Structure-Activity Relationship of Phenylpyrazolones against Trypanosoma cruzi

Article abstract of DOI:10.1002/cmdc.202000136

Chagas disease is a neglected parasitic disease caused by the parasitic protozoan Trypanosoma cruzi and currently affects around 8 million people. Previously, 2-isopropyl-5-(4-methoxy-3-(pyridin-3-yl)phenyl)-4,4-dimethyl-2,4-dihydro-3H-pyrazol-3-one (NPD-0227) was discovered to be a sub-micromolar inhibitor (pIC₅₀=6.4) of T. cruzi. So far, SAR investigations of this scaffold have focused on the alkoxy substituent, the pyrazolone nitrogen substituent and the aromatic substituent of the core phenylpyrazolone. In this study, modifications of the phenyldihydropyrazolone scaffold are described. Variations were introduced by installing different substituents on the phenyl core, modifying the geminal dimethyl and installing various bio-isosteres of the dihydropyrazolone group. The anti T. cruzi activity of NPD-0227 could not be surpassed as the most potent compounds show pIC₅₀ values of around 6.3. However, valuable additional SAR data for this interesting scaffold was obtained, and the data suggest that a scaffold hop is feasible as the pyrazolone moiety can be replaced by a oxazole or oxadiazole with minimal loss of activity.

Full text of DOI:10.1002/cmdc.202000136

Accepted Article
Title: Structure activity relationship of phenylpyrazolones against
Trypanosoma cruzi
Authors: Maarten Sijm, Geert Jan Sterk, Guy Caljon, Louis Maes, Iwan
J.P. de Esch, and Rob Leurs
This manuscript has been accepted after peer review and appears as an
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of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
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to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: ChemMedChem 10.1002/cmdc.202000136
Link to VoR: https://doi.org/10.1002/cmdc.202000136
01/2020

10.1002/cmdc.202000136
ChemMedChem
Key words:
Trypanosoma cruzi, Benznidazole, Neglected parasitic disease, Phenylpyrazolones, Structure-
activity relationships
Table of contents entry:
In a search for more active compounds against Trypanosoma cruzi the SAR of a series of
phenylpyrazolones was further investigated. Previous research yielded NPD-0227 a
phenylpyrazolone with a pIC50 of 6.4 against T. cruzi. This work continues this investigation with
substituents on the phenyl core as well as modifications and replacements of the
dihydropyrazolone group.
1
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10.1002/cmdc.202000136
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Structure activity relationship of phenylpyrazolones against
Trypanosoma cruzi
Maarten Sijm1, Dr. Geert Jan Sterk1, Prof. Guy Caljon2, Prof. Louis Maes2, Prof. Iwan J.P. de
Esch1 and Prof. Rob Leurs1*.
1
Division of Medicinal Chemistry, Faculty of Sciences, Amsterdam Institute for Molecules,
Medicines and Systems (AIMMS), Vrije Universiteit Amsterdam, De Boelelaan 1108,
1081 HZ, Amsterdam, The Netherlands
2
Laboratory for Microbiology, Parasitology and Hygiene (LMPH), University of Antwerp,
Universiteitsplein 1, 2610, Antwerpen, Belgium,
2
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10.1002/cmdc.202000136
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Abstract
Chagas disease is a neglected parasitic disease caused by the parasitic protozoan Trypanosoma
cruzi and currently affects around 8 million people. Previously, 2-isopropyl-5-(4-methoxy-3-
(pyridin-3-yl)phenyl)-4,4-dimethyl-2,4-dihydro-3H-pyrazol-3-one (NPD-0227) was discovered
as a submicromolar inhibitor (pIC50 = 6.4) of T. cruzi. So far, the SAR investigation on this scaffold
focused on the alkoxy substituent, the pyrazolone nitrogen substituent and the aromatic substituent
of the core phenylpyrazolone. In this study, modifications of the phenyldihydropyrazolone scaffold
are described. Variations were introduced by installing different substituents on the phenyl core,
modifying the geminal dimethyl and installing various bio-isosteres of the dihydropyrazolone
group. The anti T. cruzi activity of NPD-0227 could not be surpassed as the most potent
compounds show pIC50 values of around 6.3. However, valuable additional SAR data for this
interesting scaffold was obtained and the data suggest that a scaffold hop is feasible since the
pyrazolone moiety can be replaced by a oxazole or oxadiazole with minimal loss of activity.
