Discovery and structure-activity relationships of pyrrolone antimalarials

Article abstract of DOI:10.1021/jm400009c

In the pursuit of new antimalarial leads, a phenotypic screening of various commercially sourced compound libraries was undertaken by the World Health Organisation Programme for Research and Training in Tropical Diseases (WHO-TDR). We report here the detailed characterization of one of the hits from this process, TDR32750 (8a), which showed potent activity against Plasmodium falciparum K1 (EC₅₀ ～ 9 nM), good selectivity (>2000-fold) compared to a mammalian cell line (L6), and significant activity against a rodent model of malaria when administered intraperitoneally. Structure-activity relationship studies have indicated ways in which the molecule could be optimized. This compound represents an exciting start point for a drug discovery program for the development of a novel antimalarial.

Full text of DOI:10.1021/jm400009c

Article
pubs.acs.org/jmc
Discovery and Structure−Activity Relationships of Pyrrolone
Antimalarials
Dinakaran Murugesan,†,○ Alka Mital,†,○ Marcel Kaiser,#,‡ David M. Shackleford,§ Julia Morizzi,§
Kasiram Katneni,§ Michael Campbell,§ Alan Hudson,∥ Susan A. Charman,§ Clive Yeates,⊥
and Ian H. Gilbert*,†
†Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Sir James Black Centre,
Dundee, DD1 5EH, U.K.
§Centre for Drug Candidate Optimisation, Monash Institute of Pharmaceutical Sciences, Monash University (Parkville Campus), 381
Royal Parade, Parkville, Victoria 3052, Australia
#Swiss Tropical and Public Health Institute, Postfach, Socinstrasse 57, 4002 Basel, Switzerland
‡University Basel, Petersplatz 1, 4003 Basel, Switzerland
⊥InPharma Consultancy, Herts, U.K.
∥Pharmacons, Kent, U.K.
S
* Supporting Information
ABSTRACT: In the pursuit of new antimalarial leads, a phenotypic screening of various
commercially sourced compound libraries was undertaken by the World Health Organisation
Programme for Research and Training in Tropical Diseases (WHO-TDR). We report here the
detailed characterization of one of the hits from this process, TDR32750 (8a), which showed
potent activity against Plasmodium falciparum K1 (EC50 ∼ 9 nM), good selectivity (>2000-fold)
compared to a mammalian cell line (L6), and significant activity against a rodent model of malaria
when administered intraperitoneally. Structure−activity relationship studies have indicated ways in which the molecule could be
optimized. This compound represents an exciting start point for a drug discovery program for the development of a novel
antimalarial.
efficacious in a P. berghei mouse model when given intra-
peritoneally (ip), but was relatively inactive orally.
INTRODUCTION
■
In recent years, the emergence of drug-resistant pathogens has
led to treatment failures for many infectious diseases, such as
malaria. Resistance to current antimalarials is a major problem,
e.g., chloroquine, sulfadoxine-pyrimethamine, and in some areas,
mefloquine.1 To reduce the spread of resistance, the World
Health Organisation (WHO) recommends treatment for malaria
to be artemisinin combination therapy; although worryingly
there are now reports of increased parasite clearance times with
the artemisinins2−7 which may herald resistance. Thus, there is
an urgent need for novel classes of compounds that are effective
against these re-emerging infections. With the elimination of
malaria now considered as a goal, it would be useful for scientists
and policy makers to have an expanded arsenal of antimalarials
that could help make this goal a reality.8,9
As part of a program for the discovery of new starting points
for drug discovery programmes, the World Health Organisation
Programme for Research and Training in Tropical Diseases
(WHO-TDR) phenotypically screened various compound
libraries. One of these was a 5000 strong structurally diverse
compound collection from ChemDiv, which was screened
against Plasmodium falciparum, the causative organism for the
most pathogenic form of malaria. This led to the identification of
pyrrolone (8a) shown in Figure 1. This compound had potent
activity against P. falciparum K1, with an EC50 ∼9 nM, and was
The screening of several hundred commercially available
compounds related to 8a containing the pyrrolone motif gave
some indication of structure−activity relationships (SAR)
(Figure 1). Here we report the further evaluation of 8a as a
novel antimalarial lead, with further systematic SAR studies, in
parallel with studies to assess drug metabolism and pharmaco-
kinetics (DMPK), with the initial aim of achieving oral efficacy in
the P. berghei mouse model, to establish the potential for further
development of the pyrrolones as antimalarials.
RESULTS AND DISCUSSION
■
Chemistry. An efficient synthesis of 8a and congeners was
developed (Scheme 1), through acylation of ethyl-3-amino-
crotonate (1) with chloroacetyl chloride10 and subsequent
cyclization to the pyrrolone 3,10 which was not stored, but used
immediately, due to its relatively poor stability. The
condensation of 3 with 3-formyl pyrroles (7a−x) in the presence
of potassium hydrogen sulfate11,12 generated the (E)-isomer
a
predominantly. This three-step sequence could be carried out in
yields of up to ∼60%, and only required chromatography at step
Received: January 3, 2013
Published: March 21, 2013
© 2013 American Chemical Society
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Figure 1. Initial SAR of lead 8a.
