.10
Table 1
Inhibitory potency of selected phenylpyridazinones against TbrPDEB1
R1
R2
2.3. SAR analysis
H
H
X
H
H
For hit exploration of 1 a series of close analogs were prepared
or collected from an earlier PDE4 inhibitor optimization project
and screened against TbrPDEB1. Table 1 shows analogs lacking
the long tetrazole containing side chain. A direct comparison of 1
with 5 shows the impact of this tetrazole containing side chain:
an 80-fold increase in inhibitory potency at TbrPDEB1. Also the
cycloheptyl moiety is important for PDE inhibition, as demon-
strated by a comparison of 4 and 5. Removal of the cycloheptyl ring
results in a 40 fold reduction in potency on TbrPDEB1. These
results, with regards to the introduction of the cycloheptyl, are
comparable to what was reported for a series of pyrazolones with
TbrPDEB1 inhibiting activity.10
N
N
R3
O
X3
X1
X4
X2
X=
Compd
R1
R2
R3
H
Ring X
TbrPDEB1a
4
5
6
7
8
9
10
11
1
MeO
MeO
MeO
H
MeO
MeO
MeO
MeO
MeO
MeO
H
MeO
MeO
MeO
MeO
MeO
X1
X1
X1
X1
X4
X2
X3
X2
4.9
6.5
5.4
4.0
5.0
5.8
<5.0
5.7
8.4
c-Heptyl
c-Heptyl
c-Heptyl
c-Heptyl
c-Heptyl
c-Heptyl
2-Propyl
O
The results obtained with 5, 6 and 7 indicate that the two meth-
oxy groups are important for high inhibitory potency. Removal of
the 3-methoxy substituent (6) results in a 13 fold reduction of
potency while the 4-methoxy group is even more important, as
removal (7) makes the compound approximately 300 fold less
active. Comparison of 5 and 8 shows that the cyclohexene ring also
has a strong influence on the activity (approximately 30 fold). The
difference in potency between 5 and 9 is remarkable: the introduc-
tion of the double bond in the cyclohexyl ring gives a 5 fold
increase in potency. Aromatization of the cyclohexyl ring (10) is
clearly detrimental for inhibiting TbrPDEB1. Compounds 9 and
11 are equipotent showing that the cycloheptyl group at position
R3 can be replaced by an isopropyl without significant loss in activ-
ity. This finding might be important in future optimization pro-
grams when trying to reduce molecular size and lipophilicity,
O
O
N
N
N
HN
N
O
N
a
ÀlogIC50 (n = 2, 0.2).
though, evidently, it will be dependent on other substitutions on
the scaffold as was observed with a series of pyrazolones were
the effect of cycloheptyl/isopropyl replacements on TbrPDEB1
inhibiting potency varied between not significant to 31 fold.10
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J. Veerman et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
Methylation of 1 resulted in 12 and 13 of which the 2 substi-
(solubility in 0.05 M phosphate buffer at pH = 7.4, containing 1% of
DMSO, 75 M for 1 vs <2 M for 12 and 13).
tuted analog, 13, is slightly less active though still very potent.
Changing the tetrazole ring of 1 to an imidazole ring (14)
decreases TbrPDEB1 potency 3 fold while substituting it for a car-
boxylic acid (17) decreased potency approximately 80 fold. The
purine analog 15 and the phenyl derivative 16 are both signifi-
cantly less active than 1. With the compounds 18–25 small varia-
tions in potencies on TbrPDEB1 is observed; a 2–5 fold increase in
potency when compared with 5 (R@CH3).
l
l
In Figure 2 a graphical presentation of the correlation between
ÀlogIC50 on TbrPDEB1 and ÀlogEC50 on the whole parasite is
given. In this correlation we left out the compounds with cLogP > 6
(12, 13, 14, 16, 24, 25) except 1 (for of its good solubility at
pH = 7.4) as being potential outliers because of their high
lipophilicity and potential poor aqueous solubility. With this selec-
tion of compounds we calculated a fairly good correlation coeffi-
cient (R2 = 0.92).
2.4. Growth inhibition
2.5. Cytotoxicity
The compounds in Table 2 (except 17 and 22) were also
screened for anti trypanosomal activity in vitro and for cytotoxic
activity on human fetal lung fibroblasts. In all cases, except 20,
there was at least a 10-fold difference between trypanocidal effi-
cacy in the T. brucei growth inhibition assay and general cytotoxi-
city. The high efficacy of 1 in the anti trypanosomal assay, as
compared to 12 and 13, is remarkable. A potential explanation
might be related to the tetrazole; Because of its pKa of about 4.3
(calculated), 1 is present mostly in its anion form under physiolog-
ical conditions, making it much less lipophilic and much better sol-
uble under these conditions compared with its methylated analogs
For cytotoxicity assessment, selected compounds were tested
against human cell line MRC-5. The selectivity index, SI, was
expressed as the ratio between EC50 in the parasite assay and
CC50 in the MRC-5 assay. An excellent selectivity index was found
for 4 compounds, the tetrazole derivatives 1, 12, 13 and the purine
15: >125–316. However the results with very lipophilic com-
pounds might be obscured by their poor aqueous solubility
(<2 lM for the methylated tetrazoles). Only 1 compound from this
series, 20, has a very poor selectivity index of 2.5, while the other
analogs have a moderate SI between 12.6 and >63.