3
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Introduction
The protozoan parasite Trypanosoma cruzi is the causative agent of Chagas disease. This
parasite is transmitted by the triatomine bug vector that used to be endemic only in Latin-America,
but is now slowly moving towards North-America as well.[1] It is estimated that currently around
8 million people are infected and many more are at risk of being infected.[2] Upon infection, the
disease first enters into an acute phase in which symptoms are generally mild, fever-like,
uncharacteristic or even absent.[3] As a result, Chagas disease is often not diagnosed in this stage
and proceeds untreated towards a chronic phase.[4] Although the initial symptoms will disappear
after a few weeks, the parasite will persist and evolve to an indeterminate symptomatic chronic
phase.[5] This ultimately develops into progressive chronic cardiomyopathy in 30% of the patients
while another 10% develops neurological, digestive or mixed clinical symptoms. Although the
remaining 60% does not develop any symptoms, they remain infective if untreated and therefor
remain an infection risk.[6]
While currently only two drugs are on the market, these are far from optimal as they have long
treatment regimes, cause adverse drug effects and have limited efficacy during the chronic phase.[7]
Benznidazole (1, figure 1) and nifurtimox (2) are nitro-heteroaromatic drugs that were developed
in the late 1960’s. Their efficacy in the acute phase is widely accepted, however, their effectiveness
during the chronic phase is still under debate.[8] In addition, they are known to cause adverse drug
effects such as weight loss, depression and amnesia.[7c, 9]
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Figure 1 Current drugs for Chagas disease: benznidazole (1) and nifurtimox (2). NPD-0227 (3)
was obtained from earlier hit optimization against T.cruzi.[10]
With these limitations of the current drugs, it is clear that the need for novel chemotherapies is
high. The drug discovery pipeline of Chagas disease has a few potential clinical candidates, mostly
from private-public partnerships originating from the last decade. Meanwhile, drug resistance has
been reported for both benznidazole (1, figure 1) and nifurtimox (2) in in vitro strains.[11] Within
the PDE4NPD (PhosphoDiEsterase inhibitors for Neglected Parasitic Disease) consortium, an
European Union funded public-private partnership to target several neglected tropical diseases,
Chagas disease was one of the focus points. We previously reported the discovery of NPD-0227
(3, figure 1), a sub-micromolar inhibitor of T. cruzi and modifications of this hit have been
described with focus on substituents of the aromatic substituent (R1), pyrazolone nitrogen (R2) and
the the alkoxy substituent (R3).[10] In this previous work, modifications on R2 and R3 did not result
in increased activities. To further investigate the structure-activity-relationships of this scaffold,
the present work focused on the effect of modifications of the core phenyl ring replacing the gem-
dimethyl moiety of the dihydropyrazolone and replacement of the dihydropyrazolone ring with
various hetero- cyclic and aromatic moieties.
5
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Chemistry
To introduce different substituents on the phenyl moiety of the phenyldihydropyrazole scaffold,
the previously reported methodology by Sijm et al. was used, starting with the desired benzoic
acids (9-19, scheme 1).[10] Several benzoic acids had to be prepared from their precursors.
Bromination of the non-halogenated benzoic acids 4-7 resulted in the desired brominated
analogues (10-13). During the bromination which led to benzoic acid 13 (5-bromo-4-methoxy-2-
methylbenzoic acid), two regioisomers were formed in a 2:1 ratio (3-Br : 5-Br). Quenching of the
mixture with a sodium thiosulfate solution (~1M) in water gave a biphasic mixture with substantial
precipitation. Filtering off the solids resulted in collection of a single (3-Br) isomer (13). Despite
substantial recrystallization efforts the 5-Br-isomer could not be isolated with sufficient purity.
Subsequent oxidation of aldehyde 8 with potassium permanganate yielded benzoic acid 9, while
the remaining benzoic acids (14-19) were bought from commercial suppliers. The obtained
benzoic acids (9-19) were converted to the corresponding acid chlorides with oxalyl chloride,
which was followed by addition of the lithium enolate of methyl isobutyrate, yielding keto-esters
21-28 and 30-32. Exception was the 3-bromo-2-fluoro-3-methoxy keto-ester (29) which was
obtained via the Friedel-Craft acylation of anisole 20. The obtained keto-esters (21-32, scheme 1)
were subsequently condensed with hydrazine to give the core phenyl-dihydropyrazolone scaffold
with various substituents on the phenyl ring (33-44).
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Scheme 1 Preperation of pyrazolones 57-68 (table 1) with modifications on the central phenyl
ring. Reagents and conditions: a: KMnO4, KPO4H2, t-BuOH, rt, 16 h, 79%, b: Br2, dioxane, rt, 16
h-9d, 68-95%, c:, Br2, AcOH, 60oC, 4 h, 75%, d: Br2, Fe, CHCl3, rt, 8 h, 35% e: i: (COCl)2, DMF,
DCM, rt, 4 h, ii: LDA, methylisobutyrate, THF, -78oC to rt, 2 h, f: i: 3-ethoxy-2,2-dimethyl-3-
oxopropanoic acid, (COCl)2, DCM, DM, ii: AlCl3, DCM, g: N2H4, EtOH, rt, 16 h, 6-91%, h: 2-
Br-propane, NaH, DMF, rt, 16 h, 29-85% i: CuCN, DMF, 150oC, 18 h, 59% j: 3-pyridinyl-B(OH)2,
Pd(dppf)Cl2.DCM, Na2CO3, DME:H2O, 120oC, 1 h, 6-88%.
