Structure-activity relationship studies of pyrrolone antimalarial agents

Article abstract of DOI:10.1002/cmdc.201300177

Previously reported pyrrolones, such as TDR32570, exhibited potential as antimalarial agents; however, while these compounds have potent antimalarial activity, they suffer from poor aqueous solubility and metabolic instability. Here, further structure-activity relationship studies are described that aimed to solve the developability issues associated with this series of compounds. In particular, further modifications to the lead pyrrolone, involving replacement of a phenyl ring with a piperidine and removal of a potentially metabolically labile ester by a scaffold hop, gave rise to derivatives with improved in vitro antimalarial activities against Plasmodium falciparum K1, a chloroquine- and pyrimethamine-resistant parasite strain, with some derivatives exhibiting good selectivity for parasite over mammalian (L6) cells. Three representative compounds were selected for evaluation in a rodent model of malaria infection, and the best compound showed improved ability to decrease parasitaemia and a slight increase in survival.

Full text of DOI:10.1002/cmdc.201300177

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DOI: 10.1002/cmdc.201300177
Structure–Activity Relationship Studies of Pyrrolone
Antimalarial Agents
Dinakaran Murugesan,[a] Marcel Kaiser,[b] Karen L. White,[c] Suzanne Norval,[a] Jennifer Riley,[a]
Paul G. Wyatt,[a] Susan A. Charman,[c] Kevin D. Read,[a] Clive Yeates,[d] and Ian H. Gilbert*[a]
Previously reported pyrrolones, such as TDR32570, exhibited
potential as antimalarial agents; however, while these com-
pounds have potent antimalarial activity, they suffer from poor
aqueous solubility and metabolic instability. Here, further struc-
ture–activity relationship studies are described that aimed to
solve the developability issues associated with this series of
compounds. In particular, further modifications to the lead pyr-
rolone, involving replacement of a phenyl ring with a piperidine
and removal of a potentially metabolically labile ester by a scaf-
fold hop, gave rise to derivatives with improved in vitro anti-
malarial activities against Plasmodium falciparum K1, a chloro-
quine- and pyrimethamine-resistant parasite strain, with some
derivatives exhibiting good selectivity for parasite over mam-
malian (L6) cells. Three representative compounds were select-
ed for evaluation in a rodent model of malaria infection, and
the best compound showed improved ability to decrease para-
sitaemia and a slight increase in survival.
Introduction
Malaria is a serious disease endemic in tropical and subtropical
regions of the world. It is a major threat to public health in
more than 100 countries and is responsible for >200 million
clinical cases each year and probably about 1 million deaths,
the majority of whom are young children and pregnant
women.[1–4] In addition, malaria is responsible for a huge eco-
nomic impact in endemic countries.[2] With increasing globali-
sation and global warming, there is a risk of malaria spreading
to new areas.[3] No vaccine is currently available for malaria,
and the resistance of the protozoa to clinically used chemo-
therapeutic agents is increasingly common. Therefore, an
urgent need exists to develop new classes of antimalarial
drugs that operate by novel mechanisms of action.
We have previously reported on the discovery and struc-
ture–activity relationship (SAR) of a series of pyrrolones, which
have potent antimalarial activity;[5] these were initially discov-
ered through a phenotypic screening process conducted by
the World Health Organisation (WHO) Programme for Research
and Training in Tropical Diseases. The prototypical compound
TDR32750 (Figure 1a) showed potent antimalarial activity
against a panel of strains of Plasmodium falciparum and signifi-
cant depression of parasitaemia (>99%) in the Plasmodium
berghei mouse model of malaria when dosed intraperitoneally.
Unfortunately, the compound has low oral bioavailability;
good oral bioavailability is a requirement for most target prod-
uct profiles for malaria. While the precise reason for the low
bioavailability is unknown, it is most likely due to a combina-
tion of metabolic instability (as evidenced by studies in hepatic
microsomes) and low aqueous solubility.[5] The ester linkage is
also susceptible to hydrolysis in blood, which contributes to
the overall systemic clearance, as previously reported. Our pre-
vious SAR studies around TDR32750[5] encompassed changes
to the A-, B- and C-rings (Figure 1a), which led to the conclu-
sion that the A-ring was reasonably tolerant of changes, unlike
the B-ring. Modifications to the C-ring were not investigated
apart from replacement of the ester with a variety of amides,
which led to compounds with decreased activity.