Figure 2. Strategy for elaboration of SAR around 8a.
a
Scheme 1. General Synthetic Approach to Pyrrolones
a
(a) Chloroacetyl chloride, pyridine, 0 °C, 30 min, 75%; (b) KOH, EtOH, 95%; (c) p-toluenesulfonic acid bound with silica gel, 15−20 min,
microwave, 80−90%, or p-toluenesulfonic acid, toluene, 90 °C, 3 h Dean−Stark apparatus; (d) POCl3, DMF, 100 °C, 3 h, 80−95% ; (e) KHSO4,
EtOH, 3 h, reflux, 80−95%; R and R1 as defined in Tables 1 and 3.
1. The 3-formylpyrroles (7a−x) were obtained through
condensation of the appropriate aniline with 2,5-hexandione
(4) (Paal−Knorr pyrrole synthesis) and subsequent Vilsmeier−
Haack formylation.13,14 The use of silica-supported p-toluene-
sulfonic acid in the condensation of anilines with 4, in a solvent-
free procedure using microwave heating, provided a considerable
rate advantage over classical procedures (i.e., reaction complete
within 15−20 min compared to over 12 h with external heating);
even relatively non-nucleophilic amines were condensed
smoothly in the absence of Lewis acid catalysts.
Ring A. Using the approach outlined in Scheme 1, a number of
analogues at the A ring (Table 1) were prepared by varying the
aniline or amine 5a−x, with a particular aim of increasing the
solubility. This provided a means of incorporating solubilizing
groups either within the aromatic ring (e.g., pyridine analogues
8q and 8r), or as pendant amines (8y and 8aa), morpholines
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d
Table 1. LogD/LogP, Kinetic Solubility, in Vitro Intrinsic Clearance and Activity against P. falciparum for Ring A Variants
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Table 1. continued
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Table 1. continued
a
b
c
Value measured using a chromatographic gLogD technique. Estimated using nephelometry. In vitro intrinsic clearance determined in mouse
d
hepatic microsomes. Chloroquine, EC50 P. falciparum K1 0.095−0.172 μM; podophyllotoxin EC50 L6 cells 0.009−0.022 μM. The EC50 values are
the means of two independent assays and varies less than 50%. n.d = not determined.
(8u), or sulfones (8n). The aryl ring was also replaced with a
simple alkyl group (8t).
A number of B-ring variants in which an aryl ring replaces the
pyrrole (9j−m) were prepared using a Suzuki coupling to make
the requisite aldehyde intermediates (Scheme 4, Table 2).
Ring C. Concern that hydrolysis of the ethyl ester in 8a might
adversely affect oral bioavailability and the elimination half-life
led to an investigation of potentially more stable ester (Scheme
1) and amide derivatives, accessible from the carboxylic acid 10c
(Scheme 5, Table 3). Base catalyzed hydrolysis of the ethyl ester
8a to generate 10c proved problematic due to decarboxylation to
10d under the conditions required. Decarboxylation could be
reduced to a minimum (∼10%) by utilizing acid-catalyzed
deprotection of the tert-butyl ester derivative 10a, with 10d
readily removed by chromatography. The effect of methylating
the pyrrolone NH was also investigated.
Ring B. The methyl groups on the pyrrole were predicted
computationally to be likely sites of metabolic vulnerability, and
it was therefore of interest to investigate the effect of
modifications of the pyrrole, including removing the methyl
groups (9a−b) and replacing them with ethyl groups (9c). The
3-formyl pyrrole intermediates were prepared according to
Scheme 2. Variants in which the B-ring pyrrole was replaced with
other 5-ring heterocycles (imidazole, pyrazole, triazole, or
thiazole) were synthesized as in Scheme 3. For the furan (9n−
o) and chloro pyrazole (9p) derivatives, the requisite aldehydes
were commercially available.
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a
Scheme 2. Synthesis of Pyrrole B-ring Variants
a
(a) Acetic acid, 90 °C, 3 h; (b) KHSO4, EtOH, reflux, 3 h; (c) 2-trifluoromethylaniline, p-toluenesulfonic acid bound with silica gel, 90 °C, reflux, 3
h; (d) POCl3, DMF, 100 °C, 3 h; (e) MeLi (3 M) in DME, THF, 0 °C − rt, 23 h, 75 °C for 17 h.
In Vitro Biological Activity. Compounds were evaluated
against P. falciparum (K1) strain and counter-screened for
cytotoxicity against mammalian L6 cells (Tables 1−3).
Compound 8a retained activity across a variety of drug-sensitive
and -resistant strains (Table 4) and showed a high degree of
selectivity for P. falciparum relative to L6.
The activities of the compounds varied from 0.008 μM to 12
μM; the most active 8a−c, 8m and 9c had P. falciparum (K1)
EC50 values of 0.008−0.021 μM, superior to chloroquine and the
majority of the compounds showed good selectivity (>1000-
fold) for P. falciparum compared to L6 mammalian cells. From
the variations across rings A−C the following SAR was noted:
Ring A (Table 1). (1) Replacing the phenyl ring with a methyl
group (8t) led to a significant loss of activity (600-fold),
suggesting that a hydrophobic group was required. Replacing the
phenyl (8s) with a benzyl (8x) resulted in a small drop in activity
(4-fold), indicating that the phenyl ring did not need to be
attached directly to the pyrrole.
ortho (8a), meta (8c), or para (8b) derivatives. An ortho CF3
would be expected to reduce the conformational flexibility of ring
A by hindering coplanarity of the pyrrole and phenyl rings, but
this did not seem to have an effect on either activity or solubility
(see below). Compounds 8a−c were only ∼5−10-fold more
active than the unsubstituted derivative 8s, consistent with the
idea that the lipophilicity contributed by ring A is the most
significant variable in this part of the pyrrolones affecting in vitro
activity.
