Inhibitors of plasmodial serine hydroxymethyltransferase (SHMT): Cocrystal structures of pyrazolopyrans with potent blood- and liver-stage activities

Article abstract of DOI:10.1021/jm501987h

Several of the enzymes related to the folate cycle are well-known for their role as clinically validated antimalarial targets. Nevertheless for serine hydroxymethyltransferase (SHMT), one of the key enzymes of this cycle, efficient inhibitors have not been described so far. On the basis of plant SHMT inhibitors from an herbicide optimization program, highly potent inhibitors of Plasmodium falciparum (Pf) and Plasmodium vivax (Pv) SHMT with a pyrazolopyran core structure were identified. Cocrystal structures of potent inhibitors with PvSHMT were solved at 2.6 ? resolution. These ligands showed activity (IC₅₀/EC₅₀ values) in the nanomolar range against purified PfSHMT, blood-stage Pf, and liver-stage P. berghei (Pb) cells and a high selectivity when assayed against mammalian cell lines. Pharmacokinetic limitations are the most plausible explanation for lack of significant activity of the inhibitors in the in vivo Pb mouse malaria model.

Full text of DOI:10.1021/jm501987h

Article
pubs.acs.org/jmc
Inhibitors of Plasmodial Serine Hydroxymethyltransferase (SHMT):
Cocrystal Structures of Pyrazolopyrans with Potent Blood- and Liver-
Stage Activities
,
†
‡,§
∥
⊥
Matthias C. Witschel,* Matthias Rottmann, Anatol Schwab, Ubolsree Leartsakulpanich,
⊥
∥
∥
∥
†
Penchit Chitnumsub, Michael Seet, Sandro Tonazzi, Geoffrey Schwertz, Frank Stelzer,
†
#
†
‡,§
⊥
́
Thomas Mietzner, Case McNamara, Frank Thater, Celine Freymond, Aritsara Jaruwat,
∞
⊥
×
×
Chatchadaporn Pinthong, Pinpunya Riangrungroj, Mouhssin Oufir, Matthias Hamburger,
er, Laura M. Sanz-Alonso, Susan Charman, Sergio Wittlin, Yongyuth Yuthavong,⊥
‡
,§
¶
○
‡,§
Pascal Mas
Pimchai Chaiyen, and Franco
̈
∞
,∥
̧
is Diederich*
†
‡
§
∥
BASF SE, Carl-Bosch-Strasse 38, 67056 Ludwigshafen, Germany
Swiss Tropical and Public Health Institute (Swiss TPH), Socinstrasse 57, 4051 Basel, Switzerland
Universitat
̈
Basel, Petersplatz 1, 4003 Basel, Switzerland
r Organische Chemie, ETH Zurich, Vladimir-Prelog-Weg 3, 8093 Zurich, Switzerland
National Center for Genetic Engineering and Biotechnology, 113 Thailand Science Park, Phahonyothin Road, Khlong Nueng,
Khlong Luang, Pathum Thani 12120, Thailand
Laboratorium fu
̈
⊥
#
California Institute for Biomedical Research (Calibr), 11119 North Torrey Pines Road, Suite 100, La Jolla, California 92037, United
States
∞
Department of Biochemistry and Center of Excellence in Protein Structure and Function, Faculty of Science, Mahidol University,
72 Rama VI Road, Bangkok 10400, Thailand
2
×
Pharmaceutical Biology, Department of Pharmaceutical Sciences, University of Basel, Klingelbergstrasse 50, 4056 Basel, Switzerland
¶
Diseases of the Developing World (DDW), GlaxoSmithKline, C. Severo Ochoa, 2, 28760 Tres Cantos, Spain
○
Centre for Drug Candidate Optimisation, Monash Institute of Pharmaceutical Sciences, 381 Royal Parade, Parkville, Victoria 3052,
Australia
*
S Supporting Information
ABSTRACT: Several of the enzymes related to the folate cycle are
well-known for their role as clinically validated antimalarial targets.
Nevertheless for serine hydroxymethyltransferase (SHMT), one of the
key enzymes of this cycle, efficient inhibitors have not been described
so far. On the basis of plant SHMT inhibitors from an herbicide
optimization program, highly potent inhibitors of Plasmodium
falciparum (Pf) and Plasmodium vivax (Pv) SHMT with a
pyrazolopyran core structure were identified. Cocrystal structures of
potent inhibitors with PvSHMT were solved at 2.6 Å resolution. These
ligands showed activity (IC /EC values) in the nanomolar range
5
0
50
against purified Pf SHMT, blood-stage Pf, and liver-stage P. berghei (Pb) cells and a high selectivity when assayed against
mammalian cell lines. Pharmacokinetic limitations are the most plausible explanation for lack of significant activity of the
inhibitors in the in vivo Pb mouse malaria model.
INTRODUCTION
antimalarials with new modes of action to counteract the
potentially catastrophic results of widespread artemisinin
resistance, which is not unlikely based on the previous
experiences with antimalarial resistance development.
In recent years, substantial efforts have been made to identify
■
Malaria is, besides tuberculosis and AIDS, with more than 250
million clinical cases causing over 600 000 deaths per year, one of
the most devastating infectious diseases worldwide. By now,
9
−11
1
widespread resistance has developed against almost all available
antimalarials. Even for the “last resort” artemisinin derivatives,
which constitute the backbone of most new combination
therapies in development, the emergence of resistance becomes
new antimalarials using target-based approaches, which could
Received: December 23, 2014
2
−8
a major concern.
Therefore, there is an urgent need for
©
XXXX American Chemical Society
A
DOI: 10.1021/jm501987h
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
have significant advantages compared to organism-based hits due
to a much better understanding of target interaction, potential
resistance mechanisms, and toxicological risks. Unfortunately,
most of the target-based programs, in spite of potent target
activities, did not result in high activity against P. falciparum in
RESULTS AND DISCUSSION
■
Initial Screen of BASF-Pyrazolopyran SHMT Inhibitors.
A total of 338 inhibitors of plant SHMT were screened for
3
antiplasmodial activity using a 72 h H-hypoxanthine incorpo-
23
1
2
ration assay. Out of these, several plant SHMT inhibitors with
cell-based assays.
EC50 values below 100 nM in the Pf cell-based assay could be
identified, with the most active compound (±)-1 (Scheme 2)
showing an EC value of 2.8 nM.
Plasmodium parasites have several essential enzymatic path-
ways in common with plants so that herbicides potentially can
serve as lead structures for new antimalarials. This has been
shown in a systematic screen of commercial agrochemicals
against different protozoans, which resulted in a high number of
50
a
Scheme 2. Synthesis of Pyrazolopyran Ligands
1
3
promising hits. Therefore, we systematically examined the
antiplasmodial activity of lead structures from target-based
14−16
herbicide programs at BASF.
Of these, inhibitors of the
enzyme serine hydroxymethyltransferase (SHMT) raised our
particular interest. SHMT plays a key role in the dTMP synthesis
1
7,18
(
Scheme 1).
As the mode of action of the antimalarial drugs
a
Scheme 1. Overview of the dTMP Cycle
a
Reagents and conditions: (a) [PdCl (PPh ) ] (2.5 mol %), Na CO ,
2
3
2
2
3
THF/H O 4:1, 60 °C, 4 h, 78%; (b) malononitrile, TiCl , pyridine,
2
4
CHCl , 60 °C, 4 days, 68%; (c) Et N, THF/MeOH 1:4, 45 °C, 6 days.
3
3
(
(
1
±)-1: 12%. (±)-8: 43%. (d) H , Pd/C, MeOH, 25 °C, 30 min, 88%;
e) CDI, then amine, THF, 25 °C, 1−3 h, 46−83%; (f) TFA, 25 °C,
4 h, quantitative. CDI = carbonyldiimidazole. TFA = trifluoroacetic
2
acid. For atom labeling, see section S9 in the Supporting Information.
a
Serine hydroxymethyltransferase (SHMT) converts tetrahydrofolate
Synthesis and Cell-Based Activity of (± )-1 and
Analogues. In addition to (±)-1, we synthesized a series of
derivatives 2−9, focusing in particular on the introduction of
water-solubilizing and cell permeability-providing groups
(Scheme 2). Ligand 2 bears a chloroquine-like substituent,
whereas 7 features the side chain of tetrahydrofolate, the
substrate of SHMT (for stereochemical descriptors of 2 and 7,
see Scheme 2). N,N-Dimethylamide derivative (±)-10 addresses
the hydrolysis of esters (±)-1 and (±)-8 under physiological
conditions.
(
THF) to 5,10-methylenetetrahydrofolate (5,10-CH -THF), a crucial
2
component for the conversion of deoxyuridine monophosphate
(
(
dUMP) to the DNA precursor deoxythymidine monophosphate
dTMP) by thymidylate synthase (TS). Dihydrofolate (DHF) formed
by this process is converted back to THF by dihydrofolate reductase
DHFR). In Plasmodium, components of the cycle are replenished by
(
de novo synthesis: dihydropteroate synthase (DHPS) condenses
pteridine diphosphate and p-aminobenzoic acid (pABA) to dihy-
dropteroate (DHP), which is converted to DHF by dihydrofolate
synthase (DHFS).
Boronic ester 11 was prepared by esterification of the
corresponding thienylcarboxylate using phenyldiazomethane
and reacted in a Suzuki cross-coupling with aryl bromide 12 to
Cycloguanil, pyrimethamine, or sulfadoxine is also based on the
inhibition of folate production, SHMT has been presumed to be
a highly relevant target for antimalarials; however, no potent
24−26
biaryl 13.
A Knoevenagel condensation afforded dicyano-
19−22
SHMT inhibitors have been described so far.
vinyl derivative 14, and tandem Michael addition−cyclization
In a screen of 100 000 compounds at BASF, two compounds
with a pyrazolopyran scaffold showed IC50 values in the
micromolar range, which could further be optimized to activities
with an excess of pyrazolone 15 led to benzyl ester (±)-8
27
featuring the pyrazolopyrane core. The use of methanol as
solvent, which was indispensable for the solubility of 15, led to
partial transesterification of (±)-8 to the corresponding methyl
ester (±)-1. Hydrogenolysis of (±)-8 provided thienylcarboxylic
acid (±)-9, from which the targeted amides 2−6, 7 (by ester
23
in the nanomolar range against plant SHMT. These plant
SHMT inhibitors were subsequently also examined as lead
structures against malaria parasites.
B
DOI: 10.1021/jm501987h
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
a
Table 1. Biological Activity of Selected Pyrazolopyran Ligands
a
Data for all compounds tested are provided in the Supporting Information (Table S1).
cleavage of 6), and 10 were obtained by condensation of the
salts. Detailed synthesis and characterization protocols can be
28
corresponding amines with carbonyldiimidazole (CDI). To
further improve solubility for biological testing, some of the
ligands were converted into trifluoroacetate or hydrochloride
found in the Supporting Information.
The cell-based activities for selected ligands are summarized in
Table 1, which includes data for the newly prepared ligands and
C
DOI: 10.1021/jm501987h
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Figure 1. Cocrystal structure (PDB code 4TMR) showing protein−ligand interaction of pyrazolopyran (+)-1 (green) and PvSHMT (white). Left image
shows polar (hydrogen bonding) interactions, right image nonpolar contacts. The mesh surface spans the volume of the binding pocket, and cofactor
PLP is shown in yellow. Distances are given in Å. Atom coloring is the following: N blue, O red, P orange, S yellow.
other derivatives from the herbicide testing program (com-
pounds (±)-16 to (±)-21). The complete biological character-
ization for all compounds in this study is provided in the
Supporting Information (Table S1). Methyl (±)-1 and benzyl
ester (±)-8 show by far the most potent activity within the low
single-digit nanomolar range. Replacing the thienyl against a
phenyl ring (in (±)-18) reduces the activity to 80 nM. The N,N-
dimethylamide (±)-10 is 20-fold less active than (±)-1.
Carboxylic acid (±)-9 is only active in the upper nanomolar
range, similar to ligand (±)-4 with a solubilizing side chain. In
general, the thienyl ring is critical for high affinity and far superior
to phenyl rings, which can be rationalized on the basis of the
structural data shown above.
Cytotoxicity Tests. The compounds showed an excellent
selectivity against mammalian L6-cells in a 72 h cytotoxicity assay
with selectivity indexes for the purified enantiomers (+)-1 and
(+)-8 of >12 000 and >4000, respectively (Table 1). In addition,
cytotoxicity was tested for compound (±)-1 in the human
HepG2 cell line (human hepatocellular liver carcinoma cell line).
No effects were observed up to the highest concentration
(
20 000 nM) tested. Distinctive structural features and kinetic
properties of plasmodial and human SHMT support the
2
9,30
observed selectivity.
Test on Resistant Pf Strains. The most active hits were also
tested against the resistant P. falciparum strains TM90C2B
(
resistant against chloroquine, pyrimethamine, mefloquine,
atovaquone), K1 (resistant against chloroquine, sulfadoxine,
pyrimethamine, cycloguanil), and V1/S (resistant against the
antifolates pyrimethamine, sulfadoxine, dapsone, cycloguanil,
and chloroquine) in the cell-based assay, showing almost
identical activities to the sensitive strain. V1/S carries four
DHFR mutations and is highly resistant to anti-DHFR drugs
The enantiomers of (±)-1 and (±)-8 were separated
enantiomeric purity >99%) by preparative HPLC on a chiral
(
stationary phase (Daicel Chiralpak-IA 250 mm × 20 mm) using
CHCl /EtOAc (95:5, 10 mL/min) as eluent. A high degree of
3
chiral recognition was observed. The (+)-enantiomers showed a
higher activity compared to their racemic mixture, whereas the
activity of the (−)-enantiomers is markedly reduced (25- and
(
Supporting Information, Table S1). Therefore, there are likely
no general cross resistance issues with this compound class.
Test on P. berghei Liver Stage. The most active
pyrazolopyran (±)-1 has also been tested against P. berghei in
the liver-stage assay. In brief, Anopheles stephensi mosquitoes
infected with a transgenic luciferase-expressing P. berghei were
obtained from the New York University Langone Medical
Center Insectary (New York). A transgenic CD81-expressing
HepG2 cell line (donated by Dominique Mazier) was seeded
into assay plates. After ∼24 h incubation, compounds were
transferred with atovaquone and puromycin used as positive
controls for parasite inhibition and liver cell cytotoxicity,
respectively. After 3−4 h, sporozoites (from a transgenic
luciferase-expressing P. berghei) were added and plates were
incubated at 37 °C, and following a 48 h incubation, relative
luminescence was measured. Wells treated with either DMSO or
positive controls were used to normalize these data for dose−
150-fold, respectively, Table 1).
PfSHMT and PvSHMT Enzymatic Assays. Reaction assays
of PfSHMT and PvSHMT were carried out using a diode array
spectrophotometer according to the protocol previously
1
9
reported. More details are described in the Experimental
Section. Table 1 shows that the inhibition of Pf SHMT (IC50
values) correlates well with the measured whole cell in vitro
activities (EC values). In general, the intrinsic inhibition on the
50
enzyme level was slightly lower (Table 1, Supporting
Information Table S1 and Supporting Information section
S10). Many of the compounds showed solubility problems at the
highest tested concentrations, which could explain some of the
differences due to the different assay characteristics. Compound
(
+)-1 was also tested on PvSHMT, showing an IC value of 98 ±
50
2
nM.
D
DOI: 10.1021/jm501987h
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
response curve fit analysis. In this assay, (±)-1 also showed a high
of Ser184. Threonine 183 forms a strong hydrogen bond to the
pyran oxygen which is flanked by Leu124 on the opposing side.
The N(2) of the pyrazole moiety undergoes hydrogen bonding
to Glu56 and is further stabilized by van der Waals interactions
with Leu130 and His129.
potency with an IC value of 9 nM.