Table 2
Inhibitory activity against TbrPDEB1, T. b. brucei proliferation and cytotoxicity of close analogs of NPD-001
O
R
O
N
N
O
Compd
R
TbrPDEB1a
8.4
T. b. bruceib
MRC-5c
5.2
SId
tPSAe
110
cLogPf
N
N
O
O
(CH2)4-
(CH2)4-
1
7.7
316
6.0
HN
N
N
N
N
12
8.5
6.6
<4.2
>251
101
6.0
N
N
N
N
13
14
8.0
7.9
6.3
6.0
<4.2
4.4
>125
40
101
76
6.3
6.5
O
(CH2)4-
(CH2)4-
N
N
O
N
N
N
15
7.8
6.7
4.4
200
>13
92
3.9
N
(CH2)4-
(CH2)5-
N
16
17
7.2
6.5
5.3
<4.2
n.t.g
51
98
7.6
6.4
HO2C
O
(CH2)4-
n.t.g
18
19
20
21
22
23
24
25
Hydroxyethyl
Methoxyethyl
2,3-Dimethoxybenzyl
3,5-Dimethoxybenzyl
2-Pyridylmethyl
4-Pyridylmethyl
4-N(CH3)2-phenylethyl
4-MeO-phenylethyl
7.0
7.1
7.1
6.9
7.0
6.8
7.2
7.2
5.7
6.1
5.8
6.0
n.t.g
5.9
6.0
5.9
4.5
4.6
5.4
16
32
2.5
12.6
71
60
71
71
64
64
54
60
3.2
3.9
5.5
5.8
4.3
4.3
6.3
6.1
4.9
n.t.g
4.8
12.6
>63
>50
<4.2
<4.2
a
b
c
d
e
f
ÀlogIC50 on TbrPDEB1 (n = 2, 0.2).
ÀlogEC50 on T. b. brucei (n = 2, 0.2).
ÀlogCC50 on MRC-5 cells (n = 2, 0.4).
Selectivity index: CC50 MRC-5/EC50 T. b. brucei.
Topological polar surface area.
Calculated logP.
g
Not tested.
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J. Veerman et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
5
Table 3
Inhibitory potency of 1 against a panel of human PDEs and TbrPDEB1 and TbrPDEB2
a
Enzyme
pIC50
Enzyme
pIC50
TbrPDEB1
TbrPDEB2
hPDE1B1
hPDE2A3
hPDE3A1
hPDE4B1
hPDE4B2
8.4
8.5
5.4
5.2
6.9
9.2
9.3
hPDE4D3
hPDE5A1
hPDE7A1
hPDE8B
hPDE9A3
hPDE10A
hPDE11A4
9.2
5.7
5.5
5.4
<5.0
6.3
5.7
a
ÀlogIC50 (n = 2, 0.2).
Figure 2. Correlation between ÀlogIC50 TbrPDEB1 and ÀlogEC50 T. b. brucei.
100
80
60
40
20
0
1B1
2A3
3A1
4B2
5A1
7A1
8B
10A
11A4
TBrPDEB1
TBrPDEB2
2.6. The parasite specific P-pocket
A special feature of TbrPDEB1 is the so-called parasite specific
P-pocket. This pocket was first observed in the Leishmania major
phosphodiesterase LmjPDEB125 and is shown to exist in TbrPDEB1
as well,26 while it is absent in the human PDEs.25 Targeting this
pocket might lead to parasite specific PDE inhibitors.25
The high TbrPDEB1 inhibiting potency of 1 as compared with 5
can be hypothesized to originate from interaction of the tetrazole
containing side chain with this pocket. In silico docking experi-
ments, using the X-ray apo structure of TbrPDEB1,26 confirmed this
possibility (Fig. 3). In this docking experiment the phenylpyridazi-
none scaffold was positioned in the hydrophobic clamp, which
consists of phenylalanine (F877) and valine (V840), while the
two ether functions were positioned to interact with the invariant
glutamate (Q874). The tetrazole moiety moved in the direction of
the P-pocket, interacting with a tyrosine (Y845). No further
docking studies, with different ligands, were undertaken because
of multiple possible binding modes with these flexible side chains.
The next step in this research would be using X-ray crystallography
to study the interaction of PDE inhibitors with the enzyme to
assess the value of the P-pocket in designing parasite selective
PDE inhibitors.
-12
-10
-8
-6
-4
log [NPD-001], M
Figure 4. Dose response curves of 1 against a panel of human PDEs, TbrPDEB1 and
TbrPDEB2.
other human PDEs in high nanomolar and micromolar concentra-
tions, suggesting that for this series of molecules human PDE4 is
the main off-target.
2.8. Inhibition of TbrPDEB1 versus human PDE4
From the above described human PDE panel screen of 1 it is
clear that especially PDE4 inhibition will limit the usefulness of
this compound. In order to find clues for optimization towards
TbrPDEB1 selectivity over all human PDEs we tested a selection
of phenylpyridazinones against the human PDE4 subtype (Table 4).
With these 7 compounds in Table 4, a 100 fold variation in
TbrPDEB1 inhibiting potency was observed while with the same
compounds the variation on hPDE4B1 inhibition was only 8 fold.
Direct comparison of 1 and 5 shows the effect of the tetrazole
containing side chain: these 2 compounds are equipotent on
PDE4 while there is an 80 fold difference on the parasite PDE. From
these data we calculated for each individual compound the ratio
between IC50 on the parasite and this human PDE, showing varia-
tion from 6 to 1000. These variations are clearly indicative for
significant differences in SAR between TbrPDEB1 and human
PDE4B1, making it likely that optimization for TbrPDEB1 selectivity
over human PDE4 is possible.
2.7. PDE selectivity panel
As most potent TbrPDEB inhibitor, we tested NPD-001 1 on a
panel of human PDEs to verify subtype potency and the results
are summarized in Table 3 and visualized in Figure 4. Besides being
a potent low nanomolar inhibitor of TbrPDEB1 and TbrPDEB2, the
compound shows the previously reported sub-nanomolar poten-
cies at the human PDE4 isoenzymes and only interacts with the
3. Conclusions
For reasons of hit validation of 1 we collected a series of
NPD-001 analogs by synthesis and from the shelves from a project
for human PDE4 inhibitors and determined their TbrPDEB1 inhibit-
ing activities. For some analogs the in vitro antiproliferative
activity on Trypanosome brucei was determined as well. Analysis
of the structure activity relationships shows that within this series
of pyridazinones the TbrPDEB1 inhibiting potency varies 25,000
fold (compare
1 with 7). Comparison of the 3,4-dimethoxy
compound 5 with 1 shows an 80 fold difference in potency, which
is speculated to originate from interaction of the tetrazole with the
parasite specific P-pocket and is supported by molecular docking
studies.
Figure 3. Proposed binding mode of NPD-001 (blue sticks) in TbrPDEB1. The P-
pocket is indicated by the gray molecular surface.
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6
J. Veerman et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
Table 4
Inhibitory potency of some analogs of 1 against TbrPDEB1 and hPDEB1
Compd
R
TbrPDEB1a
6.5
hPDE4B1b
9.2
IC50 TbrPDEB1/IC50 hPDE4B1
501
5
CH3
N
N
HN
O
O
(CH2)4-
(CH2)4-
1
8.4
8.5
9.2
9.3
6
6
N
N
N
N
12
N
N
N
N
13
14
8.0
7.9
9.2
9.7
16
63
O
(CH2)4-
(CH2)4-
N
N
O
N
N
N
15
17
7.8
6.5
8.8
9.5
10
N
(CH2)4-
N
HO2C
O
(CH2)4-
1000
a
ÀlogIC50 on TbrPDEB1 (n = 2, 0.2).
b
ÀlogIC50 on human PDE4B1 (n = 2, 0.2).
Comparison of TbrPDEB1 IC50 with antiproliferative EC50
the chemPLP scoring function. The generated docking poses were
visually inspected for formation of hydrogen bonds between the
oxygen atom of the methoxy group of compound 1 and the nitro-
gen atom of the carboxamide moiety of Q874. Interestingly, for
both tetrazole tautomers (1H or 2H) the highest ranked pose that
displayed this anticipated interaction was in both cases pose 7 of
the 25 generated poses. Both binding poses were very similar
and in both cases the parasite-selective P-pocket was being
addressed by the tetrazolephenoxy moiety.
showed good correlation between biochemical activity on the par-
asite PDE and in vitro antiproliferative activity on the whole para-
site; this result gives further target validation of the Trypanosome
PDEs.
The most potent compound in this study (1) is however a signif-
icantly more potent inhibitor of the human PDE4 subtypes than of
TbrPDEB1. To have any potential in the treatment of human African
trypanosomiasis significant selectivity for the parasite PDE over
the human PDEs is essential. This is especially important for hPDE4
as inhibition of this subtype will compromise a patient’s immune
system. These compounds clearly do not fulfill this requirement
yet, as shown by 1, which is 6–9 times more potent on the hPDE4
subtypes. Still, preliminary analysis of the activities of selected
compounds on both TbrPDEB1 and human PDE4B1 shows signifi-
cant SAR differences which is indicative for the potential of obtain-
ing TbrPDEB1 selectivity over human PDE4.
We believe that the potencies of these compounds on
TbrPDEB1, their SAR on both the parasite PDE and human PDEs,
as well as their potency in inhibiting trypanosomal growth
in vitro and their weak cytotoxicity warrants further investigations
of this chemical class towards a novel PDE inhibition based treat-
ment of this disease.
4.2. Synthetic chemistry
4.2.1. General information
All compounds have been analyzed by 1H NMR (Bruker,
400 MHz or 500 MHz) and analytical LCMS for identity and purity.
(Apparatus: Agilent 1100 Bin. Pump: G1312A, degasser; autosam-
pler, ColCom, DAD: Agilent G1315B, 220–320 nm, MSD: Agilent LC/
MSD G1946D ESI, pos/neg 100–800, column: Waters XBridgeTM C18,
50 Â 2.1 mm, 3.5
l, Temp: 35 °C, Flow: 0.8 mL/min, Gradient:
t0 = 2% A, t3.5min = 98% A, t6min = 98% A, Posttime: 2 min; Eluent A:
0.1% formic acid in acetonitrile, Eluent B: 0.1% formic acid in
water). Exact molecular masses (HRMS) were determined on a Bru-
ker microTOF-Q mass spectrometer equipped with an electrospray
ion source using caffeine as reference. The purity of all compounds
was >95%. The synthesis of the compounds 1, 4, 5, 9, 11 and 14–18
has been described before.18,22,29,30 and the remaining compounds
(6, 7, 8, 10, 12, 13, 19–25) are prepared in an analogous manner
and shortly described below.