[a] Dr. D. Murugesan, S. Norval, J. Riley, Prof. P. G. Wyatt, Dr. K. D. Read,
Prof. I. H. Gilbert
Division of Biological Chemistry & Drug Discovery
College of Life Sciences, University of Dundee
Sir James Black Centre, Dow St, Dundee, DD1 5EH (UK)
E-mail: i.h.gilbert@dundee.ac.uk
[b] Dr. M. Kaiser
Swiss Tropical & Public Health Institute
Postfach, Socinstrasse 57, 4002 Basel (Switzerland)
and
University Basel
Petersplatz 1, 4003 Basel (Switzerland)
[c] Dr. K. L. White, Prof. S. A. Charman
Centre for Drug Candidate Optimisation
Monash Institute of Pharmaceutical Sciences, Monash University
381 Royal Parade, Parkville, Victoria 3052 (Australia)
[d] Dr. C. Yeates
In order to improve the physicochemical properties of the
molecules and the prospects for further development of the
compound series, our aim was to decrease the clogD value to
less than three. Here, we report on changes to the A-ring and
C-ring, which have led to compounds with enhanced antima-
larial activity and improved physicochemical properties. In par-
ticular, our work focused around some piperidine analogues of
InPharma Consultancy
Hertfordshire (UK)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201300177.
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This is an open access article under the terms of the Creative Commons
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Figure 1. a) Prototypical compound TDR32750, structure and in vitro activi-
ties.[5] b) Generic structure of compounds prepared in this study.
the A-ring in our lead molecule (Figure 1b). The piperidine
moiety has an amino group, which when unconjugated is
basic and should improve the solubility properties.
Scheme 1. General strategy for the synthesis of piperidine derivatives. Re-
gents and conditions: a) chloroacetyl chloride, pyridine, 08C, 30 min, 75%
yield; b) KOH, EtOH, 3 h, 08C, 95% yield; c) p-TsOH bound on silica gel, mi-
crowave (0–400 W at 2.45 GHz), 1808C, 15–20 min, 80–90% yield; or p-TsOH,
toluene, Dean–Stark apparatus, 908C, 3 h, 80–90%; d) 4m HCl/dioxane, 08C,
3 h, 66% yield; e) POCl3, DMF, 1008C, 3 h, 80–95% yield; f) ROSO2CH3, Et3N,
NaHCO3, CH3CN, 858C, 12 h, 50–70%; g) RBr, DIPEA, DMF, 858C, 3 h, 40–70%
yield; h) alkyl/aryl/heterocyclic aldehyde, sodium triacetoxyborohydride,
MeCN, RT, 8 h, 60–80% yield; i) KHSO4, EtOH, 3 h, reflux, 80–95% yield.
R groups are given in Table 1.
The lead compound, TDR32750, contains an ester in the C-
ring; we were concerned that this might be a metabolic liabili-
ty due to hydrolysis by esterases, since previous work has con-
firmed that there is gradual degradation of the ester in vivo.[5]
However, this is probably not the only mode of metabolism, as
we have also shown that the compound undergoes cyto-
chrome P450 (CYP450)-mediated degradation in hepatic micro-
somes.[5] Nevertheless, we thought it prudent to try and re-
place the ester to improve the clearance properties. Previously,
we reported efforts at replacing the ester with an amide, but
this led to loss of activity; possibly due to the amide causing
a conformational change compared to the ester.[5] In this
paper, we report the effect of cyclising the ester to form
a fused pyridone, which might lock the compound into a more
favourable conformation (Figure 1b).
3-Formylpyrrole intermediates 16a–z and 16aa–aj were pre-
pared in a four-step sequence: 1) condensation of tert-butyl-
oxycarbonyl (BOC)-protected 4-aminopiperidine (8) with 2,5-
hexandione 4 (Paal–Knorr pyrrole synthesis); 2) deprotection of
the BOC group; 3) coupling either by alkylation or reductive
amination to give a range of piperidine derivatives (11 a–15);
4) carbonylation using a Vilsmeier–Haack reaction (85–95%
yield).[8,9] The complete range of compounds prepared is
shown in Table 1.
The pyrrolo [3,2-c] pyridine-3, 4-dione series, that is the C-
ring analogues of TDR32750 incorporating a fused pyridone
ring, were prepared according to Scheme 2. Reaction of the
pyrrolone ring of TDR32750 with the diethyl acetal of N,N-di-
methylformamide (DMF) gave key intermediate 55.[10] Reaction
with an amine caused displacement of the dimethylamine and
cyclisation to the required pyrrolo[3,2-c] derivatives (56–64).
Results and Discussion
Chemistry
The starting point for the synthesis of the A-ring variants incor-
porating a piperidine substituent was pyrrolone 3, which was
prepared as reported previously (Scheme 1).[5] Pyrrolone 3
(which was not stored but used immediately) was then con-
densed with the required 3-formylpyrrole 4-aminopiperidine
(7, 16a–z, 16aa–aj) in the presence of potassium hydrogen
sulfate,[6,7] to give predominantly the (E)-isomer of the requisite
pyrrolone derivative (17–54).1
In vitro activity
Compounds were evaluated for activity against P. falciparum
K1, a chloroquine- and pyrimethamine-resistant parasite strain,
and counter-screened against mammalian L6 cells (Table 1).