5
0
Animal Model and Pharmacokinetics. In initial animal
models with P. berghei infected mice, no significant activity of
(±)-1 or (±)-8 could be observed. Therefore, pharmacokinetic
studies with (±)-8 were conducted. After oral and intraperitoneal
dosing of (±)-8 with 100 and 10 mg/kg, respectively, plasma
levels were below the limit of detection at all tested time points
The plane of the bicyclic scaffold is inclined toward the p-
aminobenzoic acid (pABA) channel (Supporting Information
Figure S1) opening up a small lipophilic pocket that is best filled
by an isopropyl group at the stereogenic center of the core. The
two methyl groups of the isopropyl residue form well aligned
interactions, one pointing at the edge of Tyr64 at close distance
and the other at the face of the hydrogen bonding array between
Arg371 and the cofactor pyridoxal 5′-phosphate (PLP). In
general, substitution of the isopropyl group by the cyclobutyl
group leads to a decrease in activity (Supporting Information
Table S1).
(
1, 4, 24 h after treatment). There was evidence of rapid
conversion of the ester group into the acid in the plasma samples
Supporting Information, Table S9). This potential lability of the
(
ester group may lead into the formation of metabolite (±)-9 with
more than 200-fold reduced activity and thus could explain the
lack of in vivo activity.
In liver microsomes, a rapid degradation of ester (±)-8 and
N,N-dimethylamide (±)-10 by ester/amide hydrolysis or mono-
oxygenation was observed (predicted hepatic extraction ratio
The side chain of ligand (+)-1 extends orthogonally from the
stereogenic center of the bicyclic core into the pABA channel. A
1,3,5-trisubstituted phenyl group constitutes the first segment of
the chain in all ligands tested. At least one of the substituents
(EH) of 0.84 and 0.94). The closely related biphenyl analogues
(
0
±)-17 and (±)-38 showed improved stability (predicted E of
.33 and 0.51, respectively, Supporting Information Tables S7−
H
S10). Caco-2 studies indicate moderate permeability of (±)-10
in the apical to basolateral direction. An efflux ratio of 5.5
suggests that (±)-10 might be subject to efflux in the Caco-2
system which could potentially limit oral absorption (Supporting
Information section S4, Table S5).
consists of a small, electron-withdrawing group (CN, Cl, CF ). In
3
(+)-1, a cyano group points into a small lateral pocket making
contact with Pro367. Leucine 124 flanks the unsubstituted
position para to the acceptor substituent. The second substituent
of the phenyl ring further extends along the pABA channel (lined
by hydrophobic residues) toward the periphery of the protein.
The binding mode rationalizes the experimental finding that
Despite its rapid metabolism, (±)-10 showed a reduction of
parasitemia of 38% (po) and 41% (ip) in a 4-day-test SCID mice
model with P. falciparum 3D7 infected SCID mice at 4 × 30 mg/
kg. Considering the very short half-life of the compound, the
significant reduction of parasitemia validates the activity of
pyrazolopyrans in vivo. This compound class displays folate
inhibitor-like characteristics in the parasite reduction ratio assay.
The profile is comparable with the DHFR inhibitor pyrimeth-
amine (Supporting Information section S6). This result is in
accordance with the observed activity decrease when reducing
the duration of drug exposure in vitro. The EC values of (±)-10
aromatic or hydrophobic groups (Br, CF ) can engage in
3
effective nonpolar interactions along the channel. The 2,5-
substituted thiophene moiety of (+)-1 undergoes (in addition to
CH−π interaction with Leu124 and parallel-shifted stacking with
Tyr63) a sulfur−sulfur interaction with Cys364.
At the periphery, a polar aprotic headgroup provides favorable
interaction with the more hydrophilic environment. The activity
of esters is found far superior to that of the corresponding acids
or secondary amides (Table 1, (±)-8, (±)-9, (±)-3).
5
0
against Pf NF54 decrease with prolonged exposure (EC of 211,
A comparison to Pf SHMT apo structures (PDB code 4O6Z)
shows that the CN group of the vinylogous cyanamide and N(2)
of the pyrazole moiety of (+)-1 replace two water molecules
present in the ligand-free structure (Supporting Information
50
6
7, and 59 nM at 24, 48, and 72 h, respectively) are similar to
what can be seen with pyrimethamine (EC of 25, 7.1, and 4.6
5
0
nM at 24, 48, and 72 h, respectively). The likely stage specificity
of the pyrazolopyrans is currently under investigation.
29
Figure S3). A third water molecule is displaced from a largely
apolar environment by the isopropyl group of (+)-1. Favorable
water displacement from a hydrophobic environment is also
expected for the nitrile at the phenyl ring of (+)-1.
Crystal Structure Determination of PvSHMT with (+)-1
and (+)-16. PvSHMT was crystallized from the ternary complex
of PvSHMT, glycine, and either (+)-1 or (+)-16 (Table 1) using
the microbatch method. The cocrystals diffracted to 2.7 and 2.2 Å
resolution, respectively, and belong to the C2 space group. The
structure was solved by molecular replacement using the
coordinates of a chain A protomer of PvSHMT (PDB code
Binding Mode of (+)-16. Overall, binding mode and
conformation of (+)-16 and (+)-1 are very similar (Figure 2,
Supporting Information Figure S4 and S5). The pyrazolopyran
core adopts a nearly superimposable position in the two
complexes; however, some polar interactions differ because of
different conformations of amino acid side chains, such as those
of Thr183 and Ser184. The biphenyl substituent in (+)-16
extending into the pABA channel also assumes a conformation
similar to the phenylthienyl residue in (+)-1. The F-substituent
points into the same direction as the CO of methyl ester (+)-1.
The lipophilic pocket, which hosts the isopropyl substituent at
the stereogenic center of (+)-1, is now filled by the cyclobutyl
substituent of (+)-16 but in a different orientation. The enzyme
inhibition is less potent for (+)-16 (IC50 = 470 nM) than for
(+)-1 (IC50 = 60 nM). One explanation could be the
conformational difference of the surface loop, in which Cys364
is located. This loop (residues 357−367) is positioned closer to
(+)-1, resulting in better van der Waals interactions with the
cyano moiety on the phenyl ring and the thiophene ring of (+)-1
31
4OYT) as the template.
Binding Mode of (+)-1. Ligand (+)-1 occupies the
tetrahydrofolate (THF) binding site as seen in the crystal
structure (Figure 1, PDB code 4TMR). On the basis of the
structure of the complexed ligand, enantiomer (+)-1 can be
assigned the (S)-absolute configuration with confidence. The
bicyclic pyrazolopyran core undergoes similar interactions to the
aminopyrimidinone moiety of THF (Supporting Information
Figure S2). A polar environment encloses the vinylogous
cyanamide of (+)-1. The side chain and backbone of Thr357
form hydrogen bonds to the cyano group, which also interacts
with Asn356. A tripod-shaped interaction is formed at the
nonbasic NH group with two hydrogen bonds donated to the
2
backbone carbonyls of Leu124 and Gly128 and one accepted
along the direction of the nitrogen lone pair from the side chain
E
DOI: 10.1021/jm501987h
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Stable ester bioisosteres or alternative peripheral heteroaromatic
systems should allow combination of favorable pharmacokinetic
properties with high activity. Ultimately, rational scaffold
variations are now possible to further extend the scope of the
antimalarial SHMT inhibitors.
EXPERIMENTAL SECTION
■
In Vitro Antimalarial Activity. Plasmodium falciparum drug-
sensitive NF54 and resistant K1, TM90C2B and V1/S strains were
cultivated in a variation of the medium previously described, consisting
of RPMI 1640 supplemented with 0.5% ALBUMAX II, 25 mM Hepes,
2
5 mM NaHCO buffer (pH = 7.3), 0.36 mM hypoxanthine, and 100
3
33,34
μg/mL neomycin.
Human erythrocytes served as host cells.
Cultures were maintained in an atmosphere of 3% O , 4% CO , and
2
2
9
3% N in humidified modular chambers at 37 °C. Compounds were
2
dissolved in DMSO (10 mg/mL), diluted in hypoxanthine-free culture
medium, and titrated in duplicate over a 64-fold range in 96-well plates.
Infected erythrocytes (1.25% final hematocrit and 0.3% final para-
sitemia) were added into the wells. After a 48 h incubation, 0.5 μCi
3
[
H]hypoxanthine was added, and plates were incubated for an
additional 24 h. Parasites were harvested onto glass-fiber filters, and
radioactivity was counted using a Betaplate liquid scintillation counter
(
Wallac, Zurich). The results were recorded and expressed as a
Figure 2. Cocrystal structure (PDB code 4TN4) showing polar
protein−ligand interaction of pyrazolopyran (+)-16 (blue) and
PvSHMT (white). Ligand (+)-1 (green) of superimposed structure
percentage of the untreated controls. Fifty percent inhibitory
concentrations (EC ) were estimated by linear interpolation.
35
50
In Vitro Cytotoxicity. Rat skeletal myoblasts (L6 cells) in RPMI
640 medium with 10% FCS and 2 mM L-glutamine were added to each
4TMR is shown for comparison. The mesh surface spans the volume of
1
the binding pocket, and cofactor PLP is shown in yellow. Distances are
given in Å. Atom coloring is the following: F cyan, N blue, O red, P
orange, S yellow.
well of a 96-well microtiter plate and incubated at 37 °C under a 5% CO2
atmosphere for 24 h. Compounds were added directly into the wells, and
subsequently serial drug dilutions were prepared covering a range from
1
00 to 0.002 μM. The plates were incubated for another 72 h. An
than with the equivalent moieties of (+)-16 (Supporting
Information Figure S5). More noteworthy is the lack in chiral
recognition of (±)-16 by the enzyme. In sharp contrast to the
large difference in binding affinity measured for (+)-1 and (−)-1,
the two enantiomers of 16 exhibit similar inhibition. In the cell-
based assay, the two ligands differ strongly, with EC values of 2
nM ((+)-1) and 2880 nM ((+)-16).
Tautomerism of the Pyrazolopyran Core. The small
molecule crystal structure of (±)-10 and theoretical calculations
amount of 10 μL of Alamar Blue (12.5 mg resazurin dissolved in 100 mL
water) was then added to each well, and incubation continued for a
further 1−4 h. The plates were read with a Spectramax Gemini XS
microplate fluorometer (Molecular Devices Cooperation, Sunnyvale,
CA, USA) using an excitation wavelength of 536 nm and an emission
wavelength of 588 nm. Data were analyzed using the software Softmax
Pro (Molecular Devices Cooperation, Sunnyvale, CA, USA). Decrease
of fluorescence (=inhibition) was expressed as percentage of the
fluorescence of control cultures and plotted against the drug
concentrations. From the sigmoidal inhibition curves, the IC50 values
were calculated. Podophyllotoxin was used as positive control in the
assay (IC50 = 0.0082 μM).
5
0
(Supporting Information Figures S7−S10) confirm that the
pyrazolopyran core is in the same tautomeric form in the free and
protein-bound state. The present tautomer bears the pyrazole
NH vicinal to the methyl group, enabling crucial hydrogen
bonding interactions to the side chain of Glu56.
Luciferase-Expressing Plasmodium berghei (Pb-luc) Liver
Stage Assay. The previously described Plasmodium yoelii sporozoite
invasion assay (PMID, 22096101) was modified and miniaturized into a
1
536-well luminescence-based assay. In brief, Anopheles stephensi
mosquitoes infected with a transgenic luciferase-expressing P. berghei
were obtained from the New York University Langone Medical Center
Insectary (New York). A transgenic CD81-expressing HepG2 cells (gift
from Dominique Mazier) was seeded at 3000 cells per well (5 μL
volume in complete culturing media) into a white, 1536-well assay plate
CONCLUSIONS
■
We have identified a novel pyrazolopyran series as potent
inhibitors of plasmodial SHMT. These compounds are
promising antimalarial leads, as they show high activity (IC50
values in the nanomolar range) on the Pf enzyme as well as the Pv
enzyme and activities down to the single-digit nanomolar range
in cell-based blood stage assays. The high activity in a P. berghei
liver stage model, combined with the activity on PvSHMT, makes
this target especially interesting for potential treatment of blood
stage malaria as well as hypnozoite stage P. vivax malaria. The
inhibitors showed a high selectivity margin relative to
mammalian cell lines and no resistance against the tested
multidrug resistant Pf strains. The main limitation so far is the
low metabolic stability of the most active inhibitors. With the
available cocrystal structures, a targeted optimization of the
pharmacokinetic properties should now be possible, using
structure-based design. Especially the variation of the exit vector
including the thiophene residue appears promising, and analogs
can easily be prepared in the described short synthetic sequence.
(
Greiner Bio One) using a MicroFlo Microplate Dispenser (1 μL
cassette; BioTek). After ∼24 h incubation at 37 °C, compounds (10 nL)
were transferred using an ECHO liquid handler (Labcyte). Atovaquone
and puromycin were used as positive controls for parasite inhibition and
liver cell cytotoxicity, respectively. After 3−4 h, parasites obtained from
freshly dissected mosquitoes were dispensed (750 parasites per well; 5
μL volume) by a BottleValve liquid dispenser (GNF Systems) equipped
with a custom single-tip dispense head. The assay plates were
centrifuged for 2 min at 150g, and then the plates were incubated at
32
3
7 °C. Following a 48 h incubation, the assay plates were inverted into
collection boxes and centrifuged to remove the assay media. This was
followed by a 2 μL dispense of Bright-Glo (Promega), and relative
luminescence was immediately measured on an Envision (PerkinElm-
er). CellTiter-Glo (Promega) was added to replicate assay plates lacking
parasites to determine cell viability on the Envision. Wells treated with
either DMSO or positive controls were used to normalize these data for
dose−response curve fit analysis.
F
DOI: 10.1021/jm501987h
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Enzymatic SHMT Assays. Assay mixtures (1 mL total volume)
intraperitoneally (ip). The solution formulation for po and ip dosing
contained 3% ethanol and 70% Tween 80. Blood samples from mice
were collected at 1, 4, and 24 h after dosing. Groups of four mice were
used for each time point. An amount of 50 μL of blood was collected
from each of the four mice, pooled, and centrifuged at 13 000 rpm for 7
min at 4 °C. Lithium heparin plasma was harvested and stored at −80 °C
until analysis. For detailed analytic procedure see Supporting
Information.
contained SHMT (∼0.5 μM or 27.5 μg), L-serine (2 mM), (6S)-THF
+
(
0.4 mM), β-NADP (0.25 mM), and the coupling enzyme methylene
tetrahydrofolate dehydrogenase (FolD, 5 μM or 155 μg) in 50 mM
HEPES, pH 7.0, containing 1 mM DTT and 0.5 mM EDTA. To
mixtures was added 10 μL of inhibitors with various concentrations
(
final concentrations from 1 to 1000 nM), and initial rates of the
reaction were monitored to measure the amount of noninhibited
enzyme. The inhibitors were dissolved in DMSO, and the control assays
without inhibitor but in the presence of 1% DMSO (final concentration)
were also carried out.
Crystallization of Recombinant PvSHMT and Compounds
+)-1 and (± )-16. PvSHMT was crystallized using microbatch method
in a 60-well plate (⌀ 1 mm at bottom of each well) covered with 6 mL of
baby oil (Johnson; a mixture of mineral oil, olive oil, and vitamin E, PZ
Johnson, Thailand). Protein−ligand complexes were prepared by
mixing 60 μL of purified PvSHMT protein (20−25 mg/mL) with 1
mM PLP, 60 mM β-mercaptoethanol, 90 mM glycine, and 3.2 mM
Chemical Synthesis: General Methods. Commercially available
chemicals (Abcr, Aldrich, Acros, Fluka, Fischer, Maybridge, Sigma, TCI)
were used without further purification. Analytic thin layer chromatog-
raphy (TLC) was conducted on SiO 60 F coated glass plates (Merck
2
254
(
Millipore) and on F254 Al O coated glass plates (Merck Millipore).