(3) Further variation of substitution on the aromatic ring
focused on the para position, as this is likely to be the most
susceptible aryl position to cytochrome P450 (CYP)-mediated
oxidation. In general, a wide variety of substituents were tolerated
with relatively slight variation in potency, with the notable
exception of the sulfoxide (8o), which was ∼100-fold less active
than the corresponding sulphide (8p) and sulfone (8n).
Similarly, the meta-substituted hydroxyl derivative (8l) was not
tolerated, while the meta-substituted methoxy (8k) was.
Solubilizing substituents were tolerated to a degree: although
the pyridine derivatives (8q−r) and dimethylamine (8aa)
(2) In varying the position of the CF3 on the aryl ring there
seemed to be relatively little difference in activity comparing the
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a
Scheme 3. Synthesis of Formyl Pyrazole, Thiazole, Imidazole, and Triazole Intermediates
a
(a) aq EtOH, HCl 90 °C, 3 h; (b) POCl3, DMF, 100 °C, 3 h; (c) acetylacetone, 90 °C, 3 h; (d) ethyl-2-chloroacetoacetate, EtOH, reflux; (e)
LiAlH4, THF, 0 °C; (f) PCC, CH2Cl2, rt; (g) PCl5 in CHCl3, reflux, 3 h; (h) tetrachloroethylene, 5-aminopyrimidine, few drops of POCl3; (i) t-
BuONO/TMSN3, propargyl alcohol, MeCN; (j) Dess−Martin Reagent, CH2Cl2.
furan (9n−o) or isoxazole (9q) gave a significant loss of activity
(∼20−1000-fold).
Scheme 4. Synthesis of Aldehyde Intermediates for B-Ring
Aryl Derivatives: (a) Tetrabutyl Ammonium Bromide
(TBAB), K2CO3, Pd(OAc)2, Dioxane/Water (1:1)
Ring C (Table 3).
1. Methylation of the NH (11a) resulted in a significant
reduction of potency (>100-fold) relative to 8a, consequently no
further N-substituted derivatives were investigated.
2. The potential for degradation of 8a through cleavage of the
ester by esterases was of concern, although 8a was fairly resistant
to chemical hydrolysis. Both the free acid 10c and the product
from decarboxylation (10d) were significantly less active (∼50−
150-fold) than 8a.
3. All modifications of the ester in 8a, including amides 10f−m,
resulted in considerably reduced activity, although with the
benzyl ester 10b the reduction in activity was relatively moderate
(∼5-fold). The amides were inactive, which may be due to them
adopting different conformations.
derivatives lost significant activity, the sulphone (8n) retained
activity, while the piperidine (8y) and the morpholine (8z) were
only ∼5−10-fold less active than 8a.
Ring B (Table 2). The methyl substituents on the pyrrole were
identified as potential points of CYP-mediated metabolism.
1. Removal of the methyl substituents (9a, 9b) resulted in a
significant (∼20−25-fold) loss in activity.
2. Replacing both methyls with ethyl (9c) did not significantly
affect activity.
3. Replacement of the pyrrole with imidazole (9e), pyrazole
(9f, 9g, 9p, 9q, and 9s), triazole (9h), thiazole (9i), aryl (9j−m),
4. Reduction of the double bond linking the pyrrolone and
pyrrole rings would be expected to improve solubility by
reducing planarity. However the reduced derivative 11b proved
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Table 2. cLogP, Kinetic Solubility, in Vitro Intrinsic Clearance and in Vitro Activity against P. falciparum for Ring B Variants
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Table 2. continued
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Table 2. continued
a
b
Estimated using nephelometry. In vitro intrinsic clearance determined in mouse hepatic microsomes, n.d. = not determined.
a
Scheme 5. Synthetic Approach to Amide Derivatives
a
(a) HCl (6 M), 3 h, reflux, 80−95% (ratio 10c/10d ∼ 90:10); (b) N,N-carbonyldiimidazole, THF, reflux, excess amine in MeOH, 0° C, 18-24 h;
(c) TBTU, DIPEA, secondary amine, DMF, 0 °C; 18−24 h.
significantly less active (>10-fold) than 8a; furthermore 11b was
less stable chemically.
of P. berghei when dosed ip at 100 mg/kg once daily for 4 days,
with >99% reduction in parasitaemia, comparable to chloroquine
at 10 mg/kg; no dose-ranging experiments were undertaken.
Chloroquine was dosed ip to give comparison to a known drug
by the same route. Both 8a and 8b were relatively inactive when
given orally (Table 5). Although full PK studies were not
undertaken in mice, the oral exposure of 8a was evaluated in mice
at a single dose of 50 mg/kg (formulated as a suspension in
PEG400). This confirmed that relatively low plasma levels of 8a
Thus there appears to be scope for modification of the aryl A-
ring, with less room for maneuver around the 2,5-dimethyl
pyrrole in ring B. As only fairly conservative modifications to ring
C have been investigated there may be further potential in
modifying the pyrrolone.