N-alkylation of the diydropyrazolones (33-44, scheme 1) was done using sodium hydride and
isopropylbromide, installing the desired isopropyl moiety (45-56). Final step was a Suzuki cross-
coupling to install a 3-pyridinyl ring on the 3-position yielding twelve analogues (57-68) of NPD-
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0227 (3, figure 1) with variations on the central phenyl ring. The 4-cyanophenyl (74) was prepared
via a similar route: starting from 4-bromo-3-chlorobenzoic acid (69, scheme 2). This benzoic acid
was transofrmed to the corresponding isopropyl-pyrazolone (72) in three steps. Key-step was the
conversion of the bromine towards the cyano moiety using CuCN in DMF, after which this
intermediate (73) was used in a Suzuki cross-couping to install the final 3-pyridinyl moiety (74).
Scheme 2 Preperation of pyrazolone 74 (table 1) with modification on the central phenyl ring.
Reagents and conditions: a: i: (COCl)2, DMF, DCM, rt, 4 h, ii: LDA, methylisobutyrate, THF, -
78oC to rt, 2 h, b: g: N2H4, EtOH, rt, 16 h, 24% over two steps, c: 2-Br-propane, NaH, DMF, rt, 16
h, 75% d: CuCN, DMF, 150oC, 18 h, 59%, e: 3-pyridinyl-B(OH)2, Pd(dppf)Cl2.DCM, Na2CO3,
DME:H2O, 120oC, 1 h, 61%.
Modifications of the gem-dimethyl moiety present in NPD-0227 (3) were installed in an early
stage of the synthesis route (scheme 3). Similar conditions were used as in schemes 1 and 2;
benzoic acid 75 was transformed to the corresponding acid chloride, after which the desired lithium
enolates were added to yield the cyclopentene (77), methylpiperidine (79) and tetrahydropyran
8
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analogues (80). Exception was the cyclopentyl substituted keto-ester (78), which was prepared by
a one-pot reaction in which carboxylic acid (75) was converted to the corresponding imidazolide,
followed by addition of ethyl potassium malonate and subsequent decarboxylation. This
unsubstituted keto-ester (76) was then dialkylated with 1,4-dibromobutane to yield the desired
cyclopentyl ring. These four keto-esters (77-80) were then exposed to the same sequence as in
scheme 1 to yield spiro-analogues 89-92.
Scheme 3 Introduction of variations on the gem-dimethyl position of NPD-0227 (3), resulting in
89-92 (table 2). Reagents and conditions: a: i: (COCl)2, DMF, DCM, rt, 4 h, ii: LDA,
methylisobutyrate, THF, -78oC to rt, 2 h, b: ethyl potassium malonate, TEA, CDI, MgCl2, ACN,
THF, 44%, c: K2CO3, 1,4-dibromobutane, DMSO, rt, 51%, d: N2H4, EtOH, rt, 16 h, 20-65% over
9
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two steps e: 2-bromopropane, NaH, DMF, rt, 16 h, 74-96%, f: 3-pyridinyl-B(OH)2,
Pd(dppf)Cl2.DCM, Na2CO3, DME:H2O, 120oC, 1 h, 22-60%.
Various bio-isosteres of the dihydropyrazolone moiety were installed via different synthetic
routes (scheme 4-11). Dihydropyrazolo-oxazole 95 (scheme 3) was prepared in 3 steps from keto-
ester 76. First this molecule was ring-closed with hydrazine resulting in pyrazole 93, followed by
an alkylation with 1,2-dibromoethane, yielding the pyrazolo-oxazole moiety (94). Final step was
a Suzuki reaction using previously shown conditions to yield the desired 3-pyridine substituted
pyrazolo-oxazole 95.
Scheme 4 Preparation of dihydropyrazolo-oxazole 95 (table 3). Reagents and conditions: a: N2H4,
EtOAc, rt, o.n., 67%, b: K2CO3, 1,2-dibromoethane, DMF, 80oC, 2 h, 37%, c: 3-pyridinyl-B(OH)2,
Pd(dppf)Cl2.DCM, Na2CO3, DME:H2O, 120oC, 1 h.
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Thiadiazole 98 (scheme 5) was prepared in two steps from 2-(3-bromo-4-
methoxyphenyl)acetonitrile 96. First step was a Suzuki cross coupling to yield 3-pyridinyl
intermediate 97. Subsequent ring closure with hydrazinecarbothioamide yielded the final
thiadiazole 98.
Scheme 5 Preparation of thiadiazole 98 (table 3). Reagents and conditions: a: 3-pyridinyl-B(OH)2,
Pd(dppf)Cl2.DCM, Na2CO3, DME:H2O, 120oC, 1 h, 91%, b: hydrazinecarbothiamide, TFA, 60oC,
6 h, 71%.
Pyrazole-3-thione 99 (scheme 6), the sulphur bio-isostere of NPD-0227 (3) was prepared in one
step, from NPD-0227 (3) using Lawesson’s reagent in THF.
Scheme 6 Preparaton of the thione analogue of previous optimized hit NPD-0227, 99 (table 3).
Reagents and conditions: Lawessons reagent, THF, reflux, 32 h, 76%.
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The dihydropyridazinone moiety (scheme 7) has some similarity to the dihydropyrazolone
moiety of NPD-0227 (3), although this ring contains one additional carbon. Installation of this
moiety started with a Friedel-Craft acylation of 2-bromoanisole 100, resulting in β-keto-acid 101.
Ring closure of this molecule with hydrazine gave the dihydropyridazinone ring (102) which was
subsequently alkylated using sodium hydride to give the N-isopropyl derivative (103). Similar to
previously described synthetic routes, last step of installing the 3-pyridinyl moiety was done using
Suzuki conditions as described earlier, resulting in dihydropyridazinone 104.
Scheme 7 Preparation of the dihydropyridazinone analogue 104 (table 3). Reagents and
conditions: a: AlCl3, succinic anhydride, PhNO2, 60oC, 4 h, 42%, b: N2H4.H2O, EtOH, reflux, 1 h,
89%, c: 2-bromopropane, NaH, DMF, 50oC, 3 h, 87%, d: 3-pyridinyl-B(OH)2, Pd(dppf)Cl2.DCM,
Na2CO3, DME:H2O, 120oC, 1 h, 63%.
Pyrolopyrazole 109 (scheme 8) is a bicyclic heterocycle with similarity to the original
dihydropyrazolone. Preparation starts from the previously reported dihydropyrazolone 105 which
was refluxed with Lawessons reagent to yield pyrazol-3-thione 106.[10] Addition of hydrazine to
this building block yielded hydrazineylidine 107, from which the second heterocyclic ring was
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formed by adding cyclopropanecarbonyl chloride, resulting to the pyrolopyrazole moiety (108). A
final Suzuki cross-coupling yielded pyrolopyrazole 109. Attempts to install an isopropyl,
cyclopentyl or n-propyl did not succeed as the ring-closure step proved problematic.
Scheme 8 Preperation of cyclopropylpyrolopyrazole 109 (table 3). Reagents and conditions: a:
Lawessons reagent, toluene, reflux, o.n., 69%, b: N2H4.H2O, THF, rt towards 70oC, 5 h, 82%, c:
cyclopropanecarbonylchloride, pyridine, 70oC, 2 h, then DMF, 150oC, 2 h, 47%, d: 3-pyridinyl-
B(OH)2, Pd(dppf)Cl2.DCM, Na2CO3, DME:H2O, 120oC, 1 h, 30%.
Oxazoles 120-124 (scheme 9) were prepared from benzoic acid 75; first step of this route was a
sodium hydride promoted nucleophilic attack of the benzoic acid on selected bromoketones,
yielding keto-esters 110-114. Subsequent ring closure with ammonium acetate yielded mixtures
of respective imidazoles and oxazoles (115-119), which were relatively easily separated by column
chromatography. Although attempts were made to isolate the imidazoles, these could not be
obtained in sufficient purity. The subsequent Suzuki cross coupling yielded the desired 3-pyridine
substituted phenyloxazoles 120-124.
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Scheme 9 Preparation of oxazoles 120-124 (table 4). Reagents and conditions: a: BrCH2COR,
NaH, DMF, rt, 30 m, 83-94%, b: NH4OAc, AcOH, 170oC, 6 h, 11-36%, c: 3-pyridinyl-B(OH)2,
Pd(dppf)Cl2.DCM, Na2CO3, DME:H2O, 120oC, 1 h, 36-74%.
Oxadiazoles 132-136 (scheme 10) were prepared from benzoic acid 75, which was transformed
to the ethyl ester by refluxing in EtOH in the presence of H2SO4. Addition of hydrazine to ester
125, yielded hydrazide 126 which was ring closed with the desired acid chlorides in the presence
of POCl3 to yield oxadiazoles 127-131. Final step, was the Suzuki cross-coupling to yield 3-
pyridinyl substituted phenyloxadiazoles 132-136.
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Scheme 10 Preparations of oxadiazoles 132-136 (table 4). Reagents and conditions: H2SO4, EtOH,
reflux, 6 h, 97%, N2H4.H2O, EtOH, reflux, 16 h, 53%, R-COOH, POCl3, reflux, 1 h, 42-80%, d:
3-pyridinyl-B(OH)2, Pd(dppf)Cl2.DCM, Na2CO3, DME:H2O, 120oC, 1 h, 32-65%.
The final heterocyclic replacement investigated the thiazole ring (scheme 11). Starting material
was benzoic acid 75, which was chlorinated using oxalyl chloride, followed by a quench of
ammonium hydroxide to yield benzamide 137. The benzamide was converted to the corresponding
thioamide 138 using Lawessons reagent. This intermediate (138) could be used to form the desired
thiazoles upon addition of bromoketones, yielding analogues 139 and 140. To finalize the
molecules a Suzuki cross-coupling was used to install the 3-pyridinyl moiety, resulting in
compounds 141 and 142.
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Scheme 11 Synthesis of thiazoles 141 and 142 (table 4). Reagents and conditions: a: (COCl)2,
DMF, DCM, rt, 18 h, b: 30% NH4OH in H2O, DCM, rt, 5 m, 75% over two steps, c: Lawessons
reagent, toluene, reflux, 18 h, 13%, d: BrCH2COR, 2-propanol, rt, 2 h, e: 3-pyridinyl-B(OH)2,
Pd(dppf)Cl2.DCM, Na2CO3, DME:H2O, 120oC, 1 h, 35-44% over two steps.
Results and discussion
In this work, the SAR around NPD-0227 (3) is further investigated with modifications on the
central phenyl ring, the gem-dimethyl moiety and the dihydropyrazolone headgroup. In Table 1,
the screening results are shown of various substituents on the core phenyl group. Attempts to move
the 4-methoxy of NPD-0227 to the 5-position (57) leads to a decrease in activity with a pIC50 value
of 5.1, while introducing a methoxy substituent on both the 4- and 5- position (58), shows an even
larger decrease in activity (pIC50 = 4.3).
Table 1 Phenotypic activity of core-phenyl modifications against intracellular amastigotes of
T. cruzi (Tulahuen strain) and MRC-5 cells
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T. cruzi
(pIC50)a
6.4
MRC-5
Cmpnd
R1
R2
R3
R4
SIb
(pIC50)a
4.4
H
H
H
H
H
H
H
H
H
H
F
H
MeO
H
H
H
H
H
H
H
H
H
H
F
100
4
3
MeO
MeO
5.1
4.5
57
58
59
60
61
62
63
64
65
66
67
68
74
MeO
4.3
< 4.2
4.6
> 1
16
-CH2CH2O-
5.8
Br
H
MeO
H
5.1
4.6
3
5.5
4.2
20
H
F
5.2
< 4.2
4.8
> 10
5
H
Cl
5.5
H
Me
5.6
< 4.2
< 4.2
< 4.2
< 4.2
< 4.2
< 4.2
> 25
> 25
> 13
> 5
> 2
> 1
H
MeO
MeO
MeO
MeO
CN
5.6
H
H
H
H
H
5.3
H
Me
H
Me
H
4.9
4.5
H
4.3
a all reported values are within a standard deviation of ±0.2 and the result of at least n=2, b The
selectivity index is calculated by dividing the cytotoxicity (IC50) by the T. cruzi activity (IC50).
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Constraining the methoxy-substituent in a heterocycle resulting in dihydrobenzofuran 59 only
resulted in a small decrease (pIC50 = 5.8) in activity compared to NPD-0227. Installing a bromine
on the phenyl ring next to the 4-methoxy substituent (60) resulted in a decrease in activity with a
pIC50-value of 5.1. Removal of the 4-methoxy led to analogue 61, which resulted in a 10-fold drop
in potency (pIC50 = 5.5), showing that a methoxy substituent on this position is beneficial.
Installation of a fluorine (62), chlorine (63), or methyl (64) group instead of the original 4-methoxy
substituent resulted in compounds with similar pIC50-values around 5.5 as the unsubstituted
analogue 61. Introduction of a cyano moiety (74) instead of a methoxy resulted in an even further
drop in activity with a pIC50-value of 4.3.
As the 4-methoxy substituted analogue (3) performed the best thus far, additional substituents
along with the methoxy were investigated. Both the 2-fluoro-4-methoxy (65) and the 6-fluoro-4-
methoxy (66) showed a ~10-fold decrease compared to NPD-0227 (3), while the 4-methoxy-5-
methyl (67) and 4-methoxy-6-methyl (68) showed an even larger decrease with pIC50-values of
4.9 and 4.5, respectively. Selectivity index between T. cruzi activity and MRC-5 cytotoxicity are
lower then seen with NPD-0227, with the best compounds showing a SI of > 25-fold. Exact SIs
are not known as the lowest concentration measured for cytotoxicity is 62 µM.
Table 2 Phenotypic activity of analogues with gem-diemethyl modifications against intracellular
amastigotes of T.cruzi (Tulahuen strain) and MRC-5 cells
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T. cruzi
MRC-5
(pIC50)a
Cmpnd
R
SIb
(pIC50)a
5.2
5.1
4.5
4.4
5
5
89
90
4.5
4.5
4.3
4.5
2
1
91
92
a all reported values are within a standard deviation of ±0.2 and the result of at least n=2, b The
selectivity index is calculated by dividing the cytotoxicity (IC50) by the T. cruzi activity (IC50).
Four compounds were prepared with variations of the gem-dimethyl moiety of NPD-0227 (3);
installing a cyclopentyl (89) and a cyclopentene (90) both resulted in more than a 10-fold loss in
activity compared to NPD-0227 (3). Introduction of the more polar methylpiperidine (91) and a
tetrahydropyran (92) resulted in an even further decrease in potency with both compounds having
a pIC50-value of 4.5. Selectivity index between T. cruzi activity and MRC-5 cytotoxicity are poor
for these series, with the best compounds (89, 90) showing 5-fold selectivity.
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Table 3 Phenotypic activity of dihydropyrazolone bioisosteres against intracellular amastigotes of
T. cruzi (Tulahuen strain) and MRC-5 cells
T. cruzi
MRC-5
(pIC50)a
Cmpnd
Headgroup
SIb
(pIC50)a
4.5
4.3
2
-
95
98
< 4.2
< 4.2
6.3
5.0
4.8
4.8
4.6
32
3
99
104
109
< 4.2
> 4
a all reported values are within a standard deviation of ±0.2 and the result of at least n=2, b The
selectivity index is calculated by dividing the cytotoxicity (IC50) by the T. cruzi activity (IC50).
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Diydropyrazolo-oxazole 95, in which the dihydropyrazolone oxygen is constrained in a second
5-membered ring shows a loss in activity with a pIC50 -value of 4.5. An even larger loss in activity
is observed with aminothiodiazole 98, which is inactive (pIC50 = <4.2). Replacing the oxygen of
the dihydropyrazolone with a sulphur atom, leads to dihydropyrazolethione 99, which had similar
activity (pIC50 = 6.3) as NPD-0227 (3, pIC50 = 6.4), although a small increase in toxicity against
human MRC-5 cells (pIC50 = 4.8) is observed, which is a 32-fold selectivity. Adding an extra
carbon to the ring, resulting in pyridazinone 104 leads to a pIC50-value of 5.0. Also
pyrazolotriazole 109, which has a bicyclic system with quite some similarity to the pyrazolone
ring of NPD-0227 (3), shows a decreased activity (pIC50 = 4.8).
Table 4 Phenotypic activity of analogues with gem-dimethyl modifications against intracellular
amastigotes of T. cruzi (Tulahuen strain) and MRC-5 cells
T. cruzi MRC-5
Cmpnd
X
Y
Z
R1
SIb
(pIC50)a (pIC50)a
O
O
CH
CH
N
N
5.7
6.0
5.2
5.5
3
3
120
121
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O
O
CH
CH
N
5.9
6.1
1
122
N
N
O
O
6.2
5.7
4.7
5.0
< 4.2
5.3
> 100
3
123
124
132
133
O CCH3
N
N
N
N
< 4.2
< 4.2
> 3
> 6
N
N
N
N
O
O
4.6
5.8
< 4.2
< 4.2
> 3
134
135
> 40
N
N
O
5.7
< 4.2
> 32
136
S
S
CH
CH
N
N
5.6
5.7
5.1
5.6
3
1
141
142
a all reported values are within a standard deviation of ±0.2 and the result of at least n=2, b The
selectivity index is calculated by dividing the cytotoxicity (IC50) by the T. cruzi activity (IC50).
To investigate if the dihydropyrazolone moiety could be replaced, three different aromatic
heterocycles were installed on this position while still being able to address the same region as the
isopropyl moiety of NPD-0227 (3). This resulted in a series of five membered heterocycles:
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oxazoles (120-124), oxadiazoles (132-136) and thiazoles (141-142). The synthesized oxazoles
(120-124) all showed fairly similar activities with pIC50-values around 6.0, although large
differences can be seen in toxicity towards MRC-5 cells. While the tert-butyl (120), phenyl (121)
and 3-fluorophenyl (122) substituted oxazole and phenyl substituted methyloxazole (124) all have
MRC-5 toxicities above 5.2, the 4-fluorophenyl (123) substituted oxazole shows no MRC-5
toxicity at 62 µM. With its low acticity and high selectivity (SI = > 100-fold) over MRC-5 cells,
4-fluorophenyloxazole 123 is the most promising of this series.
Addition of an extra nitrogen to the heteroaromatic ring lead to oxadiazoles 132-136 which
showed no toxicity towards MRC-5 cells at the lowest concentration screened, as all compounds
reported pIC50-values below 4.2 (62 µM). Aliphatic substituents (132-134) on the oxadiazole gave
some activity against T. cruzi with pIC50-values around 4.9. However, aromatic substituents (135-
136) are preferred as both phenyloxazole (135) and 3-fluorophenyl (136) showed activities around
pIC505.8. Finally, the thiazoles showed decent activities (pIC50 around 5.8) but toxicity of these
compounds against MRC-5 cells is equally high, making these compounds less favorable for future
studies.
Conclusion
Multiple approaches were explored to optimize the activity of NPD-0227 (3). Modification of
the core phenyl moiety delivered interesting SAR data, but activity was generally quite low, with
dihydrobenzofuran (59) performing best with a pIC50-value of 5.8. Replacing the gem-dimethyl
moiety with several spiro-analogues resulted in four analogues with a maximum activity of 5.2
(pIC50) hence showing substantially lower activities than NPD-0227 (pIC50 = 6.4). The sulfur
analogue of NPD-0227, dihydropyrazolethione 99 showed a similar activity as NPD-0227 (3) with
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a pIC50 of 6.3, however this was accompanied with an increase in cytotoxicity. As these
compounds could not match the activity of NPD-0227, no further screenings were done on other
strains or life stages, focusing on SAR of these series. Introduction of heterocycles instead of the
pyrazolone moiety also yielded several compounds with promising activities, amongst which
oxazole 123 showed a pIC50-value of 6.2 (SI = >100 fold over MRC-5 cytotoxicity) and oxadiazole
135 which had a pIC50-value of 5.8. Although these compounds do not show higher activities then
optimized lead NPD-0227 (3), these scaffold hops could be new starting points for future hit-to-
lead optimization, especially with the promising selectivity index seen for oxadiazole 123.