Many of the compounds showed potent antimalarial activity,
with eight compounds showing EC50 values of less than 10 nm
1
Some of the compounds had a trace, inseparable amount of (Z)-isomer. This
could not be detected in the 13C NMR spectra and was only detected using
UPLC.
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*
The phenyl ring was tolerant to different substitutions,
although the morpholine analogue (54) had greatly
decreased activity.
C-Ring modifications
Modifications to the C-ring showed interesting activities in
vitro against P. falciparum K1 (Table 2). The most active com-
pounds displayed sub-micromolar activities; compounds 56,
57, 58 and 63 were found to be the most active. The following
observations were made regarding the SAR of the C-ring-modi-
fied analogues:
Scheme 2. a) DMF–diethyl acetal, 858C, 15 min, 100% yield; b) R1NH2, iso-
propyl alcohol, microwave (0–400 W at 2.45 GHz), 1108C, 15–30 min, 30–
70% yield.
*
Compound 56 with a free NH group on the pyrrolo[3,2-c]-
pyridine ring showed an EC50 value of 0.047 mm. Small alkyl
substituents on the “pyridone” nitrogen were tolerated (57,
R=Me: EC50 =14 nm; 58, R=Et: EC50 =21 nm), whilst
a propyl substituent (59) led to a tenfold drop in activity.
and two compounds (29 and 44) showing sub-nanomolar po-
tencies. Most of the compounds showed good selectivity for
parasite over mammalian cells.
*
Basic substituents did not appear to be tolerated, with com-
pounds (60 and 61) exhibiting approximately a fivefold de-
crease in EC50 value relative to the unsubstituted analogue
56.
*
The para-trifluoromethylbenzyl (63) and para-pyridyl (64)
A-Ring modifications
analogues appeared to substantially retain potency com-
pared with TDR32750, although the straight benzyl deriva-
tive (62) showed around a fivefold loss in activity.
The following observations were made regarding the in vitro
antimalarial activity for compounds with variations around the
A-ring:
*
In general, simple alkyl substituents (18–23) led to com-
In vitro drug metabolism and pharmacokinetics (DMPK)
pounds that exhibit a large decrease in activity compared
with TDR32750. This also applied to a methylene cycloprop-
yl derivative (39). Interestingly, methylene cyclohexyl deriva-
tive 40 showed only a slight drop in activity compared with
TDR32750, although adding heteroatoms to the ring to im-
prove the physicochemical properties of the compounds
was counter-productive for activity (41 and 42). Similarly,
cyclohexyl analogues linked via an ethylene linker (37 and
38) showed decreased activity.
For key compounds, metabolic stability when incubated with
mouse liver microsomes was measured, and for some com-
pounds solubility was also assessed. For the A-ring variants,
the solubility increased, particularly at low pH, probably due to
the basic nature of the piperidine ring. In those derivatives
with an aromatic or benzyl substituent, the basicity of the
amine is decreased, which probably explains the lower solubili-
ty at pH 6.5.
*
Where the substituent was an aromatic ring directly at-
Disappointingly, the in vitro intrinsic clearance (CLint) values
were all rather high—the preferred value is <50 mLminÀ1 mgÀ1
tached to the piperidine ring (30), the compound retained
reasonably potent activity (EC50 =0.024 mm); however, this
compound would have decreased basicity at the piperidine
ring.
protein, although the aim is for
a value of less than
20 mLminÀ1 mgÀ1. The most stable compounds were 17, 20,
31, 35, 40, 48, and 50, with derivatives 20 and 31 being rela-
tively inactive. Compound 48 is particularly stable, presumably
due to the relatively low clogD value and the metabolic stabili-
ty of the isoxazole ring.
*
Compounds with a phenyl ring separated from the piperi-
dine by a methylene linker in general showed good poten-
cy (17, 26–29, 35, 36, 43, 44, 51).
*
Replacement of the phenyl ring by a heteroaromatic ring
For C-ring variants, the most active compounds (56–58)
were investigated for microsomal stability. Compound 56
showed moderate stability, whilst the N-alkyl congeners were
less stable, possibly due to CYP450-mediated metabolism of
the alkyl groups.
(to improve physicochemical properties) was generally det-
rimental to activity (24, 25, 45–47, 49, 52, 53). The marked
exception to this was compound 43, a pyridine, albeit with
an electron-withdrawing trifluoromethyl group, which is
likely to significantly decrease the basicity of the pyridine
nitrogen. The oxazole (48) and N-methylpyrrole (50) deriva-
tives also retained activity.
In vivo studies
*
Increasing the spacer length between the piperidine and
Three compounds were selected for further study in vivo.
the aromatic ring also led to a drop in activity (31–34).