In Vivo Efficacy Studies in P. berghei Mouse Model.
Compounds 8a and 8b showed good activity in the mouse model
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Table 3. LogD/LogP, Kinetic Solubility, in Vitro Intrinsic Clearance and in Vitro Activity against P. falciparum for Ring C Variants
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Table 3. continued
a
b
c
Value measured using a chromatographic gLogD technique. Estimated using nephelometry. In vitro intrinsic clearance determined in mouse
hepatic microsomes. n.d. = not measured.
Table 4. In Vitro Antiplasmodial and Cytotoxic Activities of 8a
P. falciparum EC50 (nM)
cytotoxicity EC50 (nM)
a
b
c
d
e
K1
D6
W2
TM90C2B
TM91C235
L6
8a
2−28 (n = 7)
63−430
2.2−3.8
14
8.4
260
4.9
11−14
250
8.6−9.3
140
15000
chloroquine
mefloquine
artemesinin
11
9.8
27
6.0
a
b
c
d
K1 = chloroquine and pyrimethamine resistant (STI). D6 = chloroquine sensitive. W2 = chloroquine resistant (WRAIR). TM90C2B =
e
atovaquone resistant (WRAIR). TM91C235 = multidrug-resistant + atovaquone sensitive (WRAIR).
were achieved (<0.01 μM after ∼5 h), which is consistent with
the lack of oral efficacy in the mouse P. berghei model.
(8r), this is probably due to protonation at pH 2.0, but not at pH
6.5; although the reason for the high pH dependence on
solubility is not clear for (8k) and (9c). Similarly compounds
with a pendant amine also showed greater solubility (8y, 8z, 8aa)
as did those with a pyridine replacement for the phenyl ring (8q,
8r).
Physicochemical Properties and in Vitro DMPK. The
molecules generally showed reasonable physicochemical proper-
ties (MW in the region of 350−450, 1 HBD, 5−7 HBA, PSA in
the range 60−100 Å2), compatible with good membrane
permeability, but the aqueous solubility of most compounds
was generally very low (Tables 1−3). The low solubility is likely a
consequence of the conjugated planar nature of the molecules,
and in some cases the lipophilicity. For some compounds,
particularly (8i, 8k, 8r) and (9c), there was a reduction in the
solubility between pH 2.0 and pH 6.5. In the case of (8i) and
Attempts to improve the solubility were made as follows:
• Addition of solubilizing groups such as morpholine (8u,
8z) and piperidine (8y) on ring A.
• Conversion of ester to amide linkages with various
solublizing groups such as methyl amine, piperidine,
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Table 5. In Vivo Antimalarial Activity of 8a in Mice Infected
with P. berghei
Table 6. Metabolic Stability Parameters for 8a and 8b Based
on NADPH-Dependent Degradation Profiles in Human/Rat/
Mouse Liver Microsomes
% reduction parasitaemia
dose (mg/kg/
a
T1/2
CLint
(μL/min/mg)
metabolites
detected
a
compound species
(min)
EH
day × 4)
route day 4
day 5 day 6 MSD
8a
8b
human
rat
58
44
30
40
50
49
31
53
0.62
0.56
0.68
0.73
0.50
0.70
none
P+16
none
none
none
none
Experiment 1
8a
100
100
10
ip
po
ip
97.72
25.51
99.9
98.35
5.3
99.6
0
11
7.7
11
5.7
mouse
human
rat
35
8a
35
chloroquine
control
56
mouse
32.9
Experiment 2
a
b
Half-life. In vitro intrinsic clearance.
8a
100
100
100
10
ip
99.93
10.7
11.7
7
8b
ip
99.91
37.5
8b
po
ip
bound), compounds were found to have moderate to high
plasma protein binding (93.7−98.8% bound).
chloroquine
chloroquine
control
99.97
99.91
20
100
po
>30
Compound 8a showed insignificant reactivity on incubation
with reduced glutathione ethyl ester, indicating that it does not
act as a Michael acceptor (which could adversely affect stability in
vivo and might give rise to toxicity), and no adducts resulting
from conjugation with glutathione were detected in the urine of a
rat dosed intravenously with 8a.
7
a
MSD = mean survival time (days).
morpholine, ethanolamine, dimethylethane-1,2-diamine,
diethylethane-1,2-diamine, 2-ethoxyethanamine.
The ester in both 8a and 8b is a potential point of metabolism
through cleavage by esterases (Figure 3). In rat blood and
plasma, ∼25% of 8a was hydrolyzed to the acid 10c over 4 h at 37
°C. Compound 10c was accompanied by traces of the
decarboxylated product 10d, consistent with the observed
chemical instability of 10c (although the formation of 10d
during the extraction and/or analytical procedure cannot be
ruled out).
In Vivo DMPK Studies. In order to better understand the
efficacy data, PK studies for compounds 8a and 8b were
conducted in rats, with iv, ip, and po administration (Table 7;
Figure 4; see Supporting Information for Methodology). No sign
of toxicity was observed when 8a or 8b were administered orally
or ip at doses up to ∼20 mg/kg. There was a slight degree of
hemolysis when the compounds were dosed iv, most likely
arising from the vehicle required for formulation. Both
compounds had terminal half-lives of at least 8 h, likely due to
the high volumes of distribution. Compound 8a exhibited high
plasma clearance (73 mL/min/kg), which in vitro studies suggest
is due to a combination of moderate hepatic metabolic clearance
and blood-mediated degradation, but not renal elimination given
that there was minimal recovery of 8a excreted unchanged in
urine. While plasma/blood stability studies were not conducted
with 8b, similar degradation would be expected given the
structural similarity to 8a.