4. Experimental section
4.1. Molecular docking
The 4aS,8aR enantiomer of 1 was built manually using MOE27
(version 2012.10). After energy minimization (MMF94x) the mole-
cule was stored in a MOE database file and the tetrazole moiety
was deprotonated using the MOE Wash function. A mol2 file was
generated and used as input for PLANTS.28 The A chain of the apo
TbrPDEB1 crystal structure (pdb: 4I15) was prepared for docking
using MOE (version 2012.10) by adding and minimizing the hydro-
gen atoms using the protonate 3D function. The water molecules
and metal ions were removed from the structure. The pocket of
the protein was manually defined (Y668, H669, N717, T783,
M785, N822, I823, N825, V826, S833, W836, A837, V840, T841,
F844, Y845, L859, M861, F862, N867, M868, E869, L870, G873,
Q874, F877) and both the protein and the pocket were stored in
mol2 format as input for PLANTS. The 4aS,8aR enantiomer of 1
was docked 25 times using the speed 1 setting of PLANTS and
Method A: Methoxybenzene or 1,2-dimethoxybenzene
(50 mmol) was added to a suspension of aluminiumtrichloride
(6.67 g, 50 mmol) in DCM (100 ml) at 0 °C. After complete addition,
cis-1,2,3,6-tetrahydrophthalic anhydride or phthalic anhydride
(50 mmol) was added. After 5 h at rt, the mixture was poured onto
ice. The organic layer was dried over MgSO4 and evaporated. The
residue was triturated with diethyl ether and dried; yield 40–60%.
Method B:
A solution of 3-methoxybromobenzene (3.74 g,
20 mmol) in dry THF was added slowly to a suspension of Mg(s)
(0.53 g, 22 mmol) in THF and the resulting mixture refluxed for
5 h, after which it was added slowly to a solution of cis-1,2,3,6-
tetrahydrophthalic anhydride (3.04 g, 20 mmol) in THF. The mix-
ture was stirred at room temperature for 18 h and subsequently
quenched with ammonium chloride. Next the mixture was acidi-
fied with hydrochloric acid (2 N) and the mixture extracted with
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J. Veerman et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
7
diethyl ether. The ether solution was dried over MgSO4 and
evaporated. The residue was triturated with diethyl ether; yield
40–50%.
1.45–1.69 (m, 6H). LC–MS-ESI+ m/z 353 [M+H]+; HRMS-ESI+ [M
+H]+ calcd for C22H29N2O2 353.2224, found 353.2217.
Method C: A solution of a ketocarboxylic acid (10 mmol) and
hydrazineÁH2O (0.60 g, 12 mmol) in ethanol (150 ml) was refluxed
for 6 h. After cooling to room temperature the precipitate was
filtered off and dried; yield 80–90%.
Method D: NaH (0.22 g, 5.5 mmol, 60% mineral oil) was added to
a suspension of a pyridazinone (5 mmol) in DMF and the resulting
mixture was stirred for 30 min. After addition of bromocyclohep-
tane (1.06 g, 6 mmol) the mixture was stirred at room temperature
for 18 h and subsequently poured into water. The resulting
mixture was extracted with diethyl ether, the ether layer was dried
over MgSO4 and evaporated. The residue was purified by column
chromatography (ethyl acetate hexane gradients); yield 50–80%.
4.2.2.4.
acid (7a).
cis-2-(3-Methoxybenzoyl)-1,2,3,6-tetrahydrobenzoic
Method B, from 3-methoxybromobenzene and
cis-1,2,3,6-tetrahydrophthalic anhydride, followed by column
chromatography using hexane/ethyl acetate + 1% AcOH = 19/1 to
1/1; Yield: 41%. LC–MS-ESI+ m/z 261 [M+H]+; purity 82%.
4.2.2.5.
phthalazin-1-one (7b).
benzoyl)-1,2,3,6-tetrahydrobenzoic acid (7a, 2.6 g, 10 mmol) and
hydrazine hydrate (0.60 g, 12 mmol); Yield: 67%. LC–MS-ESI+ m/z
257 [M+H]+.
cis-4-(3-Methoxy-phenyl)-4a,5,8,8a-tetrahydro-2H-
Method C, from cis-2-(3-methoxy-
Method E:
acidÁhydrate
A
mixture of 55 mmol of 4-toluenesulfonic
4.2.2.6.
cis-2-Cycloheptyl-4-(3-methoxy-phenyl)-4a,5,8,
Method D, from
(10.46 g, 55 mmol) and cis-2-cycloheptyl-
8a-tetrahydro-2H-phthalazin-1-one (7).
4-(3-cyclopentyloxy-4-methoxy-phenyl)-4a,5,8,8a-tetrahydro-2H-
phthalazin-1-one (19a) (21.83 g, 50 mmol) in toluene (250 ml) was
refluxed in a Dean–Stark as described previously.12
cis-4-(3-methoxy-phenyl)-4a,5,8,8a-tetrahydro-2H-phthalazin-1-
one (7b). (1.28 g, 5 mmol) and bromocycloheptane (1.06 g;
6 mmol); Yield: 53%. 1H NMR (CDCl3) d 8.22 (dd, J1 = 2.0 Hz,
J2 = 8.8 Hz, 1.0H), 8.04 (dd, J1 = 2.0 Hz, J2 = 8.8 Hz, 1.0H), 7.70 (m,
2H), 7.20 (m, 2H), 7.10 (m, 1H), 3.87, s, 6H), 3.61 (quintet,
J = 7.0 Hz, 1H), 1.55 (m, 12H). LC–MS-ESI+ m/z 353 [M+H]+;
HRMS-ESI+ [M+H]+ calcd for C22H29N2O2 353.2224, found
353.2225.
Method F: A mixture of cis-2-cycloheptyl-4-(3-hydroxy-4-meth-
oxy-phenyl)-4a,5,8,8a-tetrahydro-2H-phthalazin-1-one12 (0.70 g,
1.9 mmol), K2CO3 (0.53 g, 3.8 mmol) and alkylating agent
(3.4 mmol) in DMF (20 ml) was stirred at 65 °C upon completion
(2–6 h). Then the reaction was quenched with water and the
resulting mixture was extracted with ethyl acetate. The organic
layers were combined and washed with sodium bicarbonate (aq,
satd) and water and subsequently dried over Na2SO4 and evapo-
rated. The residue was purified by flash chromatography (ethyl
acetate hexane gradients).
Method G: A solution of diisopropyl azodicarboxylate (67 mg,
0.33 mmol) in THF (2 ml) was added dropwise to an ice-cooled
solution of cis-2-cycloheptyl-4-(3-hydroxy-4-methoxy-phenyl)-
4a,5,8,8a-tetrahydro-2H-phthalazin-1-one,12 (100 mg, 0.27 mmol),
an alcohol (0.27 mmol) and 0.35 mmol triphenylphosphine
(92 mg, 0.35 mmol) in THF (25 ml). After complete addition the
reaction mixture was allowed to reach room temperature and
the resulting mixture was stirred for 2 h. Next the reaction mixture
was concentrated under reduced pressure, the residue partitioned
between ethyl acetate and NaOH (aq, 1 M). The organic phase was
washed with brine and subsequently dried over Na2SO4 and evap-
orated. The residue was purified by flash chromatography (ethyl
acetate hexane gradients); yield 24%.
4.2.2.7. 2-Cycloheptyl-6-(3,4-dimethoxy-phenyl)-4,5-dihydro-
2H-pyridazin-3-one (8).
Method D, from 6-(3,4-dimethoxy-
phenyl)-4,5-dihydro-2H-pyridazin-3-one20 (1.17 g, 5 mmol) and
bromocycloheptane (1.06 g; 6 mmol); crystallized from methanol.
Yield: 64%. 1H NMR (CDCl3) d 7.47 (d, J = 2.0 Hz, 1H), 7.22 (dd,
J = 2.0, 8.4 Hz, 1H), 6.87 (d, J = 8.4 Hz, 1H), 4.78–4.86 (m, 1H),
3.94 (s, 3H), 3.92 (s, 3H), 2.87 (t, J = 7.8 Hz, 2H), 2.55 (t, J = 7.8 Hz,
2H), 1.78–1.92 (m, 6H), 1.50–1.65 (m, 6H). LC–MS-ESI+ m/z 331
[M+H]+; HRMS-ESI+ [M+H]+ calcd for C19H27N2O3 331.2016, found
331.2013.
4.2.2.8. 2-Cycloheptyl-4-(3,4-dimethoxyphenyl)-2H-phthalazin-
1-one (10).
Prepared from 4-(3,4-dimethoxyphenyl)-2-phe-
nyl-2H-phthalazin-1-one20 (1.41 g, 5 mmol) and bromocyclohep-
tane (1.06 g; 6 mmol) by method D. Crystallized from methanol;
Yield: 71%. 1H NMR (CDCl3): d 8.57–8.54 (m, 1H), 7.85–7.74 (m,
3H), 7.23–7.16 (m, 2H), 7.04 (d, J = 8.2 Hz, 1H), 5.26 (heptet,
J = 4.7 Hz, 1H), 3.99 (s, 3H), 3.96 (s, 3H), 2.14–2.02 (m, 4H), 1.90–
1.81 (m, 2H), 1.75–1.58 (m, 6H). LC–MS-ESI+ m/z 379 [M+H]+;
HRMS-ESI+ [M+H]+ calcd for C23H27N2O3 379.2016, found
379.2005.
4.2.2. Synthetic details and spectroscopic data
4.2.2.1.
cis-2-(4-Methoxybenzoyl)-1,2,3,6-tetrahydrobenzoic
acid (5a).
Method A, from 4-methoxybenzene (5.4 g,
50 mmol) and cis-1,2,3,6-tetrahydrophthalic anhydride (7.6 g,
50 mmol), followed by column chromatography using hexane/
ethyl acetate + 1% AcOH = 19/1 to 1/1; Yield: 59%. LC–MS-ESI+
m/z 261 [M+H]+.
4.2.2.9. cis-2-Cycloheptyl-4-(4-methoxy-3-(4-(4-(2-methyl-2H-
tetrazol-5yl)phenoxy)butoxy)phenyl)-4a,5,8,8a-tetrahydrophth
alazin-1(2H)-one (12) and cis-2-cycloheptyl-4-(4-methoxy-3-(4-
(4-(1-methyl-1H-tetrazol-5-yl)phenoxy)butoxy)phenyl)-4a,5,8,
4.2.2.2.
phthalazin-1-one (5b).
cis-4-(4-Methoxy-phenyl)-4a,5,8,8a-tetrahydro-2H-
Method C, from cis-2-(4-methoxy-
8a-tetrahydrophthalazin-1(2H)-one (13).
1.752 mmol) and 2 ml of DMF was added to a 5 mL MW-vial with
K2CO3 (242 mg,
benzoyl)-1,2,3,6-tetrahydrobenzoic acid (5a, 2.6 g, 10 mmol) and
hydrazine hydrate (0.60 g, 12 mmol). Yield: 74%; LC–MS-ESI+ m/z
257 [M+H]+.
1 (100 mg, 0.171 mmol) and MW irradiated at 145 °C for 20 min.
Iodomethane (12 lL, 0.193 mmol) was added through the septum
and the reaction mixture was MW irradiated at 145 °C for 30 min
and then stirred at rt for 18 h. The reaction mixture was diluted
with ethyl acetate 15 mL) and washed with brine (3 Â 8 mL) in
order to remove the DMF. The organic layer was dried over Na2SO4,
filtered and evaporated. The crude product was purified with flash
column chromatography (Biotage Silicycle 25 g, silica, ethyl acetate
in cyclohexane (10–70%, 2-10-3 CV) to give 12 (75.9 mg,
0.127 mmol, 72% yield) as the major component in the first eluted
peak and 13 (19 mg, 0.032 mmol, 18% yield) as the minor
component in the second eluted peak, resulting in a total yield of
4.2.2.3.
tetrahydro-2H-phthalazin-1-one (6).
compound cis-4-(4-methoxy-phenyl)-4a,5,8,8a-tetrahydro-2H-
phthalazin-1-one (5b, 1.28 g, 5 mmol) and bromocycloheptane
(1.06 g; 6 mmol); Yield: 52%. 1H NMR (CDCl3):
7.76 (d,
cis-2-Cycloheptyl-4-(4-methoxy-phenyl)-4a,5,8,8a-
Method D, from
d
J = 9.0 Hz, 2H), 6.94 (d, J = 9.0 Hz, 2H), 5.74–5.82 (m, 1H),
5.62–5.71 (m, 1H), 4.76–4.86 (m, 1H), 3.85 (s, 3H), 3.25–3.32 (m,
1H), 2.95–3.06 (m, 1H), 2.72 (t, J = 6.0 Hz, 1H), 1.70–2.25 (m, 9H),
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8
J. Veerman et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
95 mg (90%) with a regioisomer ratio of 4:1 major/minor. The posi-
tion of the introduced methyl group was determined with 2D NMR
experiments. 12: 1H NMR (500 MHz, CDCl3) d (ppm) 8.03 (appar. d,
J = 8.74 Hz, 2H), 7.51 (appar. s, 1H), 7.27 (appar. d, J = 8.76 Hz, 1H),
6.98 (appar. d, J = 8.87 Hz, 2H), 6.87 (appar. d, J = 8.45 Hz, 1H), 5.77
(d, J = 8.60 Hz, 1H), 5.66 (d, J = 8.40 Hz, 1H), 4.87–4.78 (m, 1H), 4.36
(s, 3H), 4.21–4.15 (m, 2H), 4.15–4.08 (m, 2H), 3.88 (s, 3H), 3.27 (dt,
J = 11.45, 5.74 Hz, 1H), 2.99 (br d, J = 17.74 Hz, 1H), 2.71 (t,
J = 5.68 Hz, 1H), 2.23–1.95 (m, 8H), 1.95–1.85 (m, 1H), 1.84–1.69
(m, 4H), 1.65–1.46 (m, 6H); LC–MS-ESI+ m/z 599 [M+H]+; HRMS-
ESI+ [M+Na]+ calcd for C34H42N6O4Na 621.3160, found 621.3162.
13: 1H NMR (500 MHz, CDCl3) d (ppm) 7.69 (d, J = 8.67 Hz, 2H),
7.53 (s, 1H), 7.29 (d, J = 10.23 Hz, 1H), 7.07 (d, J = 8.67 Hz, 2H),
6.89 (d, J = 8.44 Hz, 1H), 5.78 (d, J = 9.82 Hz, 1H), 5.67 (d,
J = 10.08 Hz, 1H), 4.91–4.78 (m, 1H), 4.22–4.13 (m, 4H), 4.16 (s,
3H), 3.90 (s, 3H), 3.29 (dt, J = 11.48, 5.72 Hz, 1H), 3.00 (d,
J = 18.31 Hz, 1H), 2.72 (t, J = 5.81 Hz, 1H), 2.25–1.96 (m, 8H),
1.96–1.86 (m, 1H), 1.84–1.71 (m, 4H), 1.70–1.46 (m, 6H); LC–MS-
ESI+ m/z 599 [M+H]+; HRMS-ESI+ [M+Na]+ calcd for C34H42N6O4Na
621.3160, found 621.3173.
1H), 7.20–7.25 (m, 1H), 6.92 (d, J = 8.5 Hz, 1H), 5.73–5.81 (m,
1H), 5.61–5.69 (m, 1H), 5.36 (s, 2H), 4.71–4.82 (m, 1H), 3.96 (s,
3H), 3.17–3.26 (m, 1H), 2.92–3.03 (m, 1H), 2.68 (t, J = 5.8 Hz, 1H),
1.80–2.23 (m, 5H), 1.40–1.80 (m, 10H). LC–MS-ESI+ m/z 460 [M
+H]+; HRMS-ESI+ [M+H]+ calcd for C28H34N3O3 460.2595, found
460.2607.
4.2.2.14. cis-2-Cycloheptyl-4-[4-methoxy-3-(pyridin-4-ylmeth-
oxy)-phenyl]-4a,5,8,8a-tetrahydro-2H-phthalazin-1-one
(23).
Method F, from 4-(chloromethyl)pyridineÁHCl (558 mg,
3.4 mmol); in this case 4 equiv of potassium carbonate were added
and the reaction mixture was stirred for 24 h at 65 °C. Then 1 equiv
of potassium carbonate was added and the reaction mixture was
stirred for additional 26 h at 90 °C; Yield: 37%. 1H NMR (CDCl3) d
8.62 (d, J = 5.6 Hz, 2H), 7.45 (d, J = 1.8 Hz, 1H), 7.39 (d, J = 5.6 Hz,
2H), 7.28 (dd, J = 8.4, 1.8 Hz, 1H), 6.92 (d, J = 8.4 Hz, 1H),
5.74–5.81 (m, 1H), 5.61–5.70 (m, 1H), 5.25 (s, 2H), 4.73–4.83 (m,
1H), 3.96 (s, 3H), 3.19–3.38 (m, 1H), 2.94–3.04 (m, 1H), 2.68 (t,
J = 5.9 Hz, 1H), 1.81–2.25 (m, 5H), 1.42–1.80 (m, 10H). LC–MS-
ESI+ m/z 460 [M+H]+; HRMS-ESI+ [M+H]+ calcd for C28H34N3O3
460.2595, found 460.2597.
4.2.2.10. cis-2-Cycloheptyl-4-[4-methoxy-3-(2-methoxyethoxy)-
phenyl]-4a,5,8,8a-tetrahydro-2H-phthalazin-1-one (19).
Method F, from 1-bromo-2-methoxyethane (473 mg, 3.4 mmol);
In this case the reaction mixture was stirred for 4 h at 65 °C and
the reaction mixture was quenched with hydrogen chloride (aq,
1 N) instead of water; Yield: 24%. 1H NMR (CDCl3) d 7.54 (d,
J = 2.0 Hz, 1H), 7.31 (dd, J = 8.5, 2.0 Hz, 1H), 6.88 (d, J = 8.5, 1H),
5.74–5.83 (m, 1H), 5.63–5.72 (m, 1H), 4.78–4.88 (m, 1H), 4.24 (t,
J = 5.0 Hz, 2H), 3.90 (s, 3H), 3.81 (t, J = 5.0 Hz, 2H), 3.47 (s, 3H),
3.23–3.33 (m, 1H), 2.95–3.06 (m, 1H), 2.72 (t, J = 5.5 Hz, 1H),
1.86–2.26 (m, 5H), 1.45–1.86 (m, 10H). LC–MS-ESI+ m/z 427 [M
+H]+; HRMS-ESI+ [M+H]+ calcd for C25H35N2O4 427.2591, found
427.2578.
4.2.2.15. cis-2-Cycloheptyl-4-{3-[2-(4-dimethylaminophenyl)-
ethoxy]-4-methoxy-phenyl}-4a,5,8,8a-tetrahydro-2H-phthalaz
in-1-one (24).
Method G, from 2-(4-dimethylaminophenyl)
ethanol (45 mg, 0.27 mmol); Yield: 25%. 1H NMR (CDCl3) d 7.50
(d, J = 1.8 Hz, 1H), 7.30 (dd, J = 8.5, 1.8 Hz, 1H), 7.19 (d, J = 8.6 Hz,
2H), 6.89 (d, J = 8.5 Hz, 1H), 6.72 (d, J = 8.6 Hz, 2H), 5.74–5.82 (m,
1H), 5.63–5.70 (m, 1H), 4.77–4.87 (m, 1H), 4.24 (t, J = 8.0 Hz, 2H),
3.92 (s, 3H), 3.24–3.31 (m, 1H), 3.10 (t, J = 7.9 Hz, 2H), 2.94–3.05
(m, 1H), 2.93 (s, 6H), 2.71 (t, J = 5.7 Hz, 1H), 1.81–2.25 (m, 5H),
1.42–1.80 (m, 10H). LC–MS-ESI+ m/z 516 [M+H]+; HRMS-ESI+ [M
+H]+ calcd for C32H42N3O3 516.3221, found 516.3228.
4.2.2.16. cis-2-Cycloheptyl-4-{3-[2-(4-methoxyphenyl)-ethoxy]-
4-methoxy-phenyl}-4a,5,8,8a-tetrahydro-2H-phthalazin-1-one
4.2.2.11.
4-methoxy-phenyl]-4a,5,8,8a-tetrahydro-2H-phthalazin-1-one
(20). Method F, from 1-(chloromethyl)-2,3-dimethoxyben-
cis-2-Cycloheptyl-4-[3-(2,3-dimethoxybenzyloxy)-
(25).
Method G, from 2-(4-methoxyphenyl)ethanol (41 mg,
0.27 mmol); Yield: 23%. 1H NMR (CDCl3) d 7.50 (d, J = 1.8 Hz, 1H),
7.29 (dd, J = 8.0, 1.9 Hz, 1H), 7.23 (d, J = 8.5 Hz, 2H), 6.83–6.93 (m,
3H), 5.73–5.83 (m, 1H), 5.63–5.71 (m, 1H), 4.76–4.87 (m, 1H),
4.26 (t, J = 7.6 Hz, 2H), 3.92 (s, 3H), 3.80 (s, 3H), 3.23–3.31 (m,
1H), 3.13 (t, J = 7.6 Hz, 2H), 2.96–3.06 (m, 1H), 2.71 (t, J = 5.8 Hz,
1H), 1.86–2.25 (m, 5H), 1.44–1.86 (m, 10H). LC–MS-ESI+ m/z 503
[M+H]+; HRMS-ESI+ [M+H]+ calcd for C31H39N2O4 503.2904, found
503.2915.