Experimental section biology
Trypanosoma cruzi in vitro assay. Bloodstream trypomastigotes (BT) of the Y strain of T. cruzi were
obtained by cardiac puncture of infected Swiss Webster mice on the parasitaemia peak.[12] For the standard
in vitro susceptibility assay on intracellular amastigotes, T. cruzi Tulahuen CL2, β-galactosidase strain
(DTU VI, nifurtimox-sensitive) was used. The strain is maintained on MRC-5SV2 (human lung fibroblast)
cells in MEM medium, supplemented with 200 mM L-glutamine, 16.5 mM NaHCO3, and 5% heat
inactivated fetal calf serum (FCSi). After incubation at 37 °C for 7 d, parasite growth was assessed by
adding the α-galactosidase substrate chlorophenol red-α-D-galactopyranoside. The color reaction was read
at 540 nm after 4 h, and absorbance values were expressed as a percentage of the blank controls. All cultures
and assays are conducted at 37°C under an atmosphere of 5% CO2.[13] Benznidazole was used as a reference
compound.
MRC-5 cytotoxicity in vitro assay. MRC-5-SV2 cells, originally from a human diploid lung cell line, were
cultivated in MEM, supplemented with L-glutamine (20 mM), 16.5 mM sodium hydrogen carbonate and
5% FCSi. For the assay, 104 cells/well were seeded onto the test plates containing the pre-diluted sample
and incubated at 37°C and 5% CO2 for 72 h. Cell viability was assessed fluorometrically 4 h after addition
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of resazurin (excitation 550 nm, emission 590 nm). The results are expressed as percentage reduction in
cell viability compared to untreated controls. Tamoxifen was used as a reference compound.
Experimental section chemistry
Chemicals and reagents were obtained from commercial suppliers and were used without further
purification. Anhydrous DMF, THF and DCM were obtained by passing them through an activated alumina
column prior to use. Microwave reactions were executed using a Biotage® Initiator microwave system. 1H
NMR spectra were recorded on a Bruker Avance 250 (250 MHz), Bruker Avance 400 (400 MHz), Bruker
Avance 500 (500 MHz) or Bruker 600 Avance (600 MHz) spectrometer. Data are reported as follows:
chemical shift, integration, multiplicity (s = singlet, d = doublet, dd = double doublet, t = triplet, dt = double
triplet, q = quartet, p = pentet, h = heptet, bs = broad singlet, m = multiplet), and coupling constants (Hz).
Chemical shifts are reported in ppm with the natural abundance of deuterium in the solvent as the internal
reference (CDCl3: δ 7.26, (CD3)2SO: δ 2.50). 13C NMR spectra were recorded on a Bruker Avance 500 (126
MHz) or Bruker Avance 600 (150 MHz). Chemical shifts are reported in ppm with the solvent resonance
resulting from incomplete deuteration as the internal reference (CDCl3: δ 77.16 or (CD3)2SO: δ 39.52).
Systematic names for molecules according to IUPAC rules were generated using the Chemdraw AutoName
program. LC-MS data was gathered using a Shimadzu HPLC/MS workstation with a LC-20AD pump
system, SPD-M20A diode array detection, and a LCMS-2010 EV mass spectrometer. The column used is
an Xbridge C18 5 μm column (100 mm × 4.6 mm). Solvents used were the following: solvent B = ACN,
0.1% formic Acid; solvent A = water, 0.1% formic acid. The analysis was conducted using a flow rate of
1.0 mL/min, start 5% B, linear gradient to 90% B in 4.5 min, then 1.5 min at 90% B, linear gradient to 5%
B in 0.5 min and then 1.5 min at 5% B, total run time of 8 min. All reported compounds have purities >95%,
measured at 254 nm, unless otherwise mentioned. All HRMS spectra were recorded on a Bruker micrOTOF
mass spectrometer using ESI in positive-ion mode. Column purifications were either carried out
automatically using Biotage equipment or manually, using 60-200 mesh silica. TLC analyses were
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performed with Merck F254 alumina silica plates using UV visualization. All reactions were done under
N2 atmosphere, unless specifically mentioned.
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Experimental data
Compounds 10, 23, 35, 47, 59, 110, 115, 120, 125-127 and 132 are reported in the main article.
The experimental data of the other compounds can be found in the supporting information.
7-bromo-2,3-dihydrobenzofuran-5-carboxylic acid (10)
2,3-dihydrobenzofuran-5-carboxylic acid (5 g, 31 mmol) was dissolved in
dioxane (100 mL) and bromine (3.14 mL, 61 mmol) was added. The reaction was stirred for 16
hours after which it was quenched with sodium bisulfate. Solids were collected and recrystallized
from MeOH yielding 7 g (29 mmol, 95%) of the title compound as a white solid.1H NMR (600
MHz, DMSO-d6) δ 12.86 (s, 1H), 7.85 (s, 1H), 7.78 (s, 1H), 4.72 (t, J = 8.8 Hz, 2H), 3.34 (app. s,
overlap with H2O peak, 3H). 13C NMR (151 MHz, DMSO-d6) δ 166.4, 161.1, 133.1, 130.2, 126.2,
125.3, 101.6, 73.1, 29.9. LC-MS (ESI) m/z found: no mass observed ; retention time: 3.67 minutes.
methyl 3-(7-bromo-2,3-dihydrobenzofuran-5-yl)-2,2-dimethyl-3-oxopropanoate (23)
Benzoic acid 10 (2.0 g, 8.2 mmol) was dissolved in DCM (100 mL). The
mixture was stirred at rt, and oxalyl dichloride (1.1 mL, 12.3 mmol) and a few drops of DMF were
added After 3 hours volatiles were evaporated and the mixture was redissolved in 30 mL of THF.