Compound 8a is predicted to be uncharged at physiological
pH as are those compounds where amines are appended directly
to the phenyl ring C (8i, 8u) or the pyridine analogues (8q, 8r).
The more basic derivatives, where the amine is appended via a
methylene linker (8y, 8z, 8aa), are predicted to have varying
degrees of ionization, which correlates with their significantly
higher aqueous solubility.
Compounds showed moderate to high rates of degradation
when incubated with mouse liver microsomes (see Supporting
Information for methodology). The main exceptions to this were
(8u, 8aa) and (10d), which appeared to be slightly more stable.
Putative metabolites having molecular weight consistent with the
products of mono-oxygenation (P+16 for 8a, 8d, 8e, 8h, 8m, 8p,
8q, 9b, 9c, 10a, 10f), bis-oxygenation (P+32 for 8q, 10a), O-
demethylation (P-14 for 8i), and/or morpholine ring cleavage
(P-12 for 8u), were detected for a number of analogues (see
Supporting Information). Compound 10c showed an increased
rate of degradation in microsomes containing the dual cofactors
NADPH and uridine-5′-diphospho-glucuronic acid (UDPGA)
(the cofactors for CYP450-mediated metabolism and glucur-
onidation, respectively) relative to NADPH alone, suggesting
that this compound is susceptible to primary glucuronidation in
the microsomal test system. A putative glucuronide metabolite
(P+176) was also detected for 10c (see Supporting Informa-
tion).
It was predicted computationally that the methyl groups on
the pyrrole (ring B) were likely to be metabolically unstable.
Hence, these were removed (9a, 9b) or replaced with an ethyl
group (9c), but these compounds were found to have even lower
metabolic stability. All attempts to replace the pyrrole with other
heterocycles resulted in much reduced antimalarial activity.
In addition to investigating their stability with mouse liver
microsomes, key compounds (8a and 8b) were investigated in
rat and human liver microsomes, in order to obtain an idea of
species variability in metabolic degradation (Table 6). These
results suggested only marginal differences in microsomal
stability between species.
Both 8a and 8b had low oral bioavailability (7 and 5%,
respectively) which is likely due to the combined effect of high
first pass clearance and poor absorption resulting from the low
aqueous solubility. There is also the potential for hydrolysis of
the ester linkage in the gut, and during passage through the
enterocytes and the liver. Despite the low oral bioavailability, the
C
max of both compounds when dosed at 20 mg/kg po was about
10-fold higher than the EC50 against parasites in vitro.
CONCLUSIONS
■
From phenotypic screening a series of pyrrolone derivatives have
been identified with good in vitro activity against P. falciparum
combined with good selectivity relative to the L6 mammalian cell
line. A lack of cross resistance with standard antimalarials
suggests they may have a novel mode of action, although this has
not been investigated. While some of the pyrrolones have shown
Protein binding was investigated for selected compounds
(Supporting Information). With the exception of the pyridine
containing compounds (8q) (80% bound) and (8r) (79%
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Figure 3. Degradation of 8a in vivo through ester hydrolysis.
good in vivo activity in a P. berghei mouse model when
administered by the intraperitoneal route, oral activity has so
far proved elusive, likely resulting from a combination of poor
absorption due to the low aqueous solubility and rapid first-pass
metabolism through cytochrome P450-mediated oxidation and/
or esterase cleavage. The lack of oral activity is a key hurdle that
needs to be overcome in establishing the potential for further
development of this series. SAR studies in conjunction with in
vitro microsomal stability studies have indicated that there is
scope for modification at several points on the lead compounds.
Table 7. Pharmacokinetic Properties of 8a and 8b in Sprague
Dawley Rats
a
8a
8b
b
c
d
b
c
oral
20
iv
ip
oral
19
iv
dose (mg/kg)
apparent t1/2 (h)
% dose in urine
4.5
12
19
9.1
1.0
87
7.9
c.n.c.
n.d.
0.08
0.11
200
6−7
0.05
0.01
3.3
n.d.
C
T
max (μM)
0.19
100
max (min)
15
oral bioavailability F%
45.7
4−5
Vdss (L/kg)
13
73
19
22
EXPERIMENTAL SECTION
■
plasma clearance
(mL/min/kg)
Parasitology and DMPK methods are described in the Supporting
Information.
Profiling Software. StarDrop (www.optibrium.com) was used to
a
b
n.d. = not determined. c.n.c = could not calculate. Oral suspension
c
formulated with aqueous HPMC. iv solution formulated in aqueous
vehicle with 40% (v/v) propylene glycol. ip suspension formulated in
d
predict the sites of metabolism of the compounds.
Chemistry. All commercially available reagents, solvents, and starting
materials were purchased from Aldrich Chemical Co. (UK). Where
necessary a Biotage FLASH 25+ column chromatography system was
used to purify mixtures; reagent-grade solvents used for chromatog-
raphy were purchased from Fisher Scientific (UK) and flash column
chromatography silica cartridges were obtained from Biotage (UK).
Analytical thin-layer chromatography (TLC) was performed on
precoated TLC plates (layer 0.20 mm silica gel 60 with fluorescent
indicator UV254, from Merck). Developed plates were air-dried and
analyzed under a UV lamp (UV 254/365 nm). Microwave irradiation
was conducted using a BIOTAGE INITIATOR unit. The machine
consists of a continuous focused microwave power delivery system with
operator-selectable power output (0−400 W at 2.45 GHz). All 1H and
13C NMR spectra were recorded on a Bruker ARX-500 spectrometer
(500 and 125 MHz for 1H and 13C NMR, respectively). Chemical shifts
(δ) are reported in ppm relative to the residual solvent peak or internal
standard (tetramethylsilane), and coupling constants (J) are reported in
hertz (Hz). Data are reported as follows: chemical shift, multiplicity (br
= broad, s = singlet, d = doublet, t = triplet, m = multiplet), integration.
LC−MS 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 Technologies
6130 quadrupole spectrometer, where both instruments were connected
to an Agilent diode array detector. LC−MS chromatographic
separations were conducted with a Waters X bridge C18 column, 50
mm × 2.1 mm, 3.5 μm particle size; mobile phase, water/acetonitrile
+0.1% HCOOH, or water/acetonitrile +0.1% NH3; linear gradient from
80:20 to 5:95 over 3.5 min and then held for 1.5 min; flow rate of 0.5 mL
min−1. All assay compounds had a measured purity of ≥95% (by TLC
and UV) as determined using this analytical LC−MS system. High
resolution electrospray measurements were performed on a Bruker
Daltonics MicrOTOF mass spectrometer.
aqueous vehicle with 10% DMSO.
Ethyl (E)-3-Amino-2-(2-chloroacetyl)but-2-enoate (2). A sol-
ution of ethyl-3-aminocrotonate (2.0 g, 0.015 mol, 1.0 equiv) and
pyridine (1.2 g, 0.015 mol, 1.0 equiv) in diethylether (10 mL) was
cooled to 0 °C, and a solution of chloroacetylchloride (4.1 g, 0.037 mol,
2.4 equiv) in diethylether (5 mL) was added dropwise over 30 min,
maintaining the temperature at 0 °C. After the mixture was stirred for a
further 3 h at 0 °C the solvent was removed in vacuo. The resultant solid
was washed with cold water to yield 2 as a cream yellow powder (2.7 g,
87% yield), mp 131−132 °C. 1H NMR (500 MHz; DMSO-d6): δ 5.92
(br s, 2H, NH2), 4.57 (s, 2H, −CH2Cl), 4.27 (q, 2H,-OCH2, J = 7.2 Hz),
Figure 4. Plasma concentrations of 8a (top) and 8b (bottom) following
iv (filled circles), oral (open triangles), and ip (open squares, 8a only)
administration to male Sprague−Dawley rats at nominal iv doses of 5
mg/kg (8a) and 1 mg/kg (8b), and oral and ip doses of 20 mg/kg. Each
profile represents the average of n = 2 rats.
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2.36 (s, 3H, CH3), 1.36 (t, 3H, OCH2CH3, J = 7.2 Hz); 13C NMR (125
MHz, DMSO-d6): δ 190.6, 169.9, 168.3, 100.8, 60.5, 49.6, 24.6, 14.3; MS
(ESI) m/z 206.1 [M + H]+ 100%.
12.6, 10.7; LCMS m/z 419.1527 (M + 1); HRMS m/z 419.1579 ([M +
H]+; calcd for C22H21F3N2O3+ 419.1577.
Ethyl (5E)-5-[[2,5-Diethyl-1-[2-(trifluoromethyl) phenyl] pyr-
rol-3-yl]methylene]-2-methyl-4-oxo-1H-pyrrole-3-carboxylate
Ethyl 5-Methyl-3-oxo-1,2-dihydropyrrole-4-carboxylate (3).
Ethyl (E)-3-amino-2-(2-chloroacetyl)but-2-enoate (2) (2.0 g, 0.0097
mol) was dissolved in absolute ethanol (5 mL) and cooled to 0 °C.
Potassium hydroxide (1.09 g, 0.019 mol) was added, and the mixture
was stirred for 3 h at 0 °C and then acidified to pH 2.0 using 2 M HCl to
afford a yellow precipitate, which was washed with cold water to yield 3
as a yellow solid (1.6 g, 99% yield), mp 215 °C. 1H NMR (500 MHz;
DMSO-d6): δ 10.7 (br s, 1H, -OH), 9.4 (br s, 1H,-NH), 7.64 (s, 1H,
-NH), 6.05 (d, 1H, J = 2.4 Hz), 4.27 (q, 2H,-OCH2, J = 7.1 Hz), 4.08 (m,
2H, -OCH2), 3.80 (d, 2H), 2.4 (t, 3H, J = 1.6 Hz), 2.3(s, 3H), 1.8 (t, 3H,
J = 7.1 Hz), 1.2 (t, 3H, J = 7.0 Hz, OCH2CH3); 13C NMR (125 MHz,
DMSO-d6): δ 189.2, 171.5, 159.9, 99.3, 86.1, 60.4, 28.02, 14.2; MS (ESI)
m/z 170.14 [M + H]+ 100%.