2
3
Fluorescence extinction was detected at 254 nm, fluorescence at 366 nm.
Preparative thin layer chromatography was conducted on glass plates
with a SiO 60 F coating of 1 mm thickness (SiliCycle). Infrared (IR)
2
254
spectra were recorded on a PerkinElmer Spectrum Two FT-IR device
−1
fitted with an ATR unit (4000−600 cm ). Absorption bands are given
(
+)-1 or (±)-16. The mixture was equilibrated on ice for 30 min to allow
−1
in wavenumbers (cm ), and signal intensities are labeled with s
for complete complex formation. The crystallization drop is composed
of 1 μL each of a crystallization solution and the protein complex.
Protein crystals of PvSHMT were grown at 293 K in 20−24% w/v
PEG4000, 0.06−0.12 M NaCl, 0.1 M Tris-HCl buffer, pH 8.5, and
additive 10% v/v trifluoroethanol.
(strong), m (medium), and w (weak). Solvents used for chromatog-
raphy and extraction were distilled prior to use. Reactions were carried
1
13
out in puriss. or p.a. grade solvents. H and C NMR spectra were
recorded on Varian Gemini 300 (300/75 MHz), Bruker AVIII 300
(
(
300/75 MHz), Bruker DRX 400 (400/100 MHz), or Bruker DRX 600
600/150 MHz). All spectra were measured at 300 K. Deuterated
PvSHMT Crystal Structure Data Collection, Structure Deter-
mination, and Refinement. A single crystal was flash-vitrified in
liquid nitrogen using 20% glycerol in crystallizing agent as a
cryoprotectant. X-ray diffraction data were collected at 100 K at
wavelength of 1 Å using ADSC Quantum-315 CCD detector at
beamline 13B1, NSRRC, Taiwan. Data were processed using HKL2000
package. X-ray diffraction data and refinement statistics are listed in
Supporting Information Table S12. The structure of PvSHMT was
determined by molecular replacement using Phaser in CCP4 suite with a
chain A protomer of PvSHMT coordinate (PDB code 4OYT) as the
template. Model building and structure refinement were carried out
using Coot and Refmac5. The ligand structure was prepared using
HYPERCHEM.
solvents were obtained from Armar Chemicals. Chemical shifts are
referenced to solvent residual peaks ((CD ) CO, δ H = 2.05, δ C = 29.84
3
2
ppm; CDCl , δ H = 7.26, δ C = 77.16 ppm; (CD ) SO, δ H = 2.50, δ C =
3
3 2
3
9.52 ppm; CD CN, δ H = 1.94, δ C = 1.32 ppm; CD OD, δ H = 3.31, δ
3
3
C = 49.00 ppm; (D )THF, δ H = 3.58, δ C = 67.57 ppm). The spin
8
multiplicity is abbreviated as s (singlet), d (doublet), t (triplet),
combinations of such, or m (multiplet). Coupling constants are denoted
1
3
by J and are given in Hz. Broad signals are annotated with br. C spectra
1
13
are broadband decoupled. Assignments of H and C signals are
supported by 2D NMR experiments (COSY, HSQC, HMBC,
INADEQUATE). The nomenclature was obtained with the computer
program ACD/Name (ACD/Labs). Medium pressure liquid chroma-
tograpy (MPLC) was carried out on a Teledyne Isco CombiFlash
Animal Models. All in vivo studies conducted at the SwissTPH were
adhering to local and national regulations of laboratory animal welfare in
Switzerland (permission numbers 1731 and 2303). P. berghei in vivo
system using Teledyne RediSep SiO cartridges or on a Bu
̈
chi Sepacore
2
system using self-packed columns (C end-capped reversed phase silica
antimalarial activity was assessed for groups of five female NMRI mice
18
7
gel, Fluka 60756). Melting points were measured on a Buchi M560
̈
(
20−22 g) intravenously infected on day 0 with 2 × 10 erythrocytes
device. High-resolution electrospray ionization mass spectrometry (HR-
ESI-MS) was conducted with a Bruker maXis ESI/Nanospray Qq TOF
instrument. All mass spectra were acquired by the MS-Service of the
Laboratory of Organic Chemistry (ETH Zurich). Liquid chromatog-
raphy/mass spectrometry (LC/MS) was performed on a Dionex
UltiMate 3000 LC instrument equipped with a Dionex MSQ Plus mass
spectrometer. Agilent ZORBAX Eclipse Plus C18 columns (30 mm × 3
mm; 3.5 μm pore size) were used.
parasitized with GFP parasites (PbGFPCON, kindly donated by Drs. A.
P. Waters and C. J. Janse, University of Glasgow and Leiden
36
University). From historical data, untreated control mice died typically
between day 6 and day 7 postinfection, but in these studies all animals
showing unabated parasitemia or malaria symptoms were euthanized on
day 4. The experimental compound was formulated in 7% v/v
Tween 80/3% v/v ethanol. Compounds were administered intra-
peritoneally or orally in a 200 μL volume of 10 mL/kg as a single dose
(
24 h postinfection) or as four consecutive doses (6, 24, 48, and 72 h
Chemical Synthesis: Purification Procedure 1 (PP1). Purifica-
tion of Amines by Acid−Base Extraction. Extraction was carried out
in two centrifuge tubes (15 mL) for quick phase separation through
centrifugation. The crude product was suspended in EtOAc (10 mL)
postinfection). We determined parasitemia at 96 h postinfection, using
standard flow cytometry techniques. Activity was calculated as the
difference between the mean percent parasitemia for the control and
treated groups expressed as a percentage of the control group. The
survival time in days was also recorded up to 30 days after infection. A
compound was considered curative if the animal survived to day 30 after
infection with no detectable parasites.
under sonication and washed twice with aqueous NH solution (10 mL,
3
pH 11). The organic phase was extracted with aqueous HCl solution (10
mL, pH 2). The organic phase was discarded. The acidic aqueous phase
was adjusted to pH 11 with aqueous NH solution (leading to
3
To exclude differences between both Plasmodium species, the in vivo
antimalarial activity was also studied in the SCID mouse P. falciparum
model. This recently established model uses SCID mice engrafted with
human erythrocytes, offering the possibility to investigate P. falciparum
precipitation) and extracted twice with EtOAc (10 mL). The basic
aqueous phase was discarded. The organic phase was concentrated, and
the residue was dried under high vacuum. The obtained product may
still contain traces of imidazole which can be removed via PP2.
Chemical Synthesis: Purification Procedure 2 (PP2). Purifica-
tion of Amides by RP-MPLC. To remove traces of imidazole, reversed
phase chromatography was carried out on a 12 mm × 55 mm column
packed with C18 end-capped reversed phase silica gel (Fluka 60756).
37,38
in vivo.
In Vivo Pharmacokinetics Description. For in vivo PK studies,
female NMRI mice (20−25 g) were obtained from Harlan Laboratories
(
The Netherlands) and were randomly assigned to cages. Mice were
allowed to acclimate before initiation of the experiments. Feed and water
were given ad libitum. Compounds were formulated at concentrations
of 10 mg/mL for a dose of 100 mg/kg administered orally (po) and at a
concentration of 1 mg/mL for a dose of 10 mg/kg given
Eluent mixtures composed of variable amounts of H O (0.01% NH , pH
2 3
9.5) and MeCN at a flow rate of 30 mL/min were used. Storing columns
under basic conditions leads to hydrolysis of the C18 modification and
should therefore be avoided.
G
DOI: 10.1021/jm501987h
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Chemical Synthesis: General Procedure 1 (GP1). CDI
synthesis of (±)-1 are given in a separate procedure. Separation of (±)-8
and (±)-1 enantiomers by chiral phase HPLC (column, Daicel,
Chiralpak-IA 250 mm × 20 mm; flow, 10 mL/min; detection, 254 nm;
2
8
Mediated Synthesis of Amides. A solution of acid (±)-9 (1.0
equiv) in dry THF (54 mg/mL) was treated with a suspension of
carbonyldiimidazole (CDI) (2.0 equiv) in dry THF (50 mg/mL) and
stirred for 30 min at 25 °C. Completion of the reaction was verified via
LC−MS by concentrating a small sample and dissolving the residue in
neat MeCN. The suspension was filtered through a syringe filter to
remove excess CDI. The solution was treated with a variable amount of a
solution of amine compound in THF and was stirred for a variable time
at a variable temperature. Completion of the reaction was verified via
LC−MS. The solution was concentrated and the residue subjected to
PP1 and PP2 for purification.
eluent, CHCl /EtOAc (95:5); sample dissolved in eluent (∼6 mg/mL)
3
2
8
and injected. (−)-8: t = 5.80 min, [α] −117° (c 0.058, CHCl ).
R
D
3
28
(+)-8: t = 6.48 min, [α] 128° (c 0.065, CHCl ). (−)-1: t = 6.20 min,
R
D
3
R
[α]28 −117° (c 0.067, CHCl ). (+)-1: t = 7.18 min, [α] 128° (c
28
D
3
R
D
0.069, CHCl3).
(± )-Methyl 5-[3-(6-Amino-5-cyano-4-isopropyl-3-methyl-
1,4-dihydropyrano[2,3-c]pyrazol-4-yl)-5-cyanophenyl]-
thiophene-2-carboxylate ((± )-1, CAS 1508291-70-0). A mixture
of 32 (405 mg, 1.1 mmol), 3-methyl-1H-pyrazol-5(4H)-one 15 (110
mg, 1.1 mmol), and 2 drops of Hunig’s base in MeOH/THF 1:1 (6 mL)
̈
was stirred for 48 h at 50 °C and evaporated. The residue was taken up in
EtOAc (50 mL) and washed with water (3 × 50 mL). The organic phase
Chemical Synthesis: General Procedure 2 (GP2). TFA Salts of
Amines. To increase the solubility and facilitate handling, insoluble
compounds containing amines were converted to the trifluoroacetates.
An excess of TFA was added to the solution/suspension/solid, followed
by distilling off excess TFA under vacuum. Drying under high vacuum
offers the pure desired salt.
Chemical Synthesis: Characterization and Spectra. Reference
data for compounds commercially available or synthesized according to
literature procedures are provided in Supporting Information section
S1.1. Full characterization of compounds reported in patent application
WO 2013182472 A1 ((±)-16 to (±)-21 and (±)-35 to (±)-48) is
provided in the Supporting Information section S1.2. For NMR spectra,
structure, and atom numbering of all reported compounds see
Supporting Information section S9.
was dried over Na
(SiO ; n-hexane/EtOAc 4:1 → 1:1) and subsequent crystallization gave
(±)-1 (40 mg, 8%) as an off-white solid. R = 0.11 (SiO ; n-hexane/
EtOAc 1:1); mp 186−187 °C. H NMR (400 MHz, CD OD): δ = 0.90
and 1.02 (2 d, J = 6.5 Hz, 6 H; CHMe ), 1.86 (s, 3 H; Me−C(3″)), 2.89
(heptet, J = 6.6 Hz, 1 H; CHMe ), 3.89 (s, 3 H; OMe), 7.52 (br d, J = 4.0
SO and evaporated. Column chromatography
2
4
2
f
2
1
3
2
2
Hz, 1 H; H−C(4)), 7.76 (br t, J = 1.5 Hz, 1 H; H−C(4′)), 7.78 (br d, J =
4.0 Hz, 1 H; H−C(3)), 7.92 (br t, J = 1.7 Hz, 1 H; H−C(2′)), 7.98 ppm
1
3
(br t, 1 H; H−C(6′)). C NMR (100 MHz, CD
OD): δ = 11.93 (Me−
3
C(3″)), 18.72 and 19.22 (CHMe
), 36.49 (CHMe ), 52.84 (OMe),
2
2
61.70 (C(5″)), 100.31 (C(3″a)), 114.56 (C(5′)), 119.19 (NC−(5′)),
121.58 (NC−(5″)), 126.77 (C(4)), 128.85 (C(6′)), 131.73 (C(2′)),
133.14 (C(4′)), 134.66 (C(2)), 135.80 (C(3)), 135.91 (C(1′)), 137.52
(C(3″)), 148.47 (C(3′)), 149.56 (C(5)), 157.50 (C(7″a)), 163.69
(
± )-Benzyl 5-[3-(6-Amino-5-cyano-3-methyl-4-isopropyl-2,4-
dihydropyrano[2,3-c]pyrazol-4-yl)-5-cyanophenyl]thiophene-
2
-carboxylate ((± )-8) and (± )-Methyl 5-[3-(6-Amino-5-cyano-4-
isopropyl-3-methyl-1,4-dihydropyrano[2,3-c]pyrazol-4-yl)-5-
cyanophenyl]thiophene-2-carboxylate ((± )-1, CAS 1508291-
(
CO ), 164.08 (C(6″)) ppm, C(4″) obscured by solvent signal of
2
CD OD. IR (ATR): v
(
̃
= 3078 (w), 2120 (w), 1714 (m), 1636 (m), 1464
3
7
0-0). A solution of 14 (1.94 g, 4.43 mmol) in dry THF (12 mL) was
m), 1434 (m), 1366 (w), 1287 (s), 1154 (w), 1101 (s), 975 (w), 953
w), 889 (w), 820 (m), 749 (s), 689 (m), 648 (m), 627 cm (m). HR-
MALDI-MS m/z (%): 460.1438 (100, [M + H] ; calcd for
C H N O S , 460.1438).
added to a solution of 3-methyl-5-pyrazolone 15 (2.17 g, 22.2 mmol) in
−1
(
dry MeOH (50 mL). Et N (0.22 g, 2.17 mmol) was added resulting in an
+
3
orange solution. The mixture was degassed, stirred under argon for 6
days at 45 °C, treated with SiO2 (10 g), and concentrated.
Chromatography on a Teledyne CombiFlash MPLC system (RediSep
+
24
22
5
3
(
4″R/S,2‴R/S)-5-[3-(6-Amino-5-cyano-3-methyl-4-isopropyl-
2
,4-dihydropyrano[2,3-c]pyrazol-4-yl)-5-cyanophenyl]-N-[5-
8
3
0 g SiO , dry loading, gradient EtOAc/cyclohexane 20:80 to 50:50 over
2
(diethylamino)pent-2-yl]thiophene-2-carboxamide (4″R/
S,2‴R/S-2). Following GP1, the active ester of (±)-9 (0.34 mmol)
and 2-amino-5-diethylaminopentane (159 mg, 1 mmol) were reacted at
25 °C over 10 min. Purification according to PP1 yielded (4″R/S,2‴R/
S)-2 (166 mg, 83%) contaminated with traces of imidazole, which were
0 min) gave (±)-8 (1.02 g, 43%) and (±)-1 (0.25 g, 12%) as yellow
foams. Data of (±)-8: R = 0.36 (SiO , EtOAc/CH Cl 40:60); mp 133−
f
2
2
2
1
1
39 °C. H NMR (600 MHz, (D )THF): δ = 0.88 and 0.98 (2 d, J = 6.6
8
Hz, 6 H; CHMe ), 1.85 (s, 3 H; Me−C(3″)), 2.89 (heptet, J = 6.6 Hz, 1
2
H; CHMe ), 5.33 (s, 2 H; CH Ph), 6.30 (br s, 2 H; NH ), 7.28−7.32 (br
separated according to PP2 (eluent H O/MeCN 65:35, change to 55:45
2
2
2
2
t, J = 7.3 Hz, 1 H; H−C(4) of Ph), 7.33−7.37 (br t, J = 7.3 Hz, 2 H; H−
C(3, 5) of Ph), 7.43−7.45 (br d, J = 7.3 Hz, 2 H; H−C(2, 6) of Ph), 7.54
after 8 min). R = 0.37 (Al O ; EtOH/EtOAc 10:90); mp 128−139 °C.
f
2
3
1
H NMR (400 MHz, CD CN): δ = 0.84 and 0.956 (2 d, J = 6.6−6.7 Hz,
3
(
(
(
(
d, J = 3.9 Hz, 1 H; H−C(4)), 7.72 (br t, J = 1.6 Hz, 1 H; H−C(4′)), 7.79
6
H; CHMe ), 0.957 (t, J = 7.1 Hz, 6 H; N(CH Me) ), 1.19 (d, J = 6.6
2
2
2
d, J = 3.9 Hz, 1 H; H−C(3)), 7.95 (br t, J = 1.8 Hz, 1 H; H−C(2′)), 8.00
Hz, 3 H; NHCHMe), 1.42−1.59 (m, 4 H; NHCHCH CH ), 1.80 (s, 3
2
2
13
br t, J = 1.6 Hz, 1 H; H−C(6′)), 11.35 ppm (br s, 1 H; NH). C NMR
H; Me−C(3″)), 2.40 (t, J = 7.0 Hz, 2 H; NCH CH ), 2.46 (q, J = 7.1 Hz,
2
2
1
150 MHz, (D )THF, INADEQUATE): δ = 12.30 ( J(C,C) = 51.5 Hz;
8
4 H; N(CH Me) ), 2.84 (heptet, J = 6.7 Hz, 1 H; CHMe ), 4.05 (br
2 2 2
1
1
Me−C(3″)), 18.90 ( J(C,C) = 36.4 Hz) and 19.41 ( J(C,C) = 36.7 Hz;
heptet, J = 6.8 Hz, 1 H; NHCH), 5.46 (s, 2 H; NH ), 6.98 (d, J = 8.4 Hz,
2
1
1
CHMe ), 36.25 ( J(C,C) = 34.4, 32.7 Hz; CHMe ), 48.86 ( J(C,C) =
2
2
1 H; CONH), 7.42 (d, J = 3.9 Hz, 1 H; H−C(4)), 7.53 (d, J = 3.9 Hz, 1
1
4
7.7, 43.9, 32.5 Hz; C(4″)), 62.60 ( J(C,C) = 87.9 Hz; C(5″)), 67.58
H; H−C(3)), 7.75 (t, J = 1.5 Hz, 1 H; H−C(4′)), 7.88 (t, J = 1.9 Hz, 1 H;
1
1
(
J(C,C) = 48.8 Hz; CH Ph), 99.65 ( J(C,C) = 72.4, 44.2 Hz; C(3″a)),
1 1
2
H−C(2′)), 7.93 (t, J = 1.6 Hz, 1 H; H−C(6′)), 10.15−10.65 ppm (br s,
13
1
14.5 ( J(C,C) = 80.2, 59.0, 58.3 Hz; C(5′)), 119.01 ( J(C,C) = 80.1
1
H; H−N(2″)). C NMR (100 MHz, CD CN): δ = 12.08 (Me−
3
1
Hz; NC−C(5′)), 120.29 (NC−C(5″)), 126.47 ( J(C,C) = 62.5, 58.3
C(3″)), 12.18 (N(CH Me) ), 18.63 and 19.07 (CHMe ), 21.20
(NHCHMe), 24.58 (CH CH CH ), 35.13 (NHCHCH ), 35.99
2
2
2
1
Hz; C(4)), 128.56 ( J(C,C) = 64.5, 58.0 Hz; C(6′)), 129.11 (C(4) of
2 2 2 2
1
Ph), 129.18 ( J(C,C) = 57.6 Hz; C(2, 6) of Ph), 129.42 (C(3, 5) of Ph),
(CHMe ), 46.66 (NHCH), 47.64 (N(CH Me) ), 48.57 (C(4″)),
2
2
2
1
1
1
31.02 ( J(C,C) = 64.2, 58.4 Hz; C(2′)), 133.13 ( J(C,C) = 59.2, 58.1
53.36 (NCH CH ), 63.48 (C(5″)), 99.99 (C(3″a)), 113.98 (C(5′)),
2
2
1
1
Hz; C(4′)), 134.58 ( J(C,C) = 87.6, 64.2 Hz; C(2)), 135.39 ( J(C,C) =
119.27 (NC−C(5′)), 120.64 (NC−C(5″)), 126.47 (C(4)), 128.65
(C(6′)), 129.28 (C(3)), 131.20 (C(2′)), 132.63 (C(4′)), 135.63
(C(1′)), 137.03 (C(3″)), 141.98 (C(2)), 146.21 (C(5)), 147.84
(C(3′)), 157.07 (C(7″a)), 161.53 (CONH), 162.89 ppm (C(6″)). IR
(ATR): ν = 3300 (w), 3168 (w), 3090 (w), 2968 (m), 2933 (m), 2874
(w), 2818 (w), 2232 (w), 2185 (m), 1630 (s), 1585 (s), 1544 (s), 1520
(s), 1486 (s), 1386 (s), 1289 (s), 1149 (m), 1069 (m), 1043 (m), 990
1
6
1
5.7, 58.9, 58.7 Hz; C(1′)), 135.50 ( J(C,C) = 64.8, 52.5 Hz; C(3)),
1
1
36.13 ( J(C,C) = 73.8, 50.3 Hz; C(3″)), 137.29 ( J(C,C) = 56.9, 49.5
1
Hz; C(1) of Ph), 148.40 ( J(C,C) = 64.1, 64.1, 42.9 Hz; C(3′)), 149.63
1
1
(
J(C,C) = 63.9, 61.8 Hz; C(5)), 157.43 (C(7″a)), 162.07 ( J(C,C) =
1
8
3
(
(
(
7
+
7.7 Hz; CO ), 163.05 ppm ( J(C,C) = 94.7 Hz; C(6″)). IR (ATR): ν =
2
319 (w), 3194 (w), 3093 (w), 2969 (w), 2879 (w), 2232 (w), 2186
−1
m), 1704 (m), 1632 (s), 1585 (s), 1535 (m), 1487 (m), 1465 (m), 1420
m), 1383 (s), 1344 (m), 1281 (s), 1243 (s), 1175 (m), 1153 (m), 1093
s), 1042 (s), 1001 (m), 940 (m), 907 (m), 865 (m), 820 (m), 783 (w),
(m), 884 (m), 817 (m), 734 (m), 700 cm (m). HR-ESI-MS m/z (%):
+
+
587.2986 (38), 586.2955 (100, [M + H] ; calcd for C H N O S ,
32 40 7 2
586.2959).
−1
45 cm (s). HR-ESI-MS m/z (%): 537.1782 (42), 536.1750 (100, [M
(± )-5-[3-(6-Amino-5-cyano-3-methyl-4-isopropyl-2,4-
dihydropyrano[2,3-c]pyrazol-4-yl)-5-cyanophenyl]-N-[3-
+
+
H] ; calcd for C H N O S , 536.1751). Data and alternative
30
26
5
3
H
DOI: 10.1021/jm501987h
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
+
(
diethylamino)propyl]thiophene-2-carboxamide ((± )-3). Fol-
52.78 (C(3, 5) of morpholinium), 55.23 (HN CH CH ), 63.41 (C(5)),
2 2
lowing GP1, the active ester of (±)-9 (0.15 mmol) and 3-
(
°
8
according to PP2 (eluent H O/MeCN 65:35, change to 60:40 after 6
min). A small fraction of (±)-3 was converted to the ditrifluoroacetate
(
(
1
64.79 (C(2, 6) of morpholinium), 100.01 (C(3a)), 114.05 (C(3′)),
diethylamino)propylamine (33 mg, 0.25 mmol) were reacted at 25
C over 20 min. Purification according to PP1 yielded (±)-3 (71 mg,
3%) contaminated with traces of imidazole, which were separated
115.57 (CF ), 119.22 (NC−C(3′)), 120.66 (NC−C(5)), 126.80
3
(C(3″)), 128.83 (C(4′)), 130.73 (C(4″)), 131.29 (C(6′)), 132.90
(C(2′)), 135.35 (C(5′)), 137.13 (C(3)), 139.82 (C(5″)), 147.39
2
(C(2″)), 147.90 (C(1′)), 157.03 (C(7a)), 160.45 (q, J(C,F) = 33.7 Hz,
2
CF CO ), 162.90 (C(6)), 164.06 ppm (CONH). IR (ATR): ν = 3312
3
2
1
±)-3a according to GP2. Data of (±)-3: mp 127−134 °C. H NMR
(w), 3202 (w), 2969 (w), 2875 (w), 2233 (w), 2187 (w), 1778 (w), 1674
(m), 1633 (s), 1587 (m), 1551 (m), 1523 (m), 1487 (m), 1423 (m),
1392 (m), 1306 (m), 1197 (s), 1171 (s), 1132 (s), 1047 (m), 985 (m),
815 (m), 798 (m), 720 (m), 705 cm (s). HR-ESI-MS m/z (%):
573.2469 (37), 572.2438 (100, [M + H] ; calcd for C H N O S ,
400 MHz, CD CN): δ = 0.85 and 0.96 (2 d, J = 6.6 Hz, 6 H; CHMe ),
3
2
.03 (t, J = 7.1 Hz, 6 H; N(CH Me) ), 1.72 (quint, J = 6.3 Hz, 2 H;
2
2
−1
CH CH CH ), 1.80 (s, 3 H; Me−C(3″)), 2.561 (q, J = 7.2 Hz, 4 H;
N(CH Me) ), 2.573 (t, J = 5.9 Hz, 2 H; NCH CH ), 2.85 (heptet, J =
2
2
2
+
+
2
2
2
2
30 34 7 3
6
.6 Hz, 1 H; CHMe ), 3.41 (td, J = 6.3, 4.8 Hz, 2 H; NHCH ), 5.46 (s, 2
572.2438).
2
2
H; NH ), 7.43 (d, J = 3.9 Hz, 1 H; H−C(4)), 7.48 (d, J = 3.9 Hz, 1 H;
(± )-5-[3-(6-Amino-5-cyano-3-methyl-4-isopropyl-2,4-
dihydropyrano[2,3-c]pyrazol-4-yl)-5-cyanophenyl]-N-[3-(4-
methylpiperazin-1-yl)propyl]thiophene-2-carboxamide
2
H−C(3)), 7.75 (t, J = 1.6 Hz, 1 H; H−C(4′)), 7.87 (t, J = 1.8 Hz, 1 H;
H−C(2′)), 7.94 (t, J = 1.6 Hz, 1 H; H−C(6′)), 8.27 (br t, J = 5.0 Hz, 1
H; CONH), 10.14−10.67 ppm (br s, 1 H; H−N(2″)). IR (ATR): ν =
(
(± )-5). Following GP1, the active ester of (±)-9 (0.34 mmol) and 3-(4-
methylpiperazino)propylamine (131 mg, 0.83 mmol) were reacted at 25
C over 20 min. Purification according to PP1 using a 10 times larger
3301 (w), 3171 (w), 3089 (w), 2968 (m), 2933 (m), 2874 (w), 2821
°
(
(
(
(
=
w), 2232 (w), 2185 (m), 1632 (s), 1585 (s), 1547 (s), 1520 (s), 1486
volume for all phases (100 mL instead of 10 mL) and conventional
separatory funnels because of lower solubility yielded (±)-5 (154 mg,
77%) contaminated with traces of imidazole, which were separated
according to PP2 (eluent H O/MeCN 75:25). R = 0.24 (Al O ; EtOH/
s), 1386 (s), 1294 (s), 1168 (m), 1044 (m), 903 (m), 883 (m), 818
−
1
m), 734 cm (m). HR-ESI-MS m/z (%): 559.2674 (34), 558.2643
+
+
100, [M + H] ; calcd for C H N O S : 558.2646). Data of (±)-3a: R
30
36
7
2
f
1
0.29 (Al O ; EtOH/EtOAc 10:90); mp 65−69 °C. H NMR (400
2
f
2
3
2
3
1
EtOAc 10:90); mp 160−172 °C. H NMR (400 MHz, CD CN): δ =
0.85 and 0.95 (2 d, J = 6.6 Hz, 6 H; CHMe ), 1.72 (quint, J = 6.5 Hz, 2 H;
MHz, CD CN): δ = 0.85 and 0.96 (2 d, J = 6.7 Hz, 6 H; CHMe ), 1.27 (t,
3
3
2
+
J = 7.3 Hz, 6 H; HN (CH Me) ), 1.80 (s, 3 H; Me−C(3″)), 1.96−2.02
2
2
2
CH CH CH ), 1.80 (s, 3 H; Me−C(3″)), 2.19 (s, 3 H; NMe), 2.31−
(
quint, 2 H; CH CH CH ), 2.85 (heptet, J = 6.7 Hz, 1 H; CHMe ), 3.10
2
2
2
2
2
2
2
+
2.41 (br, s, 8 H; N(CH CH ) N), 2.45 (t, J = 6.4 Hz, 2 H;
(
br q, J = 6.5 Hz, 2 H; HN CH CH ), 3.14−3.22 (m, 4 H;
2 2 2
2
2
+
NCH CH CH ), 2.84 (heptet, J = 6.7 Hz, 1 H; CHMe ), 3.39 (td, J =
2 2 2 2
HN (CH Me) ), 3.45 (br q, J = 6.2 Hz, 2 H; CONHCH ), 5.46 (br
2
2
2
6
.5, 5.2 Hz, 2 H; NHCH ), 5.47 (br s, 2 H; NH ), 7.45 (d, J = 3.9 Hz, 1
s, 2 H; NH ), 7.48 (d, J = 4.0 Hz, 1 H; H−C(4)), 7.58 (br t, J = 6.8 Hz, 1
2
2
2
H; H−C(4)), 7.54 (d, J = 3.9 Hz, 1 H; H−C(3)), 7.75 (t, J = 1.6 Hz, 1 H;
H−C(4′)), 7.81 (br t, J = 5.5 Hz, 1 H; CONH), 7.88 (t, J = 1.8 Hz, 1 H;
H−C(2′)), 7.94 (t, J = 1.6 Hz, 1 H; H−C(6′)), 10.20−10.80 ppm (br s,
H; CONH), 7.62 (d, J = 4.0 Hz, 1 H; H−C(3)), 7.78 (br t, J = 1.6 Hz, 1
H; H−C(4′)), 7.89 (br t, J = 1.8 Hz, 1 H; H−C(2′)), 7.95 (br t, J = 1.6
+
13
Hz, 1 H; H−C(6′)), 8.10−8.35 ppm (br s, 1 H; (CH ) NH ). C NMR
2
3
1
3
+
1 H; H−N(2″)). C NMR (100 MHz, CD CN): δ = 12.12 (Me−
(
100 MHz, CD CN): δ = 9.03 (HN (CH Me) ), 12.06 (Me−C(3″)),
8.62 and 19.05 (CHMe ), 25.41 (CH CH CH ), 36.01 (CHMe ),
3
3
2
2
C(3″)), 18.63 and 19.08 (CHMe ), 26.12 (CH CH CH ), 36.00
1
3
(
1
2
2
2
2
2
2
2
2
2
+
(CHMe ), 40.36 (NHCH ), 46.26 (NMe), 48.58 (C(4″)), 53.98
6.83 (NHCH ), 48.00 (HN (CH Me) ), 48.59 (C(4″)), 50.11
2
2
2
2
2
+
(
(
1
CH N(CH CH ) N), 55.82 (NCH CH NCH ), 57.78 (N-
HN CH CH ), 63.44 (C(5″)), 100.03 (C(3″a)), 114.08 (C(5′)),
2 2 2 2 2 2 3
2
2
1
CH CH ) CH ), 63.55 (C(5″)), 99.93 (C(3″a)), 113.99 (C(5′)),
19.26 (NC−C(5′)), 120.62 (NC−C(5″)), 126.46 (C(4)), 128.65
15.08 (q, J(C,F) ≈ 270 Hz, CF ), 119.20 (NC−C(5′)), 120.61 (NC−
2
2
2
2
3
C(5″)), 126.87 (C(4)), 128.88 (C(6′)), 130.80 (C(3)), 131.30 (C(2′)),
(
(
(
(
(
(
(
C(6′)), 129.60 (C(3)), 131.20 (C(2′)), 132.62 (C(4′)), 135.58
C(1′)), 137.02 (C(3″)), 141.53 (C(2)), 146.08 (C(5)), 147.87
C(3′)), 157.10 (C(7″a)), 161.95 (CONH), 162.87 ppm (C(6″)). IR
ATR): ν = 3301 (w), 3162 (m), 3087 (w), 2935 (m), 2878 (m), 2805
m), 2231 (w), 2185 (m), 1632 (s), 1585 (s), 1547 (s), 1520 (s), 1486
s), 1462 (m), 1421 (m), 1390 (s), 1284 (s), 1148 (s), 1103 (m), 1071
1
1
3
=
32.97 (C(4′)), 135.30 (C(1′)), 137.14 (C(3″)), 139.54 (C(2)),
2
47.59 (C(5)), 147.92 (C(3′)), 157.01 (C(7″a)), 159.51(q, J(C,F) =
8.9 Hz, CF CO ), 162.89 (C(6″)), 164.34 ppm (CONH). IR (ATR): ν
3
2
3320 (w), 2973 (w), 2234 (w), 2188 (w), 1776 (w), 1633 (m), 1587
(
(
m), 1551 (m), 1487 (m), 1392 (m), 1308 (m), 1141 (s), 1036 (m), 813
−1
m), 703 cm (s).
± )-6-Amino-5-cyano-4-[3-cyano-5-(5-{[3-(morpholin-4-ium-
-yl)propyl]carbamoyl}thiophen-2-yl)phenyl]-3-methyl-4-iso-
−1
m), 1048 (m), 1011 (m), 930 (m), 885 (m), 817 (m), 734 cm+ (m).
(
4
HR-ESI-MS m/z (%): 586.2787 (41), 585.2751 (100, [M + H] ; calcd
for C H N O S , 585.2755).
+
propyl-2,4-dihydropyrano[2,3-c]pyrazol-1-ium Bis-
31
37
8
2
(trifluoroacetate) ((± )-4a). Following GP1, the active ester of
(4″R/S)-Di-tert-Butyl N-[(5-[3-(6-Amino-5-cyano-3-methyl-4-
isopropyl-2,4-dihydropyrano[2,3-c]pyrazol-4-yl)-5-
cyanophenyl]thien-2-yl)carbonyl]-L-glutamate ((4″R/S,2‴S)-6).
Following GP1, the active ester of (±)-9 (0.34 mmol) and 29 (302 mg,
(±)-9 (0.15 mmol) and 3-morpholinopropylamine (37 mg, 0.25
mmol) were reacted at 25 °C over 20 min. Purification according to PP1
yielded (±)-4 (77 mg, 87%) contaminated with traces of imidazole,
which were separated according to PP2 (eluent H O/MeCN 70:30).
1.02 mmol) were reacted. Addition of i-Pr NEt (352 mg, 2.72 mmol) to
2
2
Note that for PP1 the precipitate in the acidic aqueous phase is insoluble
product, which dissolves again in the organic phase upon raising the pH.
For better solubility (±)-4 was converted to the ditrifluoroacetate
dissolve the formed precipitate and a reaction time of 3 h at 60 °C were
required for completion of the reaction. Chromatographic purification
on a Teledyne CombiFlash MPLC system (RediSep 40 g SiO
loading, EtOAc/cyclohexane 50:50) yielded pure (4″R/S,2‴S)-6 as a
white solid (108 mg, 46%). R = 0.44 (SiO , EtOAc/cyclohexane 70:30);
mp 133−145 °C. H NMR (400 MHz, (D )THF): δ = 0.89 and 0.99 (2
d, J = 6.6 Hz, 6 H; CHMe ), 1.42 (s, 9 H; CMe ), 1.46 (s, 9 H; CMe ),
1.87 (s, 3 H; Me−C(3″)), 1.88−1.98 (m, 1 H; CHCH H ), 2.10−2.20
, dry
2
(
±)-4a according to GP2. Data of (±)-4a: R = 0.31 (Al O ; EtOH/
f
2
3
1
EtOAc 10:90); mp 71−76 °C. H NMR (400 MHz, CD CN): δ = 0.85
f
2
3
1
and 0.96 (2 d, J = 6.5 Hz, 6 H; CHMe ), 1.80 (s, 3 H; Me−C(3)), 2.02
8
2
(
br quint, J ≈ 6.5 Hz, 2 H; CH CH CH ), 2.84 (heptet, J = 6.6 Hz, 1 H;
2
3
3
2
2
2
CHMe ), 3.02 (br t, J ≈ 12.2 Hz, 2 H; H −C(3, 5) of morpholinium),
2
ax
a
b
+
3
.15 (br t, J = 6.9 Hz 2 H; HN CH CH CH ); 3.41 (br d, J = 12.9 Hz, 2
(m, 1 H; CHCH H ), 2.30−2.38 (m, 2 H; CH CO ), 2.88 (heptet, J =
2
2
2
a
b
2
2
H; H −C(3, 5) of morpholinium), 3.42−3.49 (m, 2 H; CONHCH ),
6.7 Hz, 1 H; CHMe ), 4.50−4.58 (br td, J = 8.2, 4.4 Hz, 1 H; NHCH),
eq
2
2
3
.79 (br td, J = 12.5, 1.9 Hz, 2 H; H −C(2, 6) of morpholinium), 4.02
6.29 (s, 2 H; NH ), 7.47 (d, J = 3.9 Hz, 1 H; H−C(4)), 7.64 (d, J = 3.9
ax
2
(
2
br dd, J = 13.4, 3.0 Hz, 2 H; H −C(2, 6) of morpholinium), 5.48 (br s,
Hz, 1 H; H−C(3)), 7.69 (br t, J = 1.6 Hz, 1 H; H−C(4′)), 7.72 (br d, J =
8.3 Hz, 1 H; CONH), 7.92 (br t, J = 1.8 Hz, 1 H; H−C(2′)), 7.95 (br t, J
eq
H; NH ), 7.46 (d, J = 4.0 Hz, 1 H; H−C(3″)), 7.63 (d, J = 4.0 Hz, 1 H;
2
1
3
H−C(4″)), 7.68 (br t, J = 6.0 Hz, 1 H; CONH), 7.77 (t, J = 1.6 Hz, 1 H;
= 1.5 Hz, 1 H; H−C(6′)), 11.35 ppm (s, 1 H; NNH). C NMR (100
MHz, (D )THF): δ = 12.32 (Me−C(3″)), 18.93 and 19.44 (CHMe ),
H−C(2′)), 7.89 (t, J = 1.8 Hz, 1 H; H−C(6′)), 7.94 (t, J = 1.5 Hz, 1 H;
8
2
13
H−C(4′)), 9.82 ppm (s, 1 H; (CH ) NH+). C NMR (100 MHz,
28.32 (CHCH ), 28.36 (OCMe ), 28.45 (OCMe ), 32.60 (CH CO ),
2
3
2 3 3 2 2
CD CN): δ = 12.07 (Me−C(3)), 18.63 and 19.05 (CHMe ), 24.59
36.30 (CHMe ), 48.90 (C(4″)), 53.73 (NHCH), 62.81 (C(5″)), 80.66
3
2
2
(
CH CH CH ), 36.01 (CHMe ), 36.94 (CONHCH ), 48.58 (C(4)),
(OCMe ), 81.90 (OCMe ), 99.68 (C(3″a)), 114.56 (C(5′)), 119.12
2
2
2
2
2
3
3
I
DOI: 10.1021/jm501987h
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(
1
NC−C(5′)), 120.12 (NC−C(5″)), 125.95 (C(4)), 128.23 (C(6′)),
C(3)), 7.72 (t, J = 1.5 Hz, 1 H; H−C(4′)), 7.94 (t, J = 1.8 Hz, 1 H; H−
C(2′)), 7.98 (t, J = 1.5 Hz, 1 H; H−C(6′)), 10.8−11.8 ppm (br s, 2 H;
COOH, NH). 13C NMR (100 MHz, (D )THF): δ = 12.32 (CH −
29.80 (C(3)), 131.00 (C(2′)), 132.61 (C(4′)), 135.92 and 135.98
(
(
C(1′, 3″)), 141.25 (C(2)), 147.20 (C(5)), 148.37 (C(3′)), 157.53
8
3
C(7″a)), 161.87 (CONH), 162.99 (C(6″)), 171.97 (CHCO ), 172.60
C(3″)), 18.92 and 19.44 (CHMe ), 36.30 (CHMe ), 48.92 (C(4″)),
2
2
2
ppm (CH CO ). IR (ATR): ν = 3315 (w), 2975 (w), 2932 (w), 2232
62.78 (C(5″)), 99.65 (C(3″a)), 114.63 (C(5′)), 119.05 (NC−C(5′)),
120.12 (NC−C(5″)), 126.31 (C(4)), 128.46 (C(6′)), 131.10 (C(2′)),
132.96 (C(4′)), 135.03 (C(3)), 135.68 and 135.91 and 135.98 (C(2, 1′,
3″)), 148.45 (C(3′)), 149.16 (C(5)), 157.53 (C(7″a)), 163.00 and
2
2
(
w), 2187 (w), 1725 (m), 1632 (s), 1587 (m), 1544 (m), 1517 (m),
1
1
487 (m), 1421 (m), 1392 (m), 1367 (s), 1292 (m), 1249 (m), 1148 (s),
−1
070 (m), 1035 (m), 907 (w), 843 (m), 819 (m), 734 cm (m). HR-
+
ESI-MS m/z (%): 710.2822 (34), 709.2787 (68, [M + Na] ; calcd for
C H N NaO S , 709.2779), 575.1713 (100).
163.02 ppm (CO , C(6″)). IR (ATR): ν = 3494 (w), 3390 (w), 3341
2
+
(w), 3200 (w), 3166 (w), 3097 (w), 2966 (w), 2875 (w), 2236 (w), 2202
(m), 1704 (s), 1635 (s), 1590 (s), 1536 (w), 1487 (m), 1468 (m), 1430
(m), 1388 (s), 1337 (w), 1279 (m), 1249 (s), 1213 (m), 1190 (m), 1156
36
42
6
6
(
4R/S)-5-Cyano-4-(3-cyano-5-{5-[((S)-1,3-dicarboxypropyl)-
carbamoyl]thiophen-2-yl}phenyl)-3-methyl-4-(propan-2-yl)-
2
,4-dihydropyrano[2,3-c]pyrazol-6-aminium Trifluoroacetate
(
(
m), 1102 (m), 1067 (w), 1046 (m), 1024 (m), 932 (m), 909 (w), 869
(
(4R/S,2‴S)-7). A solution of (4″R/S,2‴S)-6 (19 mg, 0.028 mmol) in
−1
w), 839 (m), 794 (w), 751 (m), 732 (s), 706 cm (m). HR-ESI-MS m/
TFA (1 mL) was kept in a flask sealed with a glass stopper at 25 °C for 14
h. Excess TFA was distilled off. The residue was dried under high
vacuum to yield pure (4R/S,2‴S)-7 (19 mg, quant) as a yellow solid.
Prolonged exposure to high vacuum may vary the amount of
+
z (%): 447.1313 (29), 446.1283 (100, [M + H] ; calcd for
C H N O S , 446.1281).
+
23
20
5
3
5
-[3-(6-Amino-5-cyano-4-isopropyl-3-methyl-2,4-
dihydropyrano[2,3-c]pyrazol-4-yl)-5-cyanophenyl]-N,N-di-
methyl-2-thiophenecarboxamide ((± )-10). Following GP1, dime-
thylamine (2 M in THF) (0.20 mL, 0.39 mmol) was added to the active
ester of (±)-9 (56 mg, 0.11 mmol) in THF (4.9 mL). The mixture was
stirred at 25 °C for 16 h. The mixture was concentrated under reduced
pressure. Chromatography (SiO , gradient EtOAc/CH Cl 50:50 to
1
19
trifluoroacetate. The precise content was determined by H and
NMR integration and was found to be 0.8 equiv of TFA.
F
Dibromofluoromethane was used as an internal standard to correlate
1
19
H and F integral ratios. Compound (4R/S,2‴S)-7 may be converted
to the hydrochloride salt (4R/S,2‴S)-7a by dissolving in THF, adding
excess aqueous HCl solution (1 N), followed by concentrating and
drying under vacuum. Data of (4R/S,2‴S)-7: R = 0.32−0.47 (SiO ;
2
2
2
60:40) yielded the product (±)-10 as a white solid (35 mg, 66%). R =
f
1
0.12 (SiO , gradient EtOAc/CH Cl 60:40); mp 166−169 °C. H NMR
f
2
2
2
2
1
H O/THF 5:95); mp 123−131 °C. H NMR (400 MHz, (D )THF): δ
(400 MHz, (D )THF): δ = 11.35 (s, 1H; NNH), 7.95 (br t, J = 1.6 Hz,
8
2
8
=
0.89 and 0.99 (2 d, J = 6.6 Hz, 6 H; CHMe ), 1.86 (s, 3 H; Me−C(3)),
1H; H−C(6′)), 7.92 (br t, J = 1.8 Hz, 1H; H−C(2′)), 7.69 (br t, J = 1.6
Hz, 1H; H−C(4′)), 7.44 (d, J = 3.9 Hz, 1H; H−C(3)), 7.39 (d, J = 3.9
2
1
2
4
7
7
.94−2.06 (m, 1 H; CHCH H ), 2.17−2.28 (m, 1 H; CHCH H ),
a
b
a
b
.33−2.48 (m, 2 H; CH CO ), 2.88 (heptet, J = 6.7 Hz, 1 H; CHMe ),
Hz, 1H; H−C(4)), 6.28 (s, 2H; NH ), 3.14 (s, 6H; NMe ), 2.88 (p, J =
2 2
2
2
2
.63−4.70 (br td, J = 8.6, 4.8 Hz, 1 H; NHCH), 6.29 (br s, 2 H; NH ),
6.6 Hz, 1H; CHMe
CHMe ), 0.88 (d, J = 6.6 Hz, 3H; CHMe
(D )THF): δ = 163.55 (C(6″)), 162.99 (CONMe
2
), 1.86 (s, 3H; Me−C(3″)), 0.98 (d, J = 6.6 Hz, 3H;
) ppm. 13C NMR (125 MHz,
), 157.54 (C(7″a)),
2
.47 (d, J = 3.9 Hz, 1 H; H−C(4″)), 7.64 (d, J = 3.9 Hz, 1 H; H−C(3″)),
.69 (br t, J = 1.6 Hz, 1 H; H−C(2′)), 7.78 (br d, J = 8.3 Hz, 1 H;
2
2
8
2
CONH), 7.91 (br t, J = 1.8 Hz, 1 H; H−C(6′)), 7.95 ppm (br t, J = 1.5
148.36 (C(5)), 145.88 (C(3′)), 140.87 (C(3″)), 135.96, 135.87 (C(2)),
132.52 (C(3)), 130.98 (C(4′)), 130.92 (C(2′)), 128.18 (C(6′)), 125.30
(C(4)), 120.14 (NC−(5″)), 119.14 (NC−(5′)), 114.52 (C(5′)), 99.70
13
Hz, 1 H; H−C(4′)). C NMR (100 MHz, (D )THF): δ = 12.33 (Me−
8
C(3)), 18.92 and 19.44 (CHMe ), 28.30 (CHCH ), 31.04 (CH CO ),
2
2
2
2
3
6.30 (CHMe ), 48.90 (C(4)), 52.91 (NHCH), 62.80 (C(5)), 99.67
(C(3″a)), 62.83 (C(5″)), 48.89 (C(4″)), 36.30 (NMe
2
), 19.45
(CHMe obscured
by solvent residual signal of (D )THF. IR (ATR): ν = 3175 (br w), 2967
8
2
(
1
1
C(3a)), 114.56 (C(3′)), 119.12 (NC−C(3′)), 120.13 (NC−C(5)),
2
), 18.94 (CHMe
2
), 12.32 (Me−C(3″)) ppm, CHMe
2
25.94 (C(4″)), 128.22 (C(4′)), 129.77 (C(3″)), 131.01 (C(6′)),
32.59 (C(2′)), 135.95 and 136.01 (C(5′, 3)), 141.36 (C(2″)), 147.13
(w), 2932 (w), 2231 (w), 2185 (m), 1632 (s), 1585 (s), 1538 (w), 1487
(m), 1435 (w), 1420 (w), 1390 (s), 1284 (w), 1265 (w), 1204 (w), 1183
(w), 1152 (w), 1057 (br m), 877 (br w), 813 (m), 733 (m), 695 (m).
(
(
C(5″)), 148.36 (C(1′)), 157.52 (C(7a)), 161.82 (CONH), 163.00
C(6)), 173.61 (CHCO ), 174.48 ppm (CH CO ). IR (ATR): ν =
2
2
2
+
3
2
1
1
(
5
500−2200 (m), 3313 (w), 3187 (w), 3094 (w), 2967 (w), 2931 (w),
234 (w), 2188 (w), 1714 (m), 1632 (s), 1587 (s), 1545 (s), 1519 (s),
493 (m), 1421 (m), 1392 (s), 1347 (m), 1296 (m), 1259 (s), 1203 (s),
149 (s), 1096 (s), 1070 (m), 1030 (s), 935 (m), 883 (m), 816 (s), 740
HR-ESI-MS m/z (%): 473.1748 (100, [M + H] ; calcd for
+
C
C
H
N
O
S , 473.1754). Elemental analysis calcd (%) for
25
25
6
2
H
24
N
O
2
S: C, 63.54; H, 5.12; N, 17.78. Found: C, 62.10; H, 5.37;
25
6
N, 16.55.
−1
m), 695 (m), 656 cm (m). HR-ESI-MS m/z (%): 576.1736 (13),
Safety Precautions. Diazomethane and its derivatives are explosive
+
+
75.1709 (36, [M + H] ; calcd for C H N O S , 575.1707), 282.2799
and highly toxic. Experiments were carried out behind a blast shield in a
28
27
6
6
1
(
3
100). Data of (4R/S,2‴S)-7a: mp 220−250 °C (dec). IR (ATR): ν =
dedicated laboratory. Phenyldiazomethane was quantified by H NMR
310 (m), 3185 (m), 3089 (w), 2964 (m), 2929 (m), 2877 (w), 2548
measurements (CDCl
Benzyl 5-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)-
thiophene-2-carboxylate (11). A solution of Me SO (3.84 g, 49.3
3
, δ = 4.94 ppm (s, 1 H; PhCHN )).
2
(
(
w), 2234 (w), 2188 (w), 1710 (m), 1630 (s), 1586 (s), 1546 (s), 1520
s), 1492 (m), 1421 (s), 1393 (s), 1295 (s), 1259 (s), 1213 (s), 1175 (s),
2
mmol, 2.2 equiv) in Et O/CH Cl 9:1 (470 mL) was cooled to −60 °C
1
153 (s), 1095 (m), 1033 (s), 881 (m), 818 (s), 745 (m), 694 (m), 651
2
2
2
−1
in an argon filled 2 L flask, treated dropwise with oxalyl chloride (5.97 g,
7 mmol), and stirred for 20 min after which gas evolution had ceased. A
solution of Et N (9.50 g, 94.1 mmol) and 27 (5.38 g, 44.8 mmol) in
Et O/CH Cl 9:1 (150 mL) was added dropwise to the cold mixture
cm (m).
4
(
± )-5-[3-(6-Amino-5-cyano-3-methyl-4-isopropyl-2,4-
dihydropyrano[2,3-c]pyrazol-4-yl)-5-cyanophenyl]thiophene-
3
2
-carboxylic Acid ((± )-9). A solution of (±)-8 (500 mg, 0.93 mmol)
2
2
2
2
6
in MeOH (10 mL) was treated with 10% Pd/C (250 mg) and stirred
behind a blast shield over 20 min. A precipitate was formed, and the
mixture turned “peach” colored. The mixture was stirred for 50 min at
−60 °C, treated dropwise with a solution of 26 (5.69 g, 22.4 mmol) in
Et O/CH Cl 9:1 (75 mL) over 20 min, and stirred in the dark for 14 h
under H (two balloons (⌀ 25 cm) of H were passed through the
solution) for 30 min. The solid was filtered off and the filtrate
evaporated. The residue was suspended in EtOAc (50 mL) by
2
2
2
2
2
sonication and extracted twice with aqueous NH solution (50 mL,
at 25 °C. Excess phenyldiazomethane 68 was deleted by addition of a
3
pH 11). The aqueous phase was acidified to pH 5 with 1 N HCl and
extracted with EtOAc (3 × 150 mL). The precipitate should rapidly be
dissolved in the organic phase to avoid aggregation. The organic phase
was washed with aqueous saturated NaCl solution (250 mL) and
1.75 N solution of acetic acid in Et O. The mixture was treated with SiO
2
2
and evaporated. Chromatography (Teledyne CombiFlash MPLC
system, RediSep 120 g SiO , dry loading, EtOAc/cyclohexane 2:98)
2
yielded the product 11 (6.73 g, 87%) as a yellow/orange oil which
crystallized over 14 h. R = 0.53 (SiO ; EtOAc/cyclohexane 20:80); mp
evaporated. Drying under high vacuum for 2 days gave (±)-9 (367 mg,
f
2
1
1
8
(
8%) as a white powder: mp 250−270 °C (dec). H NMR (400 MHz,
79−80 °C. H NMR (400 MHz, (D )THF): δ = 1.32 (s, 12 H;
8
D )THF): δ = 0.89 and 0.99 (2 d, J = 6.6 Hz, 6 H; CHMe ), 1.87 (s, 3
(CMe ) ), 5.31 (s, 2 H; CH Ph), 7.26−7.37 (m, 3 H; H−C(3′, 4′, 5′)),
8
2
2
2
2
H; Me−C(3″)), 2.89 (heptet, J = 6.5 Hz, 1 H; CHMe ), 6.30 (br s, 2 H;
7.50−7.53 (br d, J = 7.4 Hz 2 H; H−C(2′, 6′)), 7.52 (d, J = 3.7 Hz, 1 H;
2
13
NH ), 7.51 (d, J = 3.9 Hz, 1 H; H−C(4)), 7.71 (d, J = 3.9 Hz, 1 H; H−
H−C(4)), 7.81 ppm (d, J = 3.7 Hz, 1 H; H−C(3)); C NMR (100
2
J
DOI: 10.1021/jm501987h
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
MHz, (D )THF): δ = 25.21 ((CMe ) ), 67.44 (CH Ph), 85.44
CHCl (20 mL) was treated with a solution of 1 M TiCl in toluene (9.2
8
2
2
2
3
4
(
1
(CMe ) ), 129.01 (C(2′, 6′)), 129.03 (C(4′)), 129.40 (C(3′, 5′)),
mL, 9.2 mmol) leading to a light brown color and precipitation. Pyridine
(2.3 mL, 28.4 mmol) was added leading to a darkening. The mixture was
stirred at 60 °C for 4 days and diluted with MeOH (20 mL) to get a
2
2
34.71 (C(3)), 137.47 (C(1′)), 137.83 (C(4)), 140.78 (C(2)), 162.08
ppm (CO), C(5) hidden by the noise. IR (ATR): ν = 2976 (w), 1713
(
(
9
(
s), 1525 (m), 1471 (w), 1455 (w), 1384 (m), 1358 (s), 1329 (s), 1292
s), 1235 (s), 1142 (s), 1087 (s), 1073 (s), 1057 (s), 1019 (m), 961 (m),
solution. The mixture was treated with SiO (20 g) and evaporated.
2
Chromatography on a Teledyne CombiFlash MPLC system (RediSep
19 (m), 851 (s), 824 (m), 783 (w), 740 cm−1 (s). HR-ESI-MS m/z
80 g SiO , dry loading, EtOAc/cyclohexane 10:90) yielded 14 (2.11 g,
2
+
%): 368.1181 (9), 367.1151 (43, [M + H] ; calcd for
68%) as a green tinted solid. R = 0.27 (SiO , EtOAc/cyclohexane
f
2
1
1
+
+
1
C H BNaO S , 367.1149), 366.1185 (9, [M + H] ; calcd for
C H BNaO S , 366.1182), 359.0181 (100).
20:80); mp 128−129 °C. H NMR (400 MHz, CDCl )): δ = 1.22 (d, J =
6.9 Hz, 6 H; CHMe ), 3.52 (heptet, J = 6.9 Hz, 1 H; CHMe ), 5.37 (s, 2
1
8
21
4
3
10
21
+
18
4
2
2
3
-Bromo-5-(2-methylpropanoyl)benzonitrile (12). To a sol-
H; CH Ph), 7.38 (d, J = 4.0 Hz, 1 H; H−C(4)), 7.42 (br t, J = 1.5 Hz, 1
2
ution of 3,5-dibromobenzonitrile (10.95 g, 42.0 mmol) in dry THF (313
mL) at 0 °C was added dropwise isopropylmagnesium chloride in Et O
20.98 mL of a 2 M solution, 42.0 mmol). A brown solution was
H; H−C(4′)), 7.33−7.48 (m, 5 H; Ph), 7.59 (br t, J = 1.7 Hz, 1 H; H−
C(6′)), 7.84 (d, J = 3.9 Hz, 1 H; H−C(3)), 8.01 ppm (br t, J = 1.6 Hz, 1
2
1
3
(
H; H−C(2′)). C NMR (100 MHz, CDCl )): δ = 20.67 (CHMe ),
3
2
obtained. The mixture was stirred at 25 °C for 1 h. A solution of 24 (6.06
g, 46.2 mmol) in THF (21 mL) was added dropwise at 0 °C. The
mixture was stirred at 25 °C for 15 h, treated with saturated aqueous
36.24 (CHMe ), 67.38 (CH Ph), 89.01 (C(CN) ), 110.85 and 111.29
2 2 2
(C(CN) ), 114.81 (C(3′)), 117.04 (NC−C(3′)), 126.09 (C(4)),
2
128.45 (C(6′), C(2, 6) of Ph), 128.66 (C(4) of Ph), 128.82 (C(3,5) of
Ph), 129.57 (C(4′)), 131.11 (C(2′)), 134.79 (C(3)), 135.10 (C(2)),
135.54 (C(1) of Ph), 136.02 and 136.13 (C(1′, 5′)), 146.24 (C(5)),
NH Cl (75 mL) solution, and diluted with EtOAc (75 mL). The
4
aqueous layer was extracted with EtOAc (75 mL). The combined
organic layers were washed with H O (75 mL), saturated aqueous NaCl
75 mL), dried over Na SO , filtered, and evaporated to afford an orange
1
161.47 (CO ), 182.68 ppm (CC(CN) ). H NMR (300 MHz,
2
2
2
(
(D )THF): δ = 1.21 (d, J = 6.9 Hz, 6 H; CHMe ), 3.47 (heptet, J = 6.9
2
4
8
2
oil containing a white solid. The mixture was suspended in cyclohexane
and filtered. The filtrate was evaporated and the residue subjected to
Hz, 1 H; CHMe ), 5.33 (s, 2 H; CH Ph), 7.28−7.40 (m, 3 H of Ph),
2
2
7.41−7.48 (m, 2 H of Ph), 7.66 (d, J = 4.0 Hz, 1 H; H−C(4)), 7.72 (br t,
J = 1.5 Hz, 1 H; H−C(4′)), 7.84 (d, J = 4.0 Hz, 1 H; H−C(3)), 7.91 (br t,
J = 1.7 Hz, 1 H; H−C(6′)), 8.33 ppm (br t, J = 1.6 Hz, 1 H; H−C(2′)).
chromatography (SiO , gradient EtOAc/cyclohexane 0:100 to 02:98).
2
Product 12 was obtained as a faint yellow oil (6.10 g, 58%). R = 0.30
f
1
13
(
SiO ; cyclohexane/EtOAc 95:05). H NMR (400 MHz, CDCl ): δ =
C NMR (75 MHz, (D )THF): δ = 20.60 (CHMe ), 37.18 (CHMe ),
2
3
8
2
2
1
.22 (d, J = 6.9 Hz, 6 H; CHMe ), 3.45 (heptet, J = 6.8 Hz, 1 H;
67.78 (CH Ph), 89.70 (C(CN) ), 112.27 and 112.62 (C(CN) ), 115.58
2
2
2
2
CHMe ), 7.95 (br t, J = 1.66 Hz, 1 H; H−C(2), 8.13 (br t, J = 1.47 Hz, 1
(C(3′)), 118.08 (NC−C(3′)), 127.30 (C(4)), 129.21 (C(4) of Ph),
129.31 (C(2,6) of Ph), 129.47 (C(3,5) of Ph), 129.65 (C(6′)), 131.26
(C(4′)), 131.87 (C(2′)), 135.48 (C(3)), 135.62 (C(2)), 136.47
(C(1′)), 137.27 (C(1) of Ph), 137.57 (C(5′)), 147.92 (C(5)), 161.89
2
13
H; H−C(6)), 8.27 ppm (br t, J = 1.73 Hz, 1 H; H−C(4)). C NMR
(
100 MHz, CDCl ): δ = 18.92 (CHMe ), 35.95 (CHMe ), 114.88
3 2 2
(
C(1)), 116.72 (CN), 123.79 (C(3)), 130.54 (C(6)), 135.69 (C(4)),
138.20 (C(2)), 138.54 (C(5)), 201.02 ppm (CO). IR (ATR): ν =
3073 (w), 2973 (w), 2933 (w), 2873 (w), 2234 (w), 1691 (s), 1592 (w),
1563 (m), 1466 (m), 1420 (m), 1384 (m), 1351 (m), 1279 (m), 1235
(CO), 183.04 (CC(CN) ) ppm. IR (ATR): ν = 3116 (w), 3071
2
(w), 2978 (w), 2939 (w), 2232 (w), 1707 (s), 1698 (s), 1592 (m), 1568
(w), 1539 (m), 1497 (w), 1463 (m), 1419 (m), 1388 (w), 1370 (m),
1348 (m), 1306 (m), 1270 (s), 1254 (s), 1238 (s), 1215 (s), 1158 (w),
1100 (s), 1031 (w), 1014 (w), 965 (m), 905 (m), 886 (m), 858 (m), 824
(
(
(
2
s), 1187 (m), 1141 (m), 1085 (m), 1031 (m), 1018 (s), 965 (w), 920
w), 882 (s), 851 (m), 832 (m), 770 (m), 746 (m), 669 (s), 608 cm
w). HR-EI-MS m/z (%): 250.9939 (18, [M ]; calcd for C H BrNO ,
50.9941).
−1
+
+
−1
11
10
(m), 742 cm (s). HR-ESI-MS m/z (%): 389.1317 (100), 461.1122
+
+
(4), 460.1092 (12, [M + H] ; calcd for C H N NaO S , 460.1090).
26 19 3 2
Benzyl 5-[3-Cyano-5-(2-methylpropanoyl)phenyl]-
(± )-Benzyl 4-[3-(6-Amino-5-cyano-4-isopropyl-3-methyl-1,4-
dihydropyrano[2,3-c]pyrazol-4-yl)-5-cyanophenyl]piperazine-
1-carboxylate ((± )-22). A mixture of 34 (250 mg, 0.57 mmol), 3-
methyl-1H-pyrazol-5(4H)-one (15) (56 mg, 0.57 mmol), and
piperidine (0.56 mL, 5.7 mmol) in EtOH (2 mL) was stirred for 4 h
at 70 °C in a sealed tube in a microwave oven and evaporated. Column
chromatography (SiO ; Et O → Et O/THF 1:1) gave (±)-22 (120 mg,
thiophene-2-carboxylate (13). A solution of 12 (4.20 g, 16.7
mmol), 11 (5.76 g, 16.7 mmol), and Na CO (2.12 g, 20 mmol) in
THF/H O 80:20 (250 mL) was degassed by sonication while passing
2
3
2
argon through the solution for 5 min, treated with [Pd(PPh ) Cl ] (290
3
2
2
mg, 0.42 mmol), and degassed by sonication under argon for 10 min.
The mixture was heated to 60 °C for 4 h, diluted with EtOAc, and
washed with saturated aqueous NaCl solution. The organic phase was
evaporated, and the residue was adsorbed on 20 g of SiO . Column
chromatography on a Teledyne CombiFlash MPLC system (RediSep
2
2
2
39%) as an off-white solid. R = 0.39 (SiO ; Et O/THF 2:1): mp 118−
f
2
2
1
119 °C. H NMR (400 MHz, CD OD): δ = 0.86 and 0.98 (2 d, J = 6.6
2
3
Hz, 6 H; CHMe ), 1.83 (s, 3 H; Me−C(3′)), 2.80 (heptet, J = 6.5 Hz, 1
2
120 g SiO , dry loading, EtOAc/cyclohexane 5:95) yielded pure 13
H; CHMe ), 3.19 (br t, J = 5.2 Hz, 4 H; (CH ) N−Ar), 3.64 (br s, 4 H;
2
2
2 2
(
5.09 g, 78%) as a yellow oil which solidified upon high vacuum drying.
(CH ) N−CO), 5.14 (s, 2 H; CH Ph), 7.18 (br d, J ≈ 1.9, 2 H; H−
2
2
2
1
R = 0.36 (SiO ; EtOAc/cyclohexane 20:80); mp 100−101 °C. H NMR
C(2,4)), 7.26 (br t, J = 2.1, 1 H; H−C(2)), 7.28−7.41 ppm (m, 5 H; Ph).
f
2
13
(
400 MHz, CDCl ): δ = 1.26 (d, J = 6.8 Hz, 6 H; CHMe ), 3.52 (heptet,
C NMR (100 MHz, CD OD): δ = 11.91 (Me−C(3′)), 18.80 and
3
2
3
J = 6.8 Hz, 1 H; CHMe ), 5.37 (s, 2 H; CH Ph), 7.41 (d, J = 3.7 Hz, 1 H;
19.26 (CHMe ), 36.52 (CHMe ), 44.69 (br, (CH ) N−CO), 48.96
2
2
2
2
2 2
H−C(4)), 7.33−7.48 (m, 5 H; Ph), 7.84 (d, J = 3.9 Hz, 1 H; H−C(3)),
(C(4′)), 49.78 ((CH ) N−Ar), 62.05 (C(5′)), 68.46 (CH Ph), 100.84
2
2
2
8
.04 (br t, J = 1.62 Hz, 1 H; H−C(2′)), 8.14 (br t, J = 1.45 Hz, 1 H; H−
(C(3′a)), 113.73 (C(5)), 118.51 (C(6)), 120.19 (NC−C(5)), 121.76
(NC−C(5′)), 122.88 (C(2)), 124.41 (C(4)), 128.96 (C(2,6) of Ph),
129.17 (C(4) of Ph), 129.56 (C(3,5) of Ph), 137.49 (C(3′)), 138.01
13
C(4′)), 8.36 ppm (br t, J = 1.66 Hz, 1 H; H−C(6′)). C NMR (100
MHz, CDCl )): δ = 19.02 (CHMe ), 35.99 (CHMe ), 67.28 (CH Ph),
3
2
2
2
1
1
1
14.33 (C(3′)), 117.61 (CN), 125.68 (C(4)), 128.40 (C(2″,6″)),
28.62 (C(4″)), 128.82 (C(3″,5″)), 129.79 (C(6′)), 131.39 (C(4′)),
32.85 (C(2′)), 134.56 (C(2)), 134.78 (C(3)), 135.64 and 135.68
(C(1) of Ph), 148.01 (C(3)), 153.04 (C(1)), 156.85 (CO N), 157.45
2
̃
(C(7′a)), 163.95 ppm (C(6′)). IR (ATR): v = 3339 (w), 3161 (w),
2958 (w), 2228 (w), 2191 (m), 1703 (m), 1656 (m), 1584 (s), 1490
(m), 1430 (s), 1399 (m), 1361 (m), 1288 (m), 1239 (s), 1223 (s), 1158
(m), 1123 (m), 1078 (m), 1039 (m), 997 (s), 902 (m), 860 (m), 758
(
(
(
(
C(1″, 1′)), 137.99 (C(5′)), 147.17 (C(5)), 161.61 (CO ), 201.84 ppm
2
CO). IR (ATR): ν = 3073 (w), 2977 (w), 2933 (w), 2871 (w), 2232
w), 1687 (s), 1588 (w), 1532 (w), 1499 (w), 1458 (m), 1441 (m), 1423
m), 1379 (m), 1357 (w), 1282 (s), 1222 (m), 1195 (m), 1149 (m),
−
1
(m), 729 (s), 695 (m), 673 cm (m). HR-MALDI-MS: m/z (%):
+
+
560.2379 (100, [M + Na] ; calcd for C H N NaO , 560.2381).
30
31
7
3
1
095 (s), 1073 (s), 1040 (s), 996 (m), 914 (m), 901 (m), 884 (m), 817
(± )-6-Amino-4-[3-cyano-5-(piperazin-1-yl)phenyl]-4-iso-
propyl-3-methyl-1,4-dihydropyrano[2,3-c]pyrazole-5-carboni-
trile ((± )-23). A suspension of (±)-22 (80 mg, 0.15 mmol) and Pd/C
−1
(
(
m), 746 cm (s). HR-ESI-MS m/z (%): 407.1426 (100), 391.1196
18), 390.1160 (62, [M + H] ; calcd for C H NO S , 309.1158).
+
+
23
20
3
Benzyl 5-[3-Cyano-5-(1,1-dicyano-3-methylbut-1-en-2-yl)-
phenyl]thiophene-2-carboxylate (14). A solution of 13 (2.76 g,
.09 mmol) and malononitrile (2.34 g, 35.5 mmol) in unstabilized dry
(10% Pd, 10 mg) in MeOH (2 mL) was purged with N for 30 min,
2
stirred at 25 °C under an H atmosphere for 16 h, and filtered through a
2
7
pad of Celite. Evaporation yielded (±)-23 (57 mg, 94%) as an orange
K
DOI: 10.1021/jm501987h
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
1
t
oil: mp > 191 °C (dec). H NMR (400 MHz, CD OD): δ = 0.86 and
mmol), NaO Bu (317 mg, 3.3 mmol), [Pd (dba) ] (92 mg, 0.1 mmol),
2 3
3
0
.99 (2 d, J = 6.6 Hz, 6 H; CHMe ), 1.84 (s, 3 H; Me−C(3)), 2.82
and 2-biphenyl-di-tert-butylphosphine (90 mg, 0.3 mmol) in toluene (5
mL) was stirred at 45 °C for 48 h in a sealed tube. The mixture was
filtered through a pad of Celite and evaporated. Column chromatog-
2
(
3
heptet, J = 6.5 Hz, 1 H, CHMe ), 3.31−3.36 (m, 4 H; (CH ) N−Ar)),
2
2 2
.40−3.45 (m, 4 H; (CH ) N−CO)), 7.27 (br dd, J = 2.4, 1.3 Hz, 1 H;
2
2
H−C(4′)), 7.28 (br t, J = 1.4 Hz, 1 H; H−C(2′)), 7.32 ppm (br dd, J =
2
raphy: (SiO ; n-hexane/EtOAc 4:1 → 3:1) gave 33 (768 mg, 65%) as an
2
13
.4, 1.7 Hz, 1 H; H−C(6′)). C NMR (100 MHz, CD OD): δ = 11.88
off-white powder. R = 0.34 (SiO ; n-hexane/EtOAc 4:1); mp 127−128
3
f
2
1
(
Me−C(3)), 18.78 and 19.23 (CHMe ), 36.54 (CHMe ), 44.73
°C. H NMR (400 MHz, CDCl ): δ = 1.21 (d, J = 6.8 Hz, 6 H; CHMe ),
3 2
2
2
(
(CH ) NH), 47.55 ((CH ) N−Ar), 49.03 (C(4)), 61.88 (C(5)),
3.26 (br t, J ≈ 5.0 Hz, 4 H; (CH ) N−Ar), 3.46 (heptet, J = 6.9 Hz, 1 H;
2
2
2
2
2 2
1
1
1
00.76 (C(3a)), 113.94 (C(3′)), 118.93 (C(4′)), 119.96 (NC−C(3′)),
21.81 (NC−C(5)), 123.28 (C(6′)), 125.35 (C(2′)), 137.47 (C(3)),
48.34 (C(1′)), 152.27 (C(5′)), 157.41 (C(7a)), 164.02 (C(6)). IR
CHMe ), 3.68 (br t, J = 5.2 Hz, 4 H; (CH ) N−CO), 5.17 (s, 2 H;
2
2 2
CH Ph), 7.26 (br dd, J = 2.6, 1.3 Hz, 1 H, H−C(2)), 7.30−7.41 (m, 5 H;
2
Ph), 7.62 (br t, J = 1.3 Hz, 1 H; H−C(4)), 7.66 ppm (br dd, J = 2.7, 1.4
1
3
(
ATR): v
̃
= 3100 (w), 2184 (w), 1626 (m), 1576 (s), 1380 (m), 1242
Hz, 1 H; H−C(6)). C NMR (100 MHz, CDCl ): δ = 19.12 (CHMe ),
3
2
−1
(
s), 1148 (s), 996 cm (m). HR-MALDI-MS m/z (%): 404.2193 (100,
35.87 (CHMe ), 43.47 (br, (CH ) N−CO), 48.30 ((CH ) N−Ar),
67.60 (CH Ph), 113.81 (C(3)), 118.59 (CN), 119.41 (C(6)), 122.29
2
2
2
2 2
+
+
[
M + H] ; calcd for C H N O , 404.2193).
22
26
7
2
Methyl 5-(3-Cyano-5-isobutyrylphenyl)thiophene-2-carbox-
(C(2)), 122.57 (C(4)), 128.17 (C(2,6) of Ph), 128.35 (C(4) of Ph),
128.71 (C(3,5) of Ph), 136.53 (C(1) of Ph), 138.09 (C(5)), 151.65
ylate (31). A mixture of Pd(OAc) (47 mg, 0.21 mmol) and S-Phos
2
(
172 mg, 0.42 mmol) in degassed THF (7 mL) was stirred under N at
2
(C(1)), 155.24 (CO N), 202.84 ppm (CO). IR (ATR): v
̃
= 2966 (w),
2
2
5 °C for 30 min. 3-Bromo-5-isobutyrylbenzonitrile 12 (1.765 g, 7.0
mmol) and 30 (1.475 g, 7.8 mmol) were added, and the mixture was
combined with a solution of K CO (2.900 g, 21 mmol) in degassed
2
1
934 (w), 2229 (w), 1682 (s), 1592 (m), 1428 (s), 1361 (m), 1288 (m),
241 (m), 1214 (s), 1111 (m), 1079 (m), 1032 (m), 1013 (m), 996 (m),
2
3
977 (m), 911 (w), 869 (w), 752 (s), 721 (m), 697 (m), 680 (m), 632
water (3 mL) and stirred at 45 °C for 16 h. The mixture was diluted with
water (100 mL) and EtOAc (100 mL), and the layers were separated.
The organic phase was dried over Na SO and evaporated. The crude
product was triturated with i-Pr O to yield 31 (1.860 g, 85%) as a gray
−1
+
cm (m). HR-MALDI-MS m/z (%): 392.1969 (100, [M + H] ; calcd
+
for C H N O , 392.1974). Elemental analysis (%) calcd for
2
3
26
3
3
2
4
C H N O (391.47): C 70.57, H 6.44, N 10.73. Found: C 70.50, H
23 25 3 3
2
6.45, N 10.60.
1
powder. Mp 139−140 °C. H NMR (300 MHz, CDCl ): δ = 1.26 (d, J =
3
Benzyl 4-[3-Cyano-5-(1,1-dicyano-3-methylbut-1-en-2-yl)-
6.9 Hz, 6 H; CHMe ), 3.52 (septet, J = 6.9 Hz, 1 H; CHMe ), 3.93 (s, 3
2
2
phenyl]piperazine-1-carboxylate (34). Under N , a solution of 33
2
H; OMe), 7.40 (d, J = 3.9 Hz, 1 H; H−C(4)), 7.80 (d, J = 3.9 Hz, 1 H;
(
666 mg, 1.7 mmol) and malononitrile (449 mg, 6.8 mmol) in CHCl (7
3
H−C(3)), 8.04 (t, J = 1.6 Hz, 1 H; H−C(2′)), 8.14 (t, J = 1.5 Hz, 1 H;
mL) was treated with a 1 M solution of TiCl in toluene (1.7 mL, 1.7
4
13
H−C(4′)), 8.36 ppm (t, J = 1.7 Hz, 1 H; H−C(6′)). C NMR (100
mmol). Pyridine (0.27 mL, 3.4 mmol) was added, and the mixture was
stirred at 75 °C for 48 h. 1 M aqueous HCl (100 mL) and EtOAc (100
mL) were added. The solid precipitate was filtered off, and the layers
were separated. The aqueous phase was extracted with EtOAc (3 × 50
mL). The combined organic phases were dried over Na SO and
MHz, CDCl ): δ = 19.00 (CHMe ), 35.97 (CHMe ), 52.59 (OMe),
3
2
2
1
14.29 (C(3′)), 117.61 (CN), 125.66 (C(4)), 129.78 (C(6′)), 131.36
(
(
C(4′)), 132.83 (C(2′)), 134.50 (C(2)), 134.57 (C(3)), 135.67
C(1′)), 137.96 (C(5′)), 146.97 (C(5)), 162.22 (CO ), 201.82 (C
2
2
4
O) ppm. IR (ATR): v
̃
= 3098 (w), 2972 (w), 2237 (w), 1707 (s), 1684
evaporated. Column chromatography (SiO ; n-hexane/EtOAc 4:1)
2
(
m), 1594 (w), 1538 (w), 1469 (w), 1425 (m), 1381 (w), 1351 (w),
gave 34 (390 mg, 52%) as an orange resin. R = 0.19 (SiO ; n-hexane/
f
2
1
316 (w), 1291 (m), 1270 (m), 1254 (s), 1227 (m), 1199 (m), 1186
1
EtOAc 4:1). H NMR (400 MHz, CDCl ): δ = 1.18 (d, J = 6.9 Hz, 6 H;
3
(
(
(
m), 1150 (w), 1095 (s), 1033 (w), 1008 (m), 959 (w), 910 (w), 886
w), 832 (w), 790 (w), 767 (w), 745 (s), 675 (w), 649 (w), 622 cm
m); HR-MALDI-MS: m/z (%): 314.0845 (100, [M + H] ; calcd for
CHMe ), 3.27 (br t, J = 5.1 Hz, 4 H; (CH ) N−Ar), 3.43 (heptet, J = 6.9
2
2 2
−1
Hz, 1 H; CHMe ), 3.68 (dd, J = 4.1, 6.4 Hz, 4 H; (CH ) N−CO), 5.16
2
2 2
+
(
s, 2 H; CH Ph), 6.85 (dd, J = 1.5, 2.5 Hz; H−C(6)), 6.89 (t, J = 1.3 Hz,
2
+
C H NO S , 314.0845).
17
16
3
1 H; H−C(4)), 7.22 (dd, J = 1.3, 2.5 Hz, 1 H; H−C(2)), 7.25−7.60 ppm
Methyl 5-[3-Cyano-5-(1,1-dicyano-3-methylbut-1-en-2-yl)-
13
(
(
(
(
(
m, 5 H; Ph). C NMR (100 MHz, CDCl ): δ = 20.36 (CHMe ), 35.98
3
2
phenyl]thiophene-2-carboxylate (32). Under N , a solution of 31
2
CHMe ), 43.01 (br, (CH ) N−CO), 47.68 ((CH ) N−Ar), 67.24
2
2
2
2 2
(
625 mg, 2.0 mmol) and malononitrile (528 mg, 8 mmol) in CHCl (5
3
CH Ph), 87.79 (C(CN) ), 111.06 and 111.48 (C(CN) ), 113.95
2
2
2
mL) was treated with a 1 M solution of TiCl in toluene (2.2 mL, 2.2
mmol). Pyridine (0.49 mL, 6 mmol) was added, and the mixture was
stirred at 70 °C for 18 h in a sealed tube. 1 M aqueous HCl (100 mL) and
CHCl (100 mL) were added. The solid precipitate was filtered off, and
4
C(3)), 117.82 and 117.91 (NC−C(3), C(6)), 119.76 and 119.82
C(2,4)), 127.81 (C(2,6) of Ph), 128.03 (C(4) of Ph), 128.42 (C(3,5)
of Ph), 135.84 (C(5)), 136.28 (C(1) of Ph), 150.96 (C(1)), 154.88
3
(
(
(
(
CO N), 184.15 ppm (CC(CN) ). IR (ATR): v
̃
= 2974 (w), 2232
2
2
the layers were separated. The aqueous phase was extracted with CHCl3
m), 1694 (s), 1586 (s), 1497 (w), 1427 (s), 1387 (m), 1360 (m), 1288
m), 1237 (s), 1223 (s), 1162 (m), 1121 (s), 1078 (m), 1018 (m), 993
(3 × 50 mL). The combined organic phases were dried over Na SO and
2 4
evaporated. The residue was triturated with i-Pr O to yield 32 (656 mg,
−1
2
s), 912 (w), 866 (m), 844 (m), 749 (s), 695 (s), 666 cm+ (m). HR-
1
9
1
3
1%) as a red solid: mp 189−190 °C. H NMR (400 MHz, CDCl ): δ =
3
MALDI-MS m/z (%): 462.1901 (100, [M + Na] ; calcd for
C H N NaO , 462.1900).
.22 (d, J = 6.9 Hz, 6 H; CHMe ), 3.52 (septet, J = 6.9 Hz, 1 H; CHMe ),
+
2
2
2
26
25
5
.93 (s, 3 H; OMe), 7.38 (d, J = 3.9 Hz, 1 H; H−C(4)), 7.43 (t, J = 1.5
Hz, 1 H; H−C(4′)), 7.60 (t, J = 1.7 Hz, 1 H; H−C(6′)), 7.80 (d, J = 3.9
Hz, 1 H; H−C(3)), 8.01 ppm (t, J = 1.6 Hz, 1 H; H−C(2′)). C NMR
13
ASSOCIATED CONTENT
Supporting Information
■
(
(
(
(
100 MHz, CDCl ): δ = 20.64 (CHMe ), 36.23 (CHMe ), 52.66
OMe), 88.95 (C(CN) ), 110.87 and 111.31 (C(CN) ), 114.75
C(3′)), 117.06 (NC−C(3′)), 126.08 (C(4)), 128.46 (C(6′)), 129.57
C(4′)), 131.11 (C(2′)), 134.58 (C(3)), 135.03 (C(2)), 135.99 and
3 2 2
*
S
2
2
Additional figures on ligand interactions, detailed biological
activity, synthetic procedures, compound characterization, and
NMR spectra. This material is available free of charge via the
Internet at http://pubs.acs.org.
1
36.11 (C(1′,5′)), 146.08 (C(5)), 162.09 (CO ), 182.71 (C
2
C(CN) ) ppm. IR (ATR): v
̃
= 3099 (w), 2980 (w), 2234 (w), 1708
2
(
(
1
(
s), 1699 (s), 1591 (w), 1570 (w), 1537 (w), 1467 (m), 1428 (m), 1418
m), 1366 (w), 1345 (w), 1313 (m), 1277 (s), 1245 (m), 1225 (m),
185 (w), 1162 (w), 1096 (s), 1005 (w), 992 (w), 955 (w), 918 (w), 887
s), 828 (m), 790 (m), 744 (s), 724 (m), 689 (m), 653 (w), 626 (m),
AUTHOR INFORMATION
■
Corresponding Authors
−1
6
13 cm (w). HR-MALDI-MS (negative mode) m/z (%): 360.0812
−
−
*M.C.W.: e-mail, matthias.witschel@basf.com; phone, (+49)
(
100, [M − H] ; calcd for C H N O S , 360.0812).
20
14
3
2
6
21 60-41966; fax, (+49) 621 60 664 19 66.
Benzyl 4-(3-Cyano-5-isobutyrylphenyl)piperazine-1-carbox-
ylate (33). Under N , a mixture of 3-bromo-5-isobutyrylbenzonitrile 12
*F.D.: e-mail, diederich@org.chem.ethz.ch; phone, (+41) 44
632 29 92; fax, (+41) 44 632 11 09.
2
(
756 mg, 3 mmol), benzyl piperazine-1-carboxylate (0.64 mL, 3.3
L
DOI: 10.1021/jm501987h
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Author Contributions
(6) Noedl, H.; Se, Y.; Schaecher, K.; Smith, B. L.; Socheat, D.; Fukuda,
M. M. Evidence of artemisinin resistant malaria in Western Cambodia.
N. Engl. J. Med. 2008, 359, 2619−2620.
M.C.W., M.R., F.S., P.Chi., P.Cha., and F.D. designed the
research; A.S., U.L., P.Chi., M.S., S.T., G.S., C.M., F.T., C.F., A.J.,
C.P., P.R., M.O., L.M.S.-A., S.C., S.W., and Y.Y. performed
research; M.C.W., M.R., U.L., P.Chi., T.M., A.J., M.H., P.M.,
P.Cha., and F.D. analyzed data; M.C.W., M.R., A.S., G.S., P.Cha.,
and F.D. wrote the paper.
(7) Cheeseman, I. H.; Miller, B. A.; Nair, S.; Nkhoma, S.; Tan, A.; Tan,
J. C.; Saai, S. A.; Phyo, A. P.; Moo, C. L.; Lwin, K. M.; McGready, R.;
Ashley, E.; Imwong, M.; Stepniewska, K.; Yi, P.; Dondorp, A. M.;
Mayxay, M.; Newton, P. N.; White, N. J.; Nosten, F.; Ferdig, M. T.;
Anderson, T. J. C. A major genome region underlying artemisinin
resistance in malaria. Science 2012, 336, 79−82.
Notes
(8) Phyo, A. P.; Nkhoma, S.; Stepniewska, K.; Ashley, E. A.; Nair, S.;
The authors declare no competing financial interest.
McGready, R.; Moo, C.; Al-Saai, S.; Dondorp, A. M.; Lwin, K. M.;
Singhasivanon, P.; Day, N. P. J.; White, N. J.; Anderson, T. J. C.; Nosten,
F. Emergence of artemisinin-resistant malaria on the western border of
Thailand: a longitudinal study. Lancet 2012, 379, 1960−1966.
ACKNOWLEDGMENTS
■
F. Ro
̈
hl (BASF SE) conducted the high-throughput screening
(
9) Barnett, D. S.; Guy, R. K. Antimalarials in development in 2014.
Chem. Rev. 2014, 114, 11221−11241.
10) Teixeira, C.; Vale, N.; Perez, B.; Gomes, A.; Gomes, J. R. B.;
against AtSHMT. Work at ETH Zurich was supported by a grant
from the ETH Research Council. At ETH Zurich, we thank Dr.
Bruno Bernet for help with the compound characterizations and
Dr. Nils Trapp for small molecule crystallography. We thank the
MMV Foundation, especially Jeremy Burrows and Roger
Bonnert, for their support, for giving us access to their assay
network and their great expertise. P.Cha. received support from
the Thailand Research Fund (Grant RTA5680001) and Mahidol
University. The crystal structure study was financially supported
by grants from Synchrotron Light Research InstitutePublic
Organization (Grant 2-2549/LS01 to U.L. and Grant 2551/08 to
P.Chi.) and Cluster Program and Management Office, National
Science and Technology Development Agency, Thailand
(
́
Gomes, P. “Recycling” classical drugs for malaria. Chem. Rev. 2014, 114,
11164−11220.
(11) Wiesner, J.; Ortmann, R.; Jomaa, H.; Schlitzer, M. New
antimalarial drugs. Angew. Chem., Int. Ed. 2003, 42, 5274−5293.
(12) Crowther, G. J.; Napuli, A. J.; Gilligan, J. H.; Gagaring, K.; Borboa,
R.; Francek, C.; Chen, Z.; Dagostino, E. F.; Stockmyer, J. B.; Wang, Y.;
Rodenbough, P. P.; Castaneda, L. J.; Leibly, D. J.; Bhandari, J.; Gelb, M.
H.; Brinker, A.; Engels, I. H.; Taylor, J.; Chatterjee, A. K.; Fantauzzi, P.;
Glynne, R. J.; Van Voorhis, W. C.; Kuhen, K. L. Identification of
inhibitors for putative malaria drug targets among novel antimalarial
compounds. Mol. Biochem. Parasitol. 2011, 175, 21−29.
(13) Witschel, M.; Rottmann, M.; Kaiser, M.; Brun, R. Agrochemicals
(
Grants CPMO-P-00-20029, CPMO-DD/P-10-11274, and
CPMO-P-13-00835). We thank Merck Epova AG (Schaffhau-
sen, Switzerland) and Professor Martino di Salvo (Universita di
against malaria, sleeping sickness, Leishmaniasis and Chagas disease.
PLoS Neglected Trop. Dis. 2012, 6, e1805.
(14) Mombelli, P.; Witschel, M. C.; van Zijl, A. W.; Geist, J. G.;
̀
Roma) for providing high quality THF and plasmid for
expressing MTHFD, respectively. We gratefully acknowledge
the National Synchrotron Radiation Research Center (Taiwan)
for use of beamline 13B1.
̈
Rottmann, M.; Freymond, C.; Rohl, F.; Kaiser, M.; Illarionova, V.;
Fischer, M.; Siepe, I.; Schweizer, W. B.; Brun, R.; Diederich, F.
Identification of 1,3-diiminoisoindoline carbohydrazides as potential
antimalarial candidates. ChemMedChem 2012, 7, 151−158.
(15) Kunfermann, A.; Witschel, M.; Illarionov, B.; Martin, R.;
Rottmann, M.; Ho
̈
ffken, H. W.; Seet, M.; Eisenreich, W.; Knolker, H.
̈
ABBREVIATIONS USED
J.; Fischer, M.; Bacher, A.; Groll, M.; Diederich, F. Pseudilins:
halogenated, allosteric inhibitors of the non-mevalonate pathway
enzyme IspD. Angew. Chem., Int. Ed. 2014, 53, 2235−2239.
■
CDI, carbonyldiimidazole; DHF, dihydrofolate; DHFR, dihy-
drofolate reductase; DHFS, dihydrofolate synthase; DHP,
dihydropteroate; DHPS, dihydropteroate synthase; dTMP,
deoxythymidine monophosphate; dUMP, deoxyuridine mono-
phosphate; pABA, para-aminobenzoic acid; Pb, Plasmodium
berghei; Pf , Plasmodium falciparum; PLP, pyridoxal 5′-phosphate;
Pv, Plasmodium vivax; SHMT, serine hydroxymethyltransferase;
THF, tetrahydrofolate; TS, thymidylate synthase; 5,10-CH2-
THF, 5,10-methylenetetrahydrofolate
(16) Reker, D.; Seet, M.; Pillong, M.; Koch, C. P.; Schneider, P.;
Witschel, M. C.; Rottmann, M.; Freymond, C.; Brun, R.; Schweizer, B.;
Illarionov, B.; Bacher, A.; Fischer, M.; Diederich, F.; Schneider, G.
Deorphaning pyrrolopyrazines as potent multi-target antimalarial
agents. Angew. Chem., Int. Ed. 2014, 53, 7079−7084.
(17) Nirmalan, N.; Wang, P.; Sims, P. F.; Hyde, J. E. Transcriptional
analysis of genes encoding enzymes of the folate pathway in the human
malaria parasite Plasmodium falciparum. Mol. Microbiol. 2002, 46, 179−
1
(
90.
18) Pang, C. K.; Hunter, J. H.; Gujjar, R.; Podutoori, R.; Bowman, J.;
REFERENCES
■
Mudeppa, D. G.; Rathod, P. K. Catalytic and ligand-binding character-
istics of Plasmodium falciparum serine hydroxymethyltransferase. Mol.
Biochem. Parasitol. 2009, 168, 74−83.
(19) Sopitthummakhun, K.; Thongpanchang, C.; Vilaivan, T.;
Yuthavong, Y.; Chaiyen, P.; Leartsakulpanich, U. Plasmodium serine
hydroxymethyltransferase as a potential anti-malarial target: inhibition
studies using improved methods for enzyme production and assay.
Malar. J. 2012, 11, 194.
(20) Pornthanakasem, W.; Kongkasuriyachai, D.; Uthaipibull, C.;
Yuthavong, Y.; Leartsakulpanich, U. Plasmodium serine hydroxymethyl-
transferase: indispensability and display of distinct localization. Malar. J.
2012, 11, 387.
(
1) World Health Organization. World Malaria Report 2013; World
Health Organization: Geneva, 2013; p 61.
(
2) World Health Organization. Global Plan for Artemisinin Resistance
Containment; World Health Organization: Geneva, 2011.
3) World Health Organization. Emergency Response to Artemisinin
Resistance in the Greater Mekong Subregion. Regional Framework for Action
013−2015; World Health Organization: Geneva, 2013.
4) Maude, R. J.; Pontavornpinyo, W.; Saralamba, S.; Aguas, R.; Yeung,
S.; Dondorp, A. M.; Day, N. P.; White, N. J.; White, L. J. The last man
standing is the most resistant: eliminating artemisinin-resistant malaria
in Cambodia. Malar. J. 2009, 8, 31.
(
2
(
(
5) Dondorp, A. M.; Nosten, F.; Yi, P.; Das, D.; Phyo, A. P.; Tarning, J.;
(21) Maenpuen, S.; Sopitthummakhun, K.; Yuthavong, Y.; Chaiyen, P.;
Leartsakulpanich, U. Characterization of Plasmodium falciparum serine
hydroxymethyltransferaseA potential antimalarial target. Mol. Bio-
chem. Parasitol. 2009, 168, 63−73.
(22) Sopitthummakhun, K.; Maenpuen, S.; Yuthavong, Y.;
Leartsakulpanich, U.; Chaiyen, P. Serine hydroxymethyltransferase
Lwin, K. M.; Ariey, F.; Hanpithakpong, W.; Lee, S. J.; Ringwald, P.;
Silamut, K.; Imwong, M.; Chotivanich, K.; Lim, P.; Herdman, T.; An, S.
S.; Yeung, S.; Singhasivanon, P.; Day, N. P. J.; Lindegardh, N.; Socheat,
D.; White, N. J. Artemisinin resistance in Plasmodium falciparum malaria.
N. Engl. J. Med. 2009, 361, 455−467.
M
DOI: 10.1021/jm501987h
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
from Plasmodium vivax is different in substrate specificity from its
homologues. FEBS J. 2009, 276, 4023−4036.
(
23) Witschel, M.; Stelzer, F.; Hutzler, J.; Qu, T.; Mietzner, T.; Kreuz,
̈
K.; Grossmann, K.; Aponte, R.; Hoffken, H. W.; Carlo, F.; Ehrhardt, T.;
Simon, A.; Parra, R. L. Pyrazolopyrans having herbicidal and
pharmaceutical properties. PCT Int. