Lead Optimization of Phthalazinone Phosphodiesterase Inhibitors as Novel Antitrypanosomal Compounds

Article abstract of DOI:10.1021/acs.jmedchem.9b00985

Human African trypanosomiasis is causing thousands of deaths every year in the rural areas of Africa. In this manuscript we describe the optimization of a family of phtalazinone derivatives. Phosphodiesterases have emerged as attractive molecular targets for a novel treatment for a variety of neglected parasitic diseases. Compound 1 resulted in being a potent TbrPDEB1 inhibitor with interesting activity against T. brucei in a phenotypic screen. Derivative 1 was studied in an acute in vivo mouse disease model but unfortunately showed no efficacy due to low metabolic stability. We report structural modifications to achieve compounds with an improved metabolic stability while maintaining high potency against TbrPDEB1 and T. brucei. Compound 14 presented a good microsomal stability in mouse and human microsomes and provides a good starting point for future efforts.

Full text of DOI:10.1021/acs.jmedchem.9b00985

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Article
Lead optimization of phthalazinone phosphodiesterase
inhibitors as novel antitrypanosomal compounds
Irene G. Salado, Abhimanyu K. Singh, Carlos Moreno-Cinos, Guna Sakaine, marco siderius,
Pieter Van der Veken, An Matheeussen, Tiffany Van der Meer, Payman Sadek, Sheraz
Gul, Louis Maes, Geert Jan Sterk, Rob Leurs, David G. Brown, and Koen Augustyns
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00985 • Publication Date (Web): 20 Mar 2020
Downloaded from pubs.acs.org on March 21, 2020
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Lead optimization of phthalazinone phosphodiesterase inhibitors as novel antitrypanosomal
compounds
a
b#
a
a,c
Irene G. Salado , Abhimanyu K. Singh , Carlos Moreno-Cinos , Guna Sakaine , Marco
d
a
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d
d
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Siderius , Pieter Van der Veken , An Matheeussen , Tiffany van der Meer , Payman Sadek ,
f
e
d
d
b
a
Sheraz Gul , Louis Maes , Geert-Jan Sterk , Rob Leurs , David Brown , Koen Augustyns *
a
Laboratory of Medicinal Chemistry, University of Antwerp, Universiteitsplein 1, B-2610 Antwerp,
b
c
Belgium. School of Biosciences, University of Kent, Canterbury, Kent, CT2 7NJ, United Kingdom.
d
Current address: Latvian Institute of Organic Synthesis, LV-1006 Riga, Latvia. Medicinal Chemistry,
Amsterdam Institute of Molecules, Medicines & Systems, Vrije Universiteit Amsterdam, Faculty of
e
Science, 1081 Amsterdam, The Netherlands. Laboratory for Microbiology, Parasitology and Hygiene
f
(
LMPH), University of Antwerp, Universiteitsplein 1, B-2610 Antwerp, Belgium. Fraunhofer-IME SP,
#
Schnackenburgallee 114, Hamburg, Germany. Current address: Rega Institute for Medical Research,
KU Leuven, 3000 Leuven, Belgium. *Corresponding author: Koen Augustyns, email address:
koen.augustyns@uantwerpen.be
ABSTRACT
Human African trypanosomiasis is causing thousands of deaths every year in the rural areas of
Africa. In this manuscript we describe the optimization of a family of phtalazinone derivatives.
Phosphodiesterases have emerged as attractive molecular targets for a novel treatment for a
variety of neglected parasitic diseases. Compound 1 resulted to be a potent TbrPDEB1 inhibitor
with interesting activity against T. brucei in a phenotypic screen. Derivative 1 was studied in
an acute in vivo mouse disease model but unfortunately showed no efficacy due to low
metabolic stability. We report structural modifications to achieve compounds with an improved
metabolic stability while maintaining high potency against TbrPDEB1 and T. brucei.
Compound 14, presented a good microsomal stability in mouse and human microsomes and
provides a good starting point for future efforts.
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INTRODUCTION
Human African trypanosomiasis, HAT, or African sleeping sickness, is caused by the
unicellular parasites of the species Trypanosoma brucei (T.brucei). The vector that propagates
this disease is the tsetse fly, found mainly in rural Africa. The disease is divided in two different
subtypes: gambian sleeping sickness (Trypanosoma brucei gambiense) and rhodesian sleeping
sickness (Trypanosoma brucei rhodesiense). The first one is chronic and can take years until
fatality while the second one is an acute illness causing death within weeks or months after the
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infection. In both forms, the late stage of the disease is characterized by the entrance of the
parasite into the central nervous system with fatal consequences if untreated. Although the two
forms affect human beings, 98% of the cases of sleeping sickness are caused by T.brucei
gambiense, which predominates in central and western African countries.2
HAT belongs to the family of NTDs, neglected tropical diseases, these sicknesses contribute to
a high level of morbidity and mortality in the third world countries (550000 deaths per year).3
The investment from pharmaceutical companies to achieve new molecules capable to cure those
diseases is not sufficient. For this reason research from public institutions to develop new drugs,
which are safe and easy to administer is crucial.
To develop new molecules able to cure this disease, a public-private consortium has been
established funded by the European Union. This project named ‘Parasite-specific cyclic
nucleotide phosphodiesterase inhibitors to target neglected parasitic diseases’ has validated
parasitic cyclic nucleotide phosphodiesterases as valuable targets for drug discovery and has
identified parasite-specific features of the PDE active sites.4
PDEs have emerged as attractive molecular targets for a novel treatment for a variety of
neglected parasitic diseases, including African trypanosomiasis, Chagas disease, and malaria.5
6
The genome of T.brucei presents five trypanosomal cyclic nucleotide phosphodiesterases. For
7
sleeping sickness, TbrPDEB1 and TbrPDEB2 were genetically validated as drug targets. These
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enzymes are different from the human form since they have a sub-pocket, named as the parasite
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specific pocket or the P-pocket, in the substrate binding site. This pocket potentially offers an
area that can be targeted to impart selectivity, avoiding side effects related to inhibition of the
human off-targets. For instance, inhibition of hPDE4 yields side effects such as nausea and
1
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emesis. Moreover, inhibition of human PDE4 (hPDE4) attenates the immune system via
inhibition of TNF which is certainly undesirable in rural Africa. 10, 11, 12
In previous investigations carried out by de Koning et al, a phenylpyridazinone derivative was
discovered from a high throughput screening campaign (PPS54019, compound A) and was
1
3
validated as TbrPDEB1 and TbrPDEB2 inhibitor. This compound (now renamed as NPD-
1
4
001) was previously designed as hPDE4 inhibitor. It has been used as hit compound to develop
new families of TbrPDE inhibitors with improved selectivity over hPDE4 isoforms.15, 16 From
an unpublished library of compounds belonging to this family, compound 1 (Fig. 1) was
selected as one of the more promising ones because it is a potent TbrPDEB1 inhibitor with
interesting activity against T. brucei in a phenotypic assay and low cytotoxicity. In a proof of
concept study, compound 1 was used in an acute in vivo mouse disease model on swiss mice
4
infected with 10 trypanosomes T. brucei at a daily dosing of 50 mg/kg i.p. or 50 mg/kg p.o.
However it showed no efficacy because of its low metabolic stability (Fig.1). In this paper, we
report structural modifications to achieve compounds with an improved metabolic stability
while maintaining high potency against TbrPDEB1 and T. brucei.
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OMe
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pIC (Tbr)
pIC (MRC5) pKi(TbrPDEB1) pIC (hPDE4)
mouse microsomal
stability
5
0
50
50
% parent compound
remaining
6
.35
4.35
7
9
6.7% after 30 min
Figure 1. Structure of the TbrPDEB1 inhibitor, compound 1. This compound was evaluated in
a phenotypic panel including T. brucei and MRC5 to study if the compound is selective over
human cells. In addition, it was evaluated on the isolated enzymes TbrPDEB1 and hPDE4. The
mouse microsomal stability reveals the need to improve the stability of this compound.
To get a better understanding of the metabolic hot spots in compound 1, the theoretical
metabolites resulting from phase 1 metabolism were predicted by using the Meteor program
(Figure 2).
OMe
Oxydative O-demethylation
OMe
N
Allylic Hydroxylation
N
O
O
N
N
O
O
Hydrolysis of Acyclic Carboxylic Amides
Hydroxylation alpha to a Carbonyl Group
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Figure 2. Main metabolic hotspots predicted by the Meteor program.
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RESULTS
Chemistry
Based on the metabolic hotspots of compound 1, we designed different molecules to improve
the metabolic stability (Fig. 3).
Figure 3. Derivatives of compound 1, designed to improve metabolic stability.
OH
OCHF2
OMe
F
F
OCHF2
HO
HO
Cl
F
F2HCO
OMe
N
N
NH2
N
NH2
N
N
N
N
N
N
N
N
MeO
N
N
N
N
H
N
H2N
N
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O
O
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N
N
N
N
N
N
O
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N
S
O
N
S
S
H2N
N
N
H2N
N
N
NH2
O
O
O
O
O
O
N
Compound 1
S
N
N
O
O
N
O
O
NH2
Modification of the methoxy groups to avoid O-demethylation was carried out by introducing
fluorine or chlorine atoms. Changes in the core of the molecule were also performed to study
the importance of the phthalazinone scaffold in the activity and stability. The saturated form of
the phthalazinone was obtained to avoid allylic hydroxylation. Diverse heterocycles were
introduced mimicking a purine moiety, to benefit from an active uptake in the parasite by the
nucleoside P2 transport system.17-19
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Different synthetic approaches were followed depending on the final compound. In general,
(4aS, 8aR)-4-(3,4-dimethoxyphenyl)-2-(piperidin-4-yl)-4a,5,8,8a-tetrahydrophthalazin-1(2H)-
one was used as starting material (Scheme 1), to introduce the different heterocycles.
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OMe
OMe
MeO
MeO
i
N
N
N
N
O
NH
O
N
R
Comp.
R
Yield (%)
69
S
N
2
N
N
N
3
4
15
26
N
H
H2N
N
NH2
N
N
NH2
S
5
6
7
N
N
5
N
N
60
81
N
N
N
NH2
NH2
8
9
48
36
N
N
N
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N
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S
H2N
H2N
N
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N
Scheme 1. Reaction conditions: i) heteroaryl chloride, Et N, K CO , DMF, 153 °C, 2 – 18 h.
3
2
3
Another modification concerning this part of the molecule is the ring size of the imide and its
substitution pattern. These compounds were synthesized as previously described (Scheme 2).20
OMe
OMe
OMe
MeO
MeO
MeO
O
i
Cl
ii
N
N
N
N
N
N
Cl
O
NH
O
N
O
N
Cl
R
O
O
13
14 - 17
Comp.
R
Time (h)
18
T (°C)
20
Yield (%)a
50
O
N
1
1
1
4
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O
O
N
18
18
20
60
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O
O
Ph
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a
Calculated yield for the last step of the reaction.
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Scheme 2. Reaction conditions: i) Et N, K CO , THF, rt, 2 h. ii) imide derivative, K CO , DMF.
3
2
3
2
3
It is well known that methoxyphenyl derivatives can be rapidly metabolized through oxidative
O-demethylation during Phase I metabolism. In an attempt to avoid this, the two methoxy
substituents were replaced by different halogen atoms or by introducing a difluoromethoxy
moiety. Fluorine atoms have been used extensively in medicinal chemistry programmes to
improve ADME properties of molecules.21-23
The introduction of a fluorine or chlorine atom in the phenyl ring was performed following the
reactions described in Scheme 3.14
Y
Y
X
X
X
X
Y
Y
i
ii
iii
N
N
N
N
R
O
COOH
O
NH
O
N
R2
1
8, 19, 20
21, 22, 23
24 - 32
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Yield (%)a
Comp.
X
Y
R
N
N
N
N
2
2
4
5
Cl
OMe
80
S
S
S
H2N
H2N
1
1
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H
F
F
F
F
F
16
32
22
N
26
27
H2N
N
N
S
N
O
N
2
8
H
H
H
F
F
F
F
F
F
26
26
23
44
5
O
O
O
N
29
O
O
O
O
N
N
3
3
3
0
1
2
O
O
O
O
O
N
F
O
O
a
Calculated yield for the last step of the reaction.
Scheme 3. Reaction conditions: When X = Cl, Y = OMe and R = H: i) AlCl , DCM, rt,
3
overnight; ii) 4-hydrazinylpiperidine dihychloride, Et N, EtOH, 80 °C, overnight; iii) 4-chloro-
3
2
-aminothieno[2,3-d]pyrimidine, K CO , DMF, 120 °C, 16 h.
2 3
When X = H and Y = F or X = Y = F, R = MgBr: i) (3aR,7aS)-3a,4,7,7a-
tetrahydroisobenzofuran-1,3-dione, THF, rt, 16 h; ii) 4-hydrazinylpiperidine dihydrochloride,
Et N, EtOH, 80 °C, 16 h; iii) heteroaryl chloride, K CO , DMF, 120 °C, 3 – 16 h or 2-
3
2
3
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chloroacetyl chloride, Et N, DCM, 0 °C, 30 min followed by 2,6-dione derivative, K CO ,
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7
8
9
0
Furthermore, difluoromethoxy moieties were explored instead of the methoxy groups. This
2
4
reaction was performed as previously described (Scheme 4). A demethylation in presence of
BBr was carried out, followed by difluoromethylation of the diphenol derivative.
3
OMe
OH
OCHF2
OCHF2
OCHF2
OCHF2
OMe
OH
i
ii
iii
N
N
N
N
N
N
N
N
O
N
O
N
O
N
O
N
Cl
Cl
Cl
R
O
O
O
O
13
33
34
35, 36
Comp.
R
Yield (%)a
10
O
N
O
3
3
5
6
O
N
O
6
a
Calculated yield for the last step of the reaction.
Scheme 4. Reaction conditions: i) BBr , DCM, -40 °C, 2 h; ii) diethyl
3
(bromodifluoromethyl)phosphonate, KOH, acetonitrile/water, -40 °C, 45 min; iii) 2,6-dione
derivative, K CO , DMF, 120 °C, 16 h.
2
3
This modification was also performed for some of the heterocycle substituted derivatives,
leading to three different compounds (Scheme 5).
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Journal of Medicinal Chemistry
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2
3
4
5
6
7
8
9
OMe
OH
OH
X
OMe
Y
i
ii
N
N
N
N
N
N
S
S
S
O
N
O
N
O
N
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
N
N
N
N
N
N
37
38, 39
Comp.
X
Y
Yield (%)a
3
3
7
8
OH
OH
99
2
OCHF2
OCHF2
39
OCHF2
OH
2
a
Calculated yield for the last step of the reaction.
Scheme 5. Reaction conditions: i) BBr , DCM, -40 °C, 2 h; ii) diethyl
3
(
bromodifluoromethyl)phosphonate, KOH, acetonitrile/water, 45 – 120 min at -40 °C and 2 –
1
6 h at rt
The phthalazinone core was also replaced aiming to remove the different stereocenters and to
improve synthetic feasibility. The imidazotriazinone was synthesized following the procedure
2
5
already described. After the reaction of the imidazole and the acyl chloride, the cyclization of
the compound in the presence of p-toluensulfonic acid was carried out. Once the new core
scaffold was obtained, the molecule was modified as previously (Scheme 6).
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2
3
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5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
OMe
OMe
OMe
OMe
MeO
OMe
OMe
OMe
OMe
OMe
N
i
N
ii
1) iii
2) iv
v
Cl
N
N
N
N
N
N
H
N
N
N
N
O
H
O
N
NH
N
N
O
O
NH
O
N
S
40
41
42
43
N
N
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
NH2
Scheme 6. Reaction conditions: i) Et N, pyridine, rt, 16 h. ii) p-toluensulfonic acid, toluen,
3
diphenylether, ethylhydrazinecarboxylate, 110 °C, 5 h. iii) tert-butyl 4-bromopiperidine-1-
carboxylate, NaH, DMF, 166 °C, 48 h. iv) TFA, DCM, rt, 16 h. v) 4-chlorothieno[2,3-
d]pyrimidin-2-amine, Et N, DCM, rt, 16 h.
3
Because the double bond of the phthalazinone can be sensitive to epoxidation and allylic
oxidation in Phase I metabolism, the most promising compounds were also modified by
removing this double bond by hydrogenation in the presence of Pd(C) (Scheme 7).
R
R
R
R
i
N
N
N
N
O
N
O
N
R2
R2
4
4-49
Comp.
R
R2
Yield (%)
12
N
4
4
4
4
5
6
OMe
OMe
F
S
H2N
N
O
N
80
77
O
O
O
N
O
O
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Journal of Medicinal Chemistry
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2
3
4
5
6
7
8
9
O
N
4
7
F
95
O
O
O
N
48
OMe
56
79
O
O
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
O
O
N
4
9
OCHF2
Scheme 7. Reaction conditions: i) H , Pd(C), methanol, r.t, 1 – 72 h.
2
In order to decrease the molecular weight, two other scaffolds were also explored. A
pyridazinone was synthesized as described in scheme 8. The already described pyrazolone core
was obtained using the synthesis described in scheme 9.26
OMe
OMe
OMe
OMe
OMe
OMe
i
ii
N
NH
N
N
N
N
S
O
O
NH
O
N
N
N
50
51
NH2
Scheme 8. Reaction conditions: i) tert-butyl-4-bromopiperidine-1-carboxylate, NaH, DMF,
53 °C, 2 days. ii) 4-chlorothieno[2,3-d]pyrimidin-2-amine, K CO , Et N, DMF, 130 °C, 16 h.
1
2
3
3
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2
3
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5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
OMe
OMe
OMe
OMe
MeO
MeO
MeO
ii
MeO
i
iii
OMe
O
N
N
Boc
N
Cl
NH
O
N
O
O
O
52
53
54
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
iv
OMe
MeO
OMe
S
MeO
v
N
N
N
N
N
NH
N
N
O
O
56
55
Scheme 9. Reaction conditions: i) methyl isobutyrate, LDA, THF, −45 °C, 30 min, then added
to 3,4-dimethoxybenzoyl chloride, from −55 °C to rt, 16 h; ii) N H , EtOH, 80 °C, 16 h; iii)
2
4
tert-butyl-4-bromopiperidine-1-carboxylate, NaH, DMF, 153 °C, 16 h; iv) TFA, DCM, rt, 3 h;
v) 4-chlorothieno[3,2-d]pyrimidine, NaH, DMF, 153 °C, 16 h.
The last modification was the replacement of the piperidine ring by an azetidine ring using a
very similar synthetic pathway (Scheme 10).
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
i
ii
iii
N
NH
N
N
N
N
N
N
N
NH
N
O
O
O
O
Boc
R
5
7
58
59-63
Yield (%)a
Comp.
R
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Journal of Medicinal Chemistry
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2
3
4
5
6
7
8
9
N
5
6
6
6
9
0
1
2
3
S
H N
N
2
S
N
4
N
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
N
58
58
O
O
O
N
O
O
O
N
6
3
80
O
O
a
Calculated yield for the last step of the reaction.
Scheme 10. Reaction conditions: i) tert-butyl 3-bromoazetidine-1-carboxylate, NaH, DMF, 153
C, 16 h ii) TFA, DCM, rt, 16 h; iii) heteroaryl chloride, NaH, DMF, 153 °C, 4 – 16 h or 2-
chloroacetyl chloride, Et N, DCM, 0 °C, 30 min followed by 2,6-dione derivative, K CO , 100
°
3
2
3
°C, 3 – 16 h.
In vitro evaluation of the compounds against TbrPDEB1, hPDE4, T. brucei
All obtained compounds were evaluated against a parasite panel including Trypanosoma
brucei, Trypanosoma cruzi, Leishmania Infantum, Plasmodium falciparum. Cytotoxicity was
determined against a human cell line (MRC5) and peritoneal macrophages from mouse (PMM)
(
Fig. 4 and Table 1, full panel in supplementary information).
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2
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5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Furthermore, most of the compounds were evaluated on the purified catalytic domain of
TbrPDEB1 enzyme. As a measure of the selectivity, inhibition of the catalytic domain of the
human PDE4 for compounds with pKi ˃ 6 on TbrPDEB1 was determined.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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Page 17 of 75
Journal of Medicinal Chemistry
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2
3
4
5
6
7
8
9
X OMe
OMe
Y
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
A
N
B
C
N
O
O
N
N
O
O
Figure 4. Scheme of the general structure and the parts that have been modified.
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1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
Table 1. Synthesized analogues of compound 1 and their evaluation against T.brucei, MRC5, TbrPDEB1 and hPDE4.
pKi
pKi
pIC50
pIC50
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
Comp.
X
Y
A
B
R
(
hPDE4-
(T.bruc)a
(MRC5)a,b
(
TbrPDEB1-CD)a
CD)a
O
O
N
N
1
2
3
4
5
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
N
6.35
6.43
5.77
6.06
5.75
4.35
5.14
4.75
5.13
4.19
7.36
7.18
6.98
6.19
6.47
9.91
9.63
9.06
8.61
9.17
N
N
N
N
N
O
O
N
N
S
N
N
O
O
O
O
N
N
N
N
N
H
H N
N
2
N
N
N
N
NH2
N
N
N
N
S
NH2
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Journal of Medicinal Chemistry
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2
3
4
5
6
7
8
9
N
N
N
N
6
7
8
9
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
5.77
6.58
5.44
5.73
5.64
6.33
6.09
4.19
5.52
5.41
4.76
4.19
4.19
5.11
6.19
7.41
6.59
6.96
6.16
7.37
7.61
8.61
9.65
9.29
9.50
8.59
9.45
9.81
N
N
N
N
N
N
N
O
O
O
O
O
O
O
N
N
N
N
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
N
NH2
N
N
N
N
NH2
N
N
N
N
N
N
N
1
0
1
2
S
N
N
N
1
1
S
H N
N
2
N
N
N
H2N
N
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1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
O
O
N
N
1
1
4
5
OMe
OMe
OMe
OMe
N
O
5.73
5.74
4.19
4.19
7.68
7.12
10.21
9.77
N
N
O
O
O
O
N
N
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
N
O
Ph
O
O
N
N
16
OMe
OMe
4.19
4.19
7.01
9.50
N
N
O
O
O
O
N
N
1
2
2
7
4
5
OMe
Cl
OMe
OMe
F
6.32
5.7
4.5
7.51
6.08
9.81
7.52
nd
N
N
N
N
O
O
O
O
N
N
N
4.19
4.19
S
S
H N
N
N
2
N
N
N
N
H
5.68
˂5.03
H N
2
N
N
2
6
F
F
5.72
4.19
˂5.08
nd
N
S
H N
N
2
O
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Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
N
N
S
N
2
2
7
8
F
H
H
H
F
F
F
F
F
F
F
4.88
4.19
4.99
4.49
4.48
4.42
4.53
4.19
4.39
4.19
˂5.25
7.11
nd
9.92
nd
N
N
N
N
N
N
O
O
O
O
O
O
O
N
N
N
O
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
O
O
N
N
29
30
˂5.02
˂4.78
˂5.34
N
O
O
O
N
N
N
O
nd
O
O
N
N
3
1
nd
N
O
O
O
N
N
3
3
2
5
F
N
O
4.32
6
4.51
4.19
<5,01
6.39
nd
N
N
O
O
O
O
N
N
OCHF2 OCHF2
N
O
9.52
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1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
O
O
N
N
3
6
OCHF2 OCHF2
5.12
4.19
6.11
8.79
N
N
O
O
N
N
S
N
3
3
3
4
7
8
9
1
OH
OH
5.29
5.17
5.47
4.19
4.49
4.19
4.6
6.05
7.20
nd
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
N
N
N
N
O
O
O
O
O
O
N
N
S
N
OCHF2 OCHF2
4.19
4.19
4,19
4,19
4,19
˂5.67
˂5.78
˂4.50
˂4.50
˂4.74
N
N
N
S
N
OCHF2
OMe
OH
nd
N
N
N
N
N
OMe
OMe
OMe
N
N
N
N
H
H
nd
N
N
42
OMe
H
nd
N
N
N
N
N
4
3
OMe
nd
S
H N
2
N
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N
N
N
4
4
4
4
4
5
6
7
OMe
OMe
F
OMe
OMe
F
6.02
5.82
4.34
5.12
4.54
5.07
4.37
4.19
7.03
9.18
nd
N
N
N
N
S
H N
N
2
O
O
O
O
O
N
N
O
˂5.01
˂4.65
˂5.16
N
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
O
O
O
N
N
N
nd
O
O
N
N
O
F
F
nd
N
O
O
O
N
N
4
4
8
9
OMe
OMe
N
5.71
5.19
4.25
4.19
7.07
9.79
nd
N
N
O
O
O
O
O
O
N
N
OCHF2 OCHF2
N
˂5.80
N
N
N
5
1
OMe
OMe
5.11
4.95
˂5.43
nd
N
S
H N
2
N
O
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
N
N
S
N
5
6
OMe
OMe
OMe
OMe
4.49
5.11
4.6
˂5.69
nd
N
O
N
N
N
N
59
4.19
6.56
8.93
N
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
H N
2
N
S
O
O
O
N
N
S
N
O
6
6
0
1
OMe
OMe
OMe
OMe
4.82
5.74
4.19
4.19
6.54
6.99
9.25
9.41
N
N
N
O
N
N
N
O
O
N
N
O
O
6
6
2
3
OMe
OMe
OMe
OMe
5.94
5.6
4.19
4.19
7.04
6.77
9.07
9.17
N
N
O
O
O
N
N
N
N
O
O
a
b
Each value is the mean of at least two independent determinations. Cytotoxicity measurement using human lung fibroblast MRC-5 SV cells.
2
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Structure-activity relationship (SAR)
In general, the compounds that were active in the phenotypic panel against T. brucei also
presented potency on the isolated catalytic domain of the TbrPDEB1 enzyme. This suggests
that the compounds are working through PDE inhibition. There are also some compounds that
do not present activity in the phenotypic panel but present potency on the enzyme (compounds
16 and 28) which might be caused by lack of membrane permeability.
1
1
1
1
1
1
1
1
1
1
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0
Among all the modifications that have been introduced, we observe that the preferred core
scaffold is the saturated or unsaturated phthalazinone (compound 11 vs 43 or 51). As linker
between the phthalazinone and the imide or heterocycle, a piperidine is clearly favored over an
azetidine with a 3-10 fold loss in potency against both T. brucei and TbrPDEB1 (compound 1
vs 61, 12 vs 59 or 17 vs 62).
Regarding the substituents in the phenyl ring, the methoxy groups are the most favourable ones
(Compound 1 vs 32 or 35). When the OMe moiety is substituted by Cl, the potency on PDEB1
decreases about 20 folds (compound 11 vs 24). When the substitution is H,F or F,F, compounds
are inactive on PDEB1 except compound 28; however, this compound did not show activity on
the antiparasitic panel. On the other hand, OCHF substitution, maintains antiparasitic activity
2
(compound 35 vs 1) although a one fold loss of activity on PDEB1 is observed. Furthermore,
when the chain is longer (compound 36 vs 17) the potency on both PDEB1 and on the parasitic
panel decreases when the OCHF is present. For the heterocyclic ring attached to the
2
piperidinyl, the thieno-pyrimidine-2-amino (Compound 11) maintains the activity on the
phenotypic panel and presents a lower pIC against the human cell line MRC5 comparing with
5
0
the hit compound 1, it is also as potent on PDEB1 as compound 1. When an amino substituent
is added to the quinazoline ring (compound 7 vs 12, and 4 vs 6) the activity on PDEB1 increases
or is maintained while the activity on the phenotypic panel decreases. Regarding the imide
moiety, when the 6-membered ring is changed by a five membered ring (compound 1 vs 15)
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the phenotypic activity decreases while the potency on PDEB1 is kept. Also, when another six-
member ring is fused the activity is higher on PDEB1 and it is maintained on the phenotypic
panel. This behavior is only valid when the substitution on the phenyl ring is OMe, OMe
0
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(compound 17 vs 31 or 1 vs 32).
Metabolic stability
Metabolism plays an important role in the development of a drug candidate; it impacts on
pharmacokinetic parameters such as oral bioavailability, clearance and half-life. This will affect
the efficacy and toxicology of the drug due to changes in the concentrations within plasma and
tissues.27
To evaluate Phase I and Phase II metabolism of the compounds, mouse and human microsomes
were used. Compounds with structural differences have been selected to be studied in mouse
and microsomes. With the mouse microsomes results, some of those compounds with
promising stability or important structural changes were selected to be evaluated in human
microsomes. In Figure 5, mouse and human microsomal stabilities at different time points are
represented, full data with standard deviation are shown in the Supplementary Information.
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B
Figure 5. A) Mouse microsomal stability of selected compounds, B) Human microsomal
stability of selected compounds.
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From these results, we can conclude that the different regions of the molecules contribute to
differences in stability. In general, the saturated phthalazinone core is more stable than the
unsaturated one (compounds 48 vs 1, 47 vs 31 and 49 vs 35), the piperidinyl also increases the
stability comparing it to the azetidine residue (compound 17 vs 62). Regarding the substituents
of the phenyl ring, generally, the substitution of the methoxy group by difluoromethoxy
moieties or fluorine atoms also enhances the stability (compound 5 vs 25, 26, 27, 38, and 39),
suggesting that the catechol moiety suffers from oxidative O-demethylation. Also the imide
ring size and substitution pattern influences metabolic stability.
0
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Compound 14 presented a good metabolic stability profile in mouse (43% of parent compound
remaining after 30 min) and human microsomes (89% after 30 min). It also combines potency
on the parasite (pIC (Tbr) = 5.73) and on the enzyme (pK (TbrPDEB1) = 7.68) with low
5
0
i
cytotoxicity. However, it completely lacks selectivity over the human enzyme (pK (hPDE4) =
i
1
0.21). Since this will result in off-target effects an improved selectivity based on structure-
based drug design is necessary before we can proceed to a next in vivo proof of concept study
in the acute mouse model of T. brucei.
X-ray analysis of selected inhibitors
To get a better understanding of their mode of interaction at atomic level, crystal structures of
selected derivatives (Figure 6) bound to TbrPDEB1 and hPDE4D catalytic domains were
determined by X-ray. We have chosen inhibitors which have shown good potency in
phenotypic assays or those with significant structural modifications, together with compound
1
4 as the most stable compound. Since the selected inhibitors fail to demonstrate any selectivity
over human off-target enzyme hPDE4D, we hoped structural findings may provide us design
ideas to help improve these and other chemically similar inhibitors.
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Structures of TbrPDEB1 bound to inhibitors 1, 11, 12, 14 and 35 were obtained in a resolution
range between 1.6 to 2.5 Å (Supplementary information). In all cases, (4aS,8aR)-enantiomer
form of the inhibitors were observed in the crystal structures and they display almost identical
binding modes to the parasitic enzyme (Figure 7). The phthalazinone core occupies the area
near the metal site while the substituted phenyl group engages the hydrophobic clamp, also
1
1
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7
known as the aromatic clamp in TbrPDEB1 , residues Val840 and Phe877. In addition to the
aromatic stacking, the ring substitutes further engage TbrPDEB1 via hydrogen bond
interactions involving Gln874, a residue strictly conserved across phosphodiesterase family.
The different tail groups orient themselves towards a space lined by helix-15 and the M-loop7
and points slightly away from the parasite specific P-pocket (Figure 7). They are largely
stabilised by hydrophobic interactions from residues located on helix-15 and the M-loop,
specially by Met861 and, up to some extent, Phe880.
We have also determined structures of the hPDE4D catalytic domain in complex with the same
set of inhibitors (Supplementary information). Their overall binding mode in hPDE4D is highly
similar to that in TbrPDEB1 with strict conservation of key hydrophobic clamping (with
Phe372 and Ile336; hPDE4D numbering) and H-bond interactions (with Gln369; hPDE4D
numbering) in the two enzymes (Supplementary information). Despite the overall similarity,
two important observations can be deduced from the determined inhibitor-bound structures in
TbrPDEB1 and hPDE4D, which may have implications in future design works; first, the tail,
stabilising hydrophobic stacking from Phe880 in TbrPDEB1, is absent in hPDE4D with the
substitution of Phe880 by Tyr375 in the equivalent position. Secondly, the tail is localized close
to the parasite specific P-pocket in TbrPDEB1. In TbrPDEB1, interaction with Phe880 may be
improved by the introduction of aromatic moieties to the existing heterocyclic tail while
maintaining the current vector direction, which may result in improved selectivity. On the other
hand, targeting the tail towards the parasite specific P-pocket may also result in improved
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