General Procedure for the Microwave-Accelerated Synthesis
of 2,5-Dimethyl-1-aryl-1H-pyrroles (6a-6aa). A mixture of 2,5-
hexandione (4) (1 mmol), the appropriate aniline (5a-5aa) (1.2 mmol)
and p-toluenesulfonic acid bound with silica gel (0.4 equiv) was stirred
in an oven-dried pressure vial fitted with a magnetic stir bar. The vial was
then placed in a microwave oven and heated twice (180 °C, 5 min)
under microwave irradiation (0−400 W at 2.45 GHz). After being
stirred for 15 min at room temperature, the mixture was filtered and the
residual silica was washed with DCM (10 mL). The solvent was
removed under reduced pressure, affording the pyrrole 6a-6aa (purity
>95%, 80−90% yield), which was used without further purification.
General Procedure for the Synthesis of 2,5-Dimethyl-1-aryl-
3-formylpyrroles (7a-7aa). Phosphorus oxychloride (6 mmol) was
added dropwise to ice-cooled DMF (12 mL) stirred under a N2
atmosphere. The mixture was kept at room temperature for 15 min,
and then a solution of the requisite pyrrole 6a-6aa (1 mmol) in DMF (5
mL) was added and the mixture was heated at 100 °C for 3 h. After
cooling, 30% NaOH was added dropwise to adjust to pH 10. The
resultant precipitate was filtered and washed with water, affording the
2,5-dimethyl-1-aryl-3-formylpyrroles 7a-7aa (80−95% yield), which
were used without further purification.
1
(9c). Yellow powder, mp 245−250 °C; H NMR (500 MHz, DMSO-
d6): δ 10.31 (s, 1H, -NH), 8.01 (d, 1H), 7.92 (t, 1H), 7.83 (t, 1H), 7.60
(d, 1H), 6.71 (s, 1H), 6.69 (s, 1H), 4.13 (m, 2H, J = 5.6, 1.1 Hz), 2.61 (br
s, 2H), 2.50 (s, 2H), 2.16 (m, 3H, J = 6.2, 7.6 Hz), 1.23 (t, 3H); 13C
NMR (125 MHz; DMSO-d6): δ 187.7, 180.5, 170.2, 163.3, 141.6, 137.7,
134.4, 134.0, 132.0, 132.0, 129.1, 127.6, 126.9, 112.8, 109.3, 104.5, 102.4,
58.2, 19.3, 17.6, 15.8, 15.3, 14.4, 12.4; IR (KBr) ν 3500−2000 (max at
3159.66, 2926.40, and 2358.21 N−H, and C−H st), 1661.05 and
1582.35 (CO, ar−C-C and ar−C-N st) cm−1; LCMS m/z 447.18 [M
+
+ H]+; HRMS m/z 447.1882 ([M + H]+; calcd for C24H26F3N2O3
447.1890).
tert-Butyl-(5E)-5-[[2,5-dimethyl-1-[2-(trifluoromethyl)-
phenyl]pyrrol-3-yl]methylene]-2-methyl-4-oxo-1H-pyrrole-3-
carboxylate (10a). Yellow powder (99% yield), mp 201−205 °C. 1H
NMR (500 MHz; DMSO-d6): δ 10.13 (br s, 1H, -NH), 8.02 (d, 2H, J =
7.0 Hz), 7.93 (t, 1H, J = 7.6), 7.83 (t, 1H, J = 7.7 Hz), 6.7 (s, 1H), 6.63 (s,
1H), 4.3 (m, 2H, J = 5.0 Hz), 1.98 (s, 3H), 1.9 (s, 3H), 1.4 (s, 9H), 1.07
(t, 3H, J = 7.0 Hz); 13C NMR (125 MHz, DMSO-d6): δ 181.6, 180.8,
168.6, 169.6, 162.4, 135.5, 131.7, 129.2, 127.6, 123.8, 113.6, 112.1, 108.9,
106.0, 78.1, 55.9, 28.2(3C), 18.5, 16.6, 12.0,(2C) 10.3; LCMS m/z
447.18 (M + 1); HRMS m/z 447.1888 ([M + H]+; calcd for
C24H26F3N2O3+ 447.1890).
Benzyl (5E)-5-[[2,5-Dimethyl-1-[2-(trifluoromethyl)phenyl]-
pyrrol-3-yl]methylene]-2-methyl-4-oxo-1H-pyrrole-3-carboxy-
1
late (10b). Yellow powder (95% yield); mp 233−236 °C. H NMR
(500 MHz; DMSO-d6): δ 10.36 (br s, 1H, -NH), 8.02 (d, 2H, J = 7.7
Hz), 7.9 (t, 1H, J = 7.0 Hz), 7.8 (t, 1H, J = 7.5 Hz), 7.54 (d, 2H, J = 7.5
Hz), 7.47 (d, 1H, J = 7.2 Hz), 7.39 (t, 1H, J = 7.2 Hz), 7.32 (t, 1H, J = 7.3
Hz), 6.72 (s, 1H), 6.70 (s, 1H), 5.19 (br s, 2H, CH2Ph), 2.5 (s, 3H), 2.0
(s, 3H), 1.9 (s, 9H); 13C NMR (125 MHz, DMSO-d6): δ 180.4, 170.5,
163.1, 137.2, 136.0, 134.4, 131.7(2C), 131.6, 130.5, 129.2, 128.3(3C),
127.5(2C), 127.3(3C), 113.6, 109.8, 106.0, 102.2, 63.7, 15.8, 12.0, 10.3;
LCMS m/z 481.17 (M + 1); HRMS m/z 481.1736 ([M + H]+; calcd for
C27H24F3N2O3+ 481.1734).
General Procedure for the Synthesis of Ethyl (5E)-5-[[2,5-
dimethyl-1-[Substituted phenyl]pyrrol-3-yl]methylene]-2-
methyl-4-oxo-1H-pyrrole-3-carboxylates (8a-8aa, 9a-9s). To a
solution of ethyl 5-methyl-3-oxo-1,2-dihydropyrrole-4-carboxylate 3
(1.0 equiv/mol) in absolute ethanol (3 mL) was added the requisite 2,5-
dimethyl-1-aryl-3-formylpyrrole 7a-7aa (1.0 equiv/mol) and potassium
hydrogen sulfate (0.2 equiv/mol). The mixture was heated at 70−80 °C
for 3 h resulting in the formation of a yellow precipitate. The mixture was
poured onto ice and filtered to afford the ethyl (5E)-5-[[2,5-dimethyl-1-
[Substituted phenyl]pyrrol-3-yl]methylene]-2-methyl-4-oxo-1H-pyr-
role-3-carboxylate 8a-8aa as a yellow powder (80−95% yield). In
general no further purification by column chromatography was required.
Ethyl (5E)-5-[[2,5-dimethyl-1-[2-(trifluoromethyl)phenyl]-
pyrrol-3-yl]methylene]-2-methyl-4-oxo-1H-pyrrole-3-carboxy-
ASSOCIATED CONTENT
* Supporting Information
Synthetic routes for the synthesis of intermediates, methods for
physicochemical evaluation of the compounds and the data
generated, methods for the drug metabolism, pharmacokinetics,
in vitro and in vivo parasite testing; chemistry experimental for
compounds not included in the main text. This material is
available free of charge via the Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
*E-mail: i.h.gilbert@dundee.ac.uk; telephone +44 1382 386 240.
■
1
late (8a). Yellow powder (0.22 g, 89%); mp 230−235 °C. H NMR
(500 MHz, DMSO-d6): δ 10.3 (s, 1H, -NH), 8.01 (t, 1H, J = 7.7 Hz)
7.91 (t, 1H), 7.8 (t, 1H, J = 7.7 Hz), 7.51 (t, 1H, J = 7.7 Hz), 6.71 (s, 1H),
6.69 (s, 1H), 4.13−4.09 (q, 2H, J = 6.9 Hz), 2.58 (s, 3H), 1.99 (s, 3H),
1.90 (s, 3H), 1.23 (t, 3H, J = 7.1 Hz); 13C NMR (125 MHz, DMSO-d6):
δ 180.5, 170.2, 163.3, 135.9, 134.36 (2C), 131.6, 131.6, 130.43 (2C),
129.17 (2C), 127.5, 113.5, 169.7, 105.9, 102.4, 58.2, 15.7, 14.4, 11.9,
10.3. IR (KBr) ν 3500−2000 (max at 3176.47, 2926.66, and 2340.11 N−
H, and C−H st), 1661.80 and 1583.65 (CO, ar−C-C and ar−C-N st)
cm−1. LCMS m/z 419.15 (M + 1); HRMS m/z calculated
(C22H22N2O3F3): 419.1577 (M + H)+; found: 419.1566. (Delta PPM:
2.40 ppm; acquired on Microtof; mass resolution: 10000 (fwhm)).
Ethyl (5E)-5-[[2,5-Dimethyl-1-[4-(trifluoromethyl)phenyl]-
pyrrol-3-yl]methylene]-2-methyl-4-oxo-1H-pyrrole-3-carboxy-
Author Contributions
○D.M. and A.M. contributed equally to the work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
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.
1
late (8b). Yellow powder, mp 252−255 °C; H NMR (500 MHz,
DMSO-d6): δ 10.27 (s, 1H, -NH), 7.96 (d, 2H) 7.65 (d, 2H), 6.75 (s,
1H), 6.70 (s, 1H), 4.16 (q, 2H, J = 5.4 Hz), 2.59 (s, 3H), 2.13 (s, 3H),
2.04 (s, 3H), 1.23 (t, 3H, J = 1.0, 1.1 Hz); 13C NMR (125 MHz; DMSO-
d6): δ 180.4, 170.2, 163.2, 140.6, 134.5, 130.4, 129.2, 128.9, 128.6,
126.67, 126.64, 124.9, 122.8, 113.9, 109.4, 106.6, 102.4, 58.1, 15.8, 14.4,
ABBREVIATIONS
CLint, intrinsic clearance; CYP, cytochrome P450; DMPK, drug
metabolism and pharmacokinetics; SAR, structure−activity
■
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relationship; UDPGA, uridine-5′-diphospho-glucuronic acid;
WHO, World Health Organisation; WHO-TDR, World Health
Organisation Programme for Research and Training in Tropical
Diseases
ADDITIONAL NOTE
■
a
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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