Compound 43 was selected from the A-ring-modified series, as
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Table 1. Activity data for A-ring variants against Plasmodium falciparum.
[e]
Compd[a]
R
EC50[b] [mm]
L6 cells
cLogD[c]
(pH 7.4)
Solubility[d] [mm]
CLint
P. falc.
pH 2.0
15–30
pH 6.5
water
[mLminÀ1 mgÀ1
]
TDR32750
0.014
34
20
5.0
2.1
7–15
>125
50#
17
0.035
56–110
14–28
>125
60#
18
19
20
21
22
23
-H
-CH3
-C2H5
-(CH2)2C(CH3)2
-(CH2)2CH3
-CH2C(CH3)3
12
1.4
2.4
0.19
1.1
>250
0.7
0.9
1.2
2.0
1.4
2.0
n.d.
n.d.
>250
n.d.
n.d.
n.d.
n.d.
n.d.
>250
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
11#
n.d.
n.d.
n.d.
7.8
7.6
22
6.6
88
0.058
24
25
0.38
0.46
5.9
1.6
1.6
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
27
26
27
0.024
0.023
51
23
5
2.2
2.2
n.d.
n.d.
n.d.
n.d.
>125
>125
117#
149#
28
29
0.002
2.7
2.6
48-97
n.d.
6–12
n.d.
>125
>125
217§
952§
0.0005
1.7
30
31
0.024
0.32
10
2.7
2.2
n.d.
n.d.
n.d.
n.d.
>125
n.d.
76#
107
n.d.
32
33
34
0.083
0.13
72
19
22
2.2
2.6
2.3
100–200
n.d.
25–51
n.d.
n.d.
n.d.
n.d.
120#
215#
114#
0.096
52–100
13–26
35
36
0.007
0.001
4.26
2.9
2.9
n.d.
n.d.
n.d.
n.d.
n.d.
88§
3
>125
190§
37
38
5.3
2.4
89
97
1.3
1.5
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
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Table 1. (Continued)
[e]
Compd[a]
R
EC50[b] [mm]
L6 cells
cLogD[c]
(pH 7.4)
Solubility[d] [mm]
CLint
P. falc.
pH 2.0
n.d.
pH 6.5
water
n.d.
[mLminÀ1 mgÀ1
]
39
40
3.5
14
13
1.7
2.1
n.d.
n.d.
78§
0.016
0.11
n.d.
n.d.
n.d.
n.d.
>125
41
42
43
81
1.6
1.4
n.d.
n.d.
n.d.
1.4
n.d.
n.d.
n.d.
n.d.
n.d.
43
44
0.004
49
2.2
2.6
>125
177§
248§
0.0004
1.2
n.d.
n.d.
>125
45
46
0.15
1.2
36
28
1.2
1.1
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
47
48
0.10
42
1.5
1.3
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
0.018
0.16
>125
<10§
49
50
1.0
16
3
1.0
1.4
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
55§
0.012
51
52
0.001
1.5
11
50
2.4
2.2
n.d.
n.d.
n.d.
n.d.
>125
n.d.
n.d.
n.d.
53
54
0.075
2.6
28
53
1.8
2.5
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
[a] Overall yields: 40–85%; reference compounds: chloroquine, EC50 =0.095–0.172 mm (P. falciparum K1); podophyllotoxin, EC50 =0.009–0.022 mm (L6 cells).
[b] The EC50 values are the mean of two independent assays, which varied less than Æ50%; n.d=not determined. [c] Calculated using StarDrop (http://
www.optibrium.com). [d] Measured using nephelometry. [e] Intrinsic clearance determined in vitro using mouse liver microsomes at Monash University (#)
or at the University of Dundee (UK) (§).
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Both via po and ip administra-
tion, compound 57 was the
most efficacious in terms of de-
crease of parasitaemia. At
a dose of 50 mgkgÀ1 ip, com-
pound 57 decreased the level of
parasitaemia by 99.9%, and
there was a significant increase
in survival time. When dosed
orally, the decrease in parasitae-
mia was less at 50.5%. Com-
pound 57 shows limited micro-
somal stability, so the decrease
in oral activity of the compound
is likely to be due to poorer ex-
Table 2. Activity data for C-ring variants against Plasmodium falciparum.
[d]
Compd[a]
R1
EC50[b] [mm]
cLogD[c]
Solubility [mm]
CLint
P. falc.
L6 cells
(pH 7.4)
water
[mLminmgÀ1 protein]
55
56
57
58
59
–
H
0.061
0.047
0.014
0.021
0.15
56
54
29
36
34
2.8
4.0
4.2
4.5
4.7
>125
>125
>125
>125
n.d.
n.d.
69
286
381
n.d.
CH3-
CH3CH2
CH3CH2CH2
60
0.23
65
2.8
n.d.
n.d.
posure as
a consequence of
61
62
(CH3)2NCH2CH2-
0.25
0.23
35
43
2.4
5.5
n.d.
n.d.
n.d.
n.d.
first-pass metabolism, but fur-
ther work is required to confirm
this hypothesis. Compound 43
failed to show significant activity
in vivo, despite showing good
potency in vitro. This might be
due to poor pharmacokinetics,
but this needs to be further in-
vestigated.
63
64
0.046
0.077
54
38
5.9
4.5
89
n.d.
n.d.
>125
[a] Overall yields: 50–90%; reference compounds: chloroquine, EC50 =0.019–0.066 mm (P. falciparum K1); podo-
phyllotoxin, EC50 =0.012 mm (L6 cells). [b] The EC50 values are the mean of two independent assays, which
varied less than Æ50%; n.d=not determined. [c] Calculated using StarDrop (http://www.optibrium.com). [d] In-
trinsic clearance determined in vitro using mouse liver microsomes at the University of Dundee (UK).
Conclusions
it represents a compound with good potency against the ma-
laria parasite and very good selectivity compared with mam-
malian cells, albeit with a slightly increased microsomal insta-
bility. Compounds 56 and 57 were chosen as C-ring-modified
derivatives, and also for their potency, selectivity and clogD
value. Compounds 57 and 58 have very similar properties;
compound 57 was chosen as it has marginally better proper-
ties. Multidose oral efficacy studies of compounds 43, 56, and
57 against green fluorescent protein (GFP)-transfected P. ber-
ghei ANKA-infected mice were conducted according to the
standard protocol (Peters), with test compounds administered
either per oral (po) or via intraperitoneal (ip) injection (Table 3).
Modifications to our prototypical lead compound TDR32750
have been shown to yield compounds that retain in vitro activ-
ity. These changes include removal of the ester functionality
that might be a point of metabolism and addition of a basic
centre that should be helpful in increasing the solubility. Al-
though these changes have not yet provided compounds with
good oral in vivo activity, the results do suggest that there is
further scope for the optimisation of the metabolic and physi-
cochemical properties of this series, which could potentially
lead to orally active antimalarial compounds.
Experimental Section
Table 3. In vivo antimalarial activity against green fluorescent protein
(GFP)-transfected Plasmodium berghei ANKA.[a]
Profiling software: StarDrop version 5.3 with the P450 metabolism
plug-in module (http://www.optibrium.com) was used to predict
the sites of metabolism of the compounds. The same software was
used to calculate the cLogD values for all compounds.
Compd
Dose
[mgkgÀ1 dayÀ1
Route [%] Reduction Survival
]
parasitaemia
[days]
43
56
57
50
50
50
ip
ip
ip
10.0
21.8
99.9
4
4
13.7
Chemistry: Chemicals and solvents were purchased from Sigma–
Aldrich or Fluka and were used as received unless otherwise
stated. Air- and moisture-sensitive reactions were carried out under
an inert atmosphere of argon in oven-dried glassware. Analytical
thin-layer chromatography (TLC) was performed on precoated TLC
plates (layer 0.20 mm silica gel 60 with fluorescent indicator
UV254; Merck). Developed plates were air-dried and analysed
under a UV lamp (254/365 nm). Flash column chromatography was
performed using prepacked silica gel cartridges (230–400 mesh,
40–63 mm; SiliCycle) using a Teledyne ISCO Combiflash Companion
or Combiflash Retrieve. Microwave irradiation was conducted using
a Biotage Initiator unit. The machine consists of a continuous fo-
cused microwave power delivery system with operator-selectable
43
56
57
100
100
100
po
po
po
0.0
13.6
50.5
4
4
8
Chloroquine
TDR32570
Untreated control
10
100
–
ip
po
–
99.97
25.5
–
20
7.7
7
[a] Animals were dosed four times a day at the stated dose. Route of ad-
ministration: per oral (po); intraperitoneal (ip). Formulation: 10% DMSO
in water.
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power output (0–400 W at 2.45 GHz). H NMR and 13C NMR spectra
were recorded on a Bruker Avance II 500 spectrometer (1H at
500.1 MHz; 13C at 125.8 MHz) or a Bruker DPX300 spectrometer (1H
at 300.1 MHz). Chemical shifts (d) are expressed in ppm recorded
using the residual solvent as the internal reference in all cases.
Signal splitting patterns are described as singlet (s), doublet (d),
triplet (t), quartet (q), pentet (p), multiplet (m), broad (br), or a com-
bination thereof. Coupling constants (J) are quoted to the nearest
0.1 Hz. LCMS analyses were performed with either an Agilent HPLC
1100 series connected to a Bruker Daltonics MicroTOF or an Agilent
Technologies 1200 series HPLC connected to an Agilent Technolo-
gies 6130 quadrupole spectrometer, where both instruments were
connected to an Agilent diode array detector. Liquid chromatogra-
phy–mass spectrometry (LCMS chromatographic separations were
conducted with a Waters X bridge C18 column (50 mmꢁ2.1 mm,
3.5 mm particle size), with a mobile phase of water/acetonitrile+
0.1% HCOOH, or water/acetonitrile+0.1% NH3, using a linear gra-
dient from 80:20 to 5:95 over 3.5 min and then held for 1.5 min, at
a flow rate of 0.5 mLminÀ1. All compound samples evaluated in
biological assays had a measured purity of ꢀ95% (by total ion cur-
rent (TIC) and UV) as determined using this analytical LCMS
system. High-resolution mass spectrometry (HRMS) using electro-
spray ionisation was performed on a Bruker Daltonics MicrOTOF
mass spectrometer.
Alternate method for the synthesis of 4-(2,5-Dimethylpyrrol-1-
yl)piperidine (10): A solution of 1-benzyl-4-(2,5-dimethyl-1H-pyrrol-
1-yl)piperidine (6) (3.8 g, 0.014 mol) in MeOH (10 mL) was treated
with a catalytic amount of Pd(OH)2. The mixture was stirred under
a hydrogen atmosphere at RT overnight. Upon completion, the re-
action mixture was filtered through Celite, and the filtrate was con-
centrated in vacuo to afford product 10 as a colourless oil, which
was used without further purification (2.4 g, 96%); 1H NMR
(500 MHz, CD3OD): d=5.79 (s, 2H), 3.94–3.88 (m, 1H), 3.17–3.13
(m, 2H), 2.66–2.60 (m, 2H), 2.23 (s, 6H), 2.10–2.02 (m, 2H), 1.77–
1.74 ppm (m, 2H); MS (ESI+): m/z (%): 179.5 [M+H]+ (100).
1
General procedure A for the preparation of 2,5-dimethyl-1-(1-
substituted-piperidine)-3-formylpyrroles (16a–z, 16aa–aj): POCl3
(6 mmol, 6 equiv) was added dropwise to ice-cooled DMF (12 mL)
under a N2 atmosphere. The mixture was allowed to warm to RT
over 15 min, then a solution of the appropriate 2,5-dimethyl-1-(1-
substituted-piperidine)-1H-pyrrole (11 a, 12a–s, 13a-i, 14a–e, 15)
(1 mmol, 1 equiv) in DMF (5 mL) was added, and the mixture was
heated at 1008C for 3 h. After cooling, 30% aq NaOH was added
dropwise to adjust the solution to approximately pH 10. The result-
ing precipitate was isolated by filtration, and washed with water,
and dried in vacuo to afford the desired 2,5-dimethyl-1-aryl/substi-
tuted-aryl-3-formylpyrrole (16a–z, 16aa–aj) (80–95% yield).
General procedure B for the preparation of (E)-ethyl 5-((1-substi-
tuted-piperidin-4-yl)-2,5-dimethyl-1H-pyrrol-3-yl)methylene)-2-
1-Benzyl-4-(2,5-dimethyl-1H-pyrrol-1-yl)piperidine (6): 2,5-Hexan-
dione (4) (2.0 g, 17.5 mmol), 4-amino-1-benzylpiperidine (5) (4.0 g,
21.0 mmol) and para-toluenesulfonic acid (p-TsOH) bound to silica
gel (0.4 equivmolÀ1) were mixed in an oven-dried pressure vials
with magnetic stir bars, and heated twice (1808C, 15 min) under
microwave irradiation (0–400 W at 2.45 GHz) and then stirred for
15 min at RT. The reaction mixture was filtered, and the silica gel
residue was washed with CH2Cl2 (10 mL). The solvent was removed
in vacuo to give compound 6 as a light brown oil (3.8 g, 90%):
1H NMR (500 MHz, CDCl3): d=7.43–7.33 (m, 5H), 5.82 (s, 2H), 4.01–
3.94 (m, 1H), 3.64 (s, 2H), 3.11–3.09 (m, 2H), 2.39 (br s, 6H), 2.38–
2.32 (m, 2H), 2.19–2.12 (dt, 2H, J=11.9 Hz), 1.89–1.85 ppm (m,
2H); MS (ESI+): m/z (%): 269.4 [M+H]+ (100).
methyl-4-oxo-4,5-dihydro-1H-pyrrole-3-carboxylates (17–54):
A
solution of 3 (1.0 molar equiv) in abs EtOH (3 mL) was treated with
the appropriate 2,5-dimethyl-1-(1-substituted-piperidine)-3-formyl-
pyrrole (16a–z, 16aa–aj) (1.0 molar equiv) and KHSO4 (0.2 molar
equiv). The reaction mixture was heated at 70–808C for 3 h and
then poured onto crushed ice and filtered to afford the desired
product (17–54) as a yellow powder (80–95% yield).
(E)-Ethyl 5-((1-(1-benzylpiperidin-4-yl)-2,5-dimethyl-1H-pyrrol-3-
yl)methylene)-2-methyl-4-oxo-4,5-dihydro-1H-pyrrole-3-carbox-
1
ylate (17): Yellow powder (0.100 g, 40%): mp: 150–1558C; H NMR
(500 MHz, CD3OD): d=10.13 (s, 1H, NH), 7.36–7.30 (m, 5H), 6.65 (s,
1H), 6.51 (s, 1H), 4.12–4.07 (m, 3H), 3.55 (br s, 2H), 2.95 (d, 2H, J=
7.0 Hz), 2.53 (s, 3H), 2.43 (t, 2H, J=5.7 Hz), 2.36 (s, 3H), 2.32 (s,
3H), 2.16 (d, 2H, J=9.1 Hz), 1.75 (d, 2H, J=10.4, Hz), 1.21 ppm (t,
3H, J=7.1 Hz); 13C NMR (125 MHz, [D6]DMSO): d=180.3, 178.3,
169.2, 164.2, 163.7, 163.3, 134.6, 130.1, 128.8, 128.5, 128.1, 126.9,
117.4, 113.0, 110.1, 102.4, 64.0, 61.7, 58.1, 57.8, 55.1, 54.6, 52.6, 30.5,
15.7, 14.4, 11.0 ppm; MS (ESI+): m/z (%): 448.3 [M+H]+ (100);
HRMS–ESI: m/z [M+H]+ calcd for C28H34N3O3: 448.2469, found:
448.2468.
1-(1-Benzylpiperidin-4-yl)-2,5-dimethyl-1H-pyrrole-3-carbalde-
hyde (7): General procedure A (below) was used to give desired
product 7 as a dark brown oil (3.5 g, 92%): 1H NMR (500 MHz,
CDCl3): d=9.78 (s, 1H, CHO), 7.36–7.25 (m, 5H), 6.25 (s, 1H), 4.00–
3.93 (m, 1H), 3.56 (m, 2H), 3.08–3.01 (m, 2H), 2.58 (s, 3H), 2.39–
2.33 (m, 2H), 2.31 (s, 3H), 2.13–2.08 (dt, 2H, J=11.8 Hz), 1.80–
1.77 ppm (m, 2H); MS (ESI+): m/z (%): 297.9 [M+H]+ (100).
tert-Butyl 4-(2,5-dimethyl-1H-pyrrol-1-yl)piperidine-1-carboxylate
(9): The procedure for 6 (see above) was used to give compound 9
(E)-Ethyl
5-((2,5-dimethyl-1-(2-(trifluoromethyl)phenyl)-1H-
1
as a brown oil (3.0 g, 62%): H NMR (500 MHz, CDCl3): d=5.68 (s,
pyrrol-3-yl)methylene)-2-((E)-2-(dimethylamino)vinyl)-4-oxo-4,5-
dihydro-1H-pyrrole-3-carboxylate (55): A mixture of TDR32750
(0.02 g, 0.004 mmol) and DMF–diethyl acetal (3 mL) was heated at
reflux for 15 min. After cooling, the precipitate was isolated by fil-
tration, washed with ice-cold water, and dried in vacuo to afford
desired enamino ester 55 as a yellow powder (0.22 g, 89%): mp:
270–2758C; 1H NMR (500 MHz, [D6]DMSO): d=9.05 (s, 1H, NH),
8.14 (d, 1H, J=13.3 Hz), 8.00 (d, 1H, J=7.4 Hz), 7.91 (t, 1H, J=
7.4 Hz), 7.81 (t, 1H, J=7.7 Hz), 7.47 (d, 1H, J=7.8 Hz), 6.64 (s, 1H),
6.32 (s, 1H), 6.09 (d, 1H, J=13.3 Hz), 4.13–4.07 (m, 2H), 2.51 (q,
6H, J=7.1 Hz), 1.95 (s, 3H), 1.91 (s, 3H), 1.23–121 ppm (m, 3H, J=
2.4, 7.05 Hz); 13C NMR (125 MHz, [D6]DMSO): d=179.9, 170.2, 165.7,
164.2, 163.2, 151.7, 134.2, 132.4, 131.8, 130.2, 114.2, 113.5, 109.3,
106.4, 105.9, 102.4, 102.3, 96.8, 84.8, 57.6, 15.8, 14.5, 14.4, 12.0,
2H), 4.20 (br s, 2H), 3.96 (tt, 1H, J=7.5, 4.5 Hz), 2.67 (t, 2H, J=
7.5 Hz), 2.21 (s, 6H), 2.04 (td, 2H, J=12.4 Hz), 1.74 (d, 2H, J=
13.5 Hz), 1.41 ppm (br s, 9H); MS (ESI+): m/z (%): 279.4 [M+H]+
(100).
4-(2,5-Dimethylpyrrol-1-yl)piperidine (10): A solution of 9 (3.0 g,
0.010 mol) in 4m HCl/dioxane (30 mL) was stirred at 08C for 3 h.
Once the reaction was complete, the solvent was removed in
vacuo. The residue was redissolved in CH2Cl2 (30 mL), washed with
10% aq NaOH (30 mL), dried over MgSO4, filtered and concentrat-
ed in vacuo to afford product 10 as a colourless oil (1.8 g, 94%):
1H NMR (500 MHz, CD3OD): d=5.79 (s, 2H), 3.94–3.88 (m, 1H),
3.17–3.13 (m, 2H), 2.66–2.60 (m, 2H), 2.23 (s, 6H), 2.10–2.02 (m,
2H), 1.77–1.74 ppm (m, 2H); MS (ESI+): m/z (%): 179.5 [M+H]+
(100).
ꢀ 2013 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2013, 8, 1537 – 1544 1543

CHEMMEDCHEM
FULL PAPERS
www.chemmedchem.org
10.2 ppm; HRMS–ESI: m/z [M+H]+ calcd for C25H27F3N3O3:
474.1999, found: 474.2002.
This material is available free of charge on the WWW under http://
dx.doi.org/10.1002/cmdc.201300177.
General procedure for the preparation of pyrrolones 56–64: A
mixture of enamino ester 55 (1.0 equiv) and the appropriate pri-
mary amine (4.0 equiv) in iso-propanol was heated at reflux for 6 h
or irradiated by microwave (0–400 W at 2.45 GHz) at 1108C for 15–
30 min and then cooled. The resultant precipitate was isolated by
filtration, washed with ice-cold MeOH or EtOH, and dried in vacuo
to afford the desired product (56–64).
Acknowledgements
This investigation was done for and received support from the
UNICEF/UNDP/World Bank/WHO Special Programme for Research
and Training in Tropical Diseases (TDR). The University of Dundee
would also like to acknowledge the Wellcome Trust (grant
083481) for support.
(E)-5-Benzyl-2-((2,5-dimethyl-1-(2-(trifluoromethyl)phenyl)-1H-
pyrrol-3-yl)methylene)-1H-pyrrolo[3,2-c]pyridine-3,4(2H,5H)-
dione (62): Isolated as a yellow powder (0.22 g, 89%): mp: 195–
1
2008C; H NMR (500 MHz, [D6]DMSO): d=9.45 (s, 1H, NH), 8.00 (d,
Keywords: antiprotozoal agents
· malaria · Plasmodium
1H, J=7.8 Hz), 7.93 (t, 2H, J=7.6 Hz), 7.83 (t, 1H, J=7.2 Hz), 7.68
(d, 2H, J=8.2 Hz), 7.59 (d, 2H, J=8.2 Hz), 7.49 (d, 1H, J=7.8 Hz),
6.83 (br s, 2H), 6.54 (s, 1H), 6.38 (d, 1H, J=7.2 Hz), 5.27–5.26 (m,
2H), 1.82 ppm (s, 6H); 13C NMR (125 MHz, [D4]acetone): d=181.7,
170.8, 162.3, 161.0, 158.2, 145.2, 143.6, 136.0, 134.8, 132.6, 132.3
(2C), 131.0 (2C), 130.8, 130.3, 129.3, 129.2, 128.3, 126.3, 126.2, 115.6,
108.7, 106.5, 95.6, 50.6, 12.3, 10.7 ppm; MS (ESI+): m/z (%): 490.18
[M+H]+ (100); HRMS–ESI: m/z [M+H]+ calcd for C28H23F3N3O2:
490.1737, found: 490.1752.
falciparum · pyrrolones · structure–activity relationships
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In vivo efficacy studies: The protocols are described in the Sup-
porting Information. In vivo efficacy studies in mice were conduct-
ed at the Swiss Tropical & Public Health Institute (Basel, Switzer-
land) according to the rules and regulations for the protection of
animal rights (“Tierschutzverordnung”) of the Swiss “Bundesamt fꢂr
Veterinꢃrwesen”. They were approved by the veterinary office of
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Supporting Information
Soc. Perkin Trans. 2 1989, 713–717.
Supporting Information contains: synthetic routes for the synthesis
of intermediates and characterisation data for compounds not in-
cluded in the main text; methods for physicochemical evaluation
of the compounds and the data generated; methods for assessing
metabolic stability; in vitro and in vivo parasite testing.
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Received: April 21, 2013
Published online on August 5, 2013
ꢀ 2013 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2013, 8, 1537 – 1544 1544