In a separate flask methyl isobutyrate (1.41 mL, 12.3 mmol) was stirred in THF (100 mL) at -78°C
and 2M LDA (4.94 mL, 9.87 mmol) was added. After stirring for 30 minutes the previously
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prepared acid chloride in THF was added dropwise, maintaining the temperate at -78°C. The
mixture was allowed to warm up to rt after which the mixture was quenched with sat. aq. NH4Cl
(200 mL), extracted with Et2O (300 mL) and dried over MgSO4.The resulting crude was used in
the next step without further purification. Crude 1H NMR (600 MHz, Chloroform-d) δ 7.85 (d, J
= 1.4 Hz, 1H), 7.61 (d, 1H), 4.75 (t, J = 8.8 Hz, 2H), 3.67 (s, 3H), 3.34 (t, J = 8.8 Hz, 2H), 1.52 (s,
7H). 13C NMR (151 MHz, Chloroform-d) δ 194.9, 175.6, 161.1, 133.3, 129.7, 128.6, 124.6, 102.7,
72.6, 53.1, 52.7, 30.1, 24.1.
3-(7-bromo-2,3-dihydrobenzofuran-5-yl)-4,4-dimethyl-1H-pyrazol-5(4H)-one (35)
Crude keto-ester 23 (1.0 g, 3.0 mmol) was dissolved in ethanol (10 mL) and
hydrazine hydrate (1.6 mL, 31 mmol) was added. The mixture was stirred overnight, after which
50 mL of water was added and the precipitate was collected yielding 800 mg (2.6 mmol, 83% over
two steps) of the title compound as a white solid. 1H NMR (500 MHz, DMSO-d6) δ 11.47 (s, 1H),
7.71 – 7.65 (m, 2H), 4.68 (t, J = 8.8 Hz, 2H), 3.36 (t, J = 8.8 Hz, 2H), 1.34 (s, 6H). 13C NMR (126
MHz, DMSO-d6) δ 181.0, 161.0, 158.5, 130.4, 128.6, 125.8, 122.6, 102.3, 72.6, 46.8, 30.4, 22.3.
LC-MS (ESI) m/z found: 309 [M+H]+; retention time: 3.91 minutes. HRMS-ESI [M+H]+
calculated for C13H14BrN2O2: 309.0233 , found: 309.0232.
3-(7-bromo-2,3-dihydrobenzofuran-5-yl)-1-isopropyl-4,4-dimethyl-1H-pyrazol-5(4H)-one (47)
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Dihydropyrazolone 35 (500 mg, 1.62 mmol) was stirred in DMF (5 mL) and
sodium hydride (60% in mineral oil) (68 mg, 1.70 mmol) was added. Stirred for 10 minutes, after
which 2-bromopropane (0.15 mL, 1.62 mmol) was added and the reaction was stirred for 18 hours.
The reaction was quenched with water, extracted with EtOAc (40 mL), which was washed with
water (2x 20 mL) and brine (20 mL). The resulting crude was purified over SiO2 using a gradient
of 80% c-hexane in EtOAc towards EtOAc to yield 475 mg (1.3 mmol, 80%) of the title compound
as a white solid. 1H NMR (600 MHz, Chloroform-d) δ 7.74 (s, 1H), 7.63 (s, 1H), 4.75 (t, J = 8.8
Hz, 2H), 4.52 (hept, J = 6.7 Hz, 1H), 3.39 (t, J = 8.8 Hz, 2H), 1.47 (s, 6H), 1.38 (d, J = 6.7 Hz,
6H). 13C NMR (151 MHz, Chloroform-d) δ 177.7, 160.5, 158.6, 129.5, 128.9, 125.7, 121.9, 102.7,
72.1, 48.8, 45.3, 30.5, 22.6, 20.8. LC-MS (ESI) m/z found: 351 [M+H]+; retention time: 4.97
minutes. HRMS-ESI[M+H]+ calculated for C16H20BrN2O2: 351.0703, found: 351.0686.
1-isopropyl-4,4-dimethyl-3-(7-(pyridin-3-yl)-2,3-dihydrobenzofuran-5-yl)-1H-pyrazol-5(4H)-
one (59)
Dihydropyrazolone 47 (100 mg, 0.28 mmol) and pyridin-3-ylboronic acid
(53 mg, 0.43 mmol) were charged to a microwave vial after which DME (3.5 mL) and 1M Na2CO3
(1.1 mL, 1.1 mmol) were added. The mixture was degassed with N2 for 5 m after which
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