zene (634 mg, 3.4 mmol). Yield 69%. 1H NMR (CDCl3) d 7.56 (d,
J = 1.8 Hz, 1H), 7.28 (dd, J = 1.8, 8.4 Hz, 1H), 7.05–7.11 (m, 2H),
6.89 (d, J = 8.5 Hz, 2H), 5.74–5.81 (m, 1H), 5.62–5.70 (m, 1H),
5.27 (s, 2H), 4.76–4.83 (m, 1H), 3.92 (s, 3H), 3.89 (s, 3H), 3.87 (s,
3H), 3.23–3.29 (m, 1H), 2.95–3.05 (m, 1H), 2.70 (t, J = 5.7 Hz,),
1.82–2.23 (m, 5H), 1.69–1.81 (m, 4H), 1.47–1.65 (m, 6H). LC–MS-
ESI+ m/z 519 [M+H]+; HRMS-ESI+ [M+H]+ calcd for C31H39N2O5
519.2853, found 519.2842.
4.3. Determination of PDE activity
4.2.2.12.
methoxy-phenyl]-4a,5,8,8a-tetrahydro-2H-phthalazin-1-one
(21). Method F, from 1-(chloromethyl)-3,5-dimethoxyben-
cis-2-Cycloheptyl-4-[3-(3,5-dimethoxybenzyloxy)-4-
The standard scintillation proximity assay (SPA) for determina-
tion of PDE activities was used to analyze effects of test compounds
on the enzymatic activities of TbrPDEB1 and TbrPDEB21 and full
length human recombinant PDE1B1, 2A3, 3A1, 4B1, 4B2, 4D3,
5A1, 7A1, 8B, 10A and 11A4.31 In these assays, the cAMP or cGMP
zene (634 mg, 3.4 mmol). Yield: 59%. 1H NMR (CDCl3) d 7.48 (d,
J = 1.9 Hz, 1H), 7.29 (dd, J = 1.9, 8.5 Hz, 1H), 6.90 (d, J = 8.5 Hz,
1H), 6.62 (d, J = 2.2 Hz, 2H), 6.39 (t, J = 2.2 Hz, 1H), 5.73–5.81 (m,
1H), 5.61–5.69 (m, 1H), 5.17 (d, J = 12.9 Hz, 2H), 4.73–4.85 (m,
1H), 3.93 (s, 3H), 3.78 (s, 6H), 3.15–3.25 (m, 1H), 2.93–3.03 (m,
1H), 2.69 (t, J = 5.8 Hz, 1H), 1.81–2.23 (m, 5H), 1.66–1.81 (m, 4H),
1.42–1.66 (m, 6H). LCMS found 519.2 [M+H]+. HRMS-ESI+ [M+H]+
calcd for C31H39N2O5 519.2853, found 519.2844.
(PDE5A1) substrate concentration was 0.5 lM.
4.4. In vitro susceptibility testing of trypanosomes and MRC-5
fibroblasts
T. b. brucei (Squib-427 strain, suramin-sensitive) trypomastig-
otes or MRC-5 fibroblasts were cultured at 37 °C and 5% CO2 in Hir-
umi-9 medium, supplemented with 10% fetal calf serum (FCS) as
described previously.23 Compound stock solutions were prepared
in 100% DMSO at 20 mM. The compounds were serially pre-diluted
(2-fold or 4-fold) in DMSO followed by a further (intermediate)
dilution in demineralized water to assure a final in-test DMSO con-
centration of <1%.
4.2.2.13.
ylmethoxy)-phenyl]-4a,5,8,8a-tetrahydro-2H-phthalazin-1-one
(22).
3.4 mmol); in this case 4 equiv of potassium carbonate were added
and the reaction mixture was stirred for 6 h at 65 °C; Yield: 47%. 1H
NMR (CDCl3) d 8.61 (d, J = 4.0 Hz, 1H), 7.72 (t, J = 7.6 Hz, 1H), 7.57
(d, J = 7.8 Hz, 1H), 7.48 (d, J = 1.8 Hz, 1H), 7.31 (dd, J = 8.4, 1.8 Hz,
cis-2-Cycloheptyl-4-[4-methoxy-3-(pyridin-2-
Method F, from 2-(chloromethyl)pyridineÁHCl (558 mg,
Please cite this article in press as: Veerman, J.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.02.032

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Author contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript. All authors contributed equally.
Acknowledgements
This research was performed under the framework of the Top
Institute Pharma project ‘Phosphodiesterase inhibitors for
Neglected Tropical Diseases’ with partners the VU University Ams-
terdam, University of Bern, The Royal Tropical Institute, Nether-
lands, Mercachem BV, Nycomed:
a Takeda Company, IOTA
Pharmaceuticals Ltd, the Drugs for Neglected Diseases initiative
(DNDi) and TI Pharma. DNDi is grateful to its donors, public and
private, who have provided funding to DNDi since its inception
in 2003. With the support of these donors, DNDi is well on its
way to achieving the objectives of building a robust pipeline and
delivering 11–13 new treatments by 2018. A full list of DNDi’s
donors can be found at http://www.dndi.org/donors/donors.html.
For the work described in this paper, DNDi allocated non-
earmarked funding from the following donors: The Bill & Melinda
Gates Foundation (BMGF)/United States of America, Reconstruc-
tion Credit Institution-Federal Ministry of Education and Research
(KfW-BMBF)/Germany and Médecins Sans Frontières (Doctors
without Borders)/International. The donors had no role in study
design, data collection and analysis, decision to publish, or prepa-
ration of the manuscript.
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Please cite this article in press as: Veerman, J.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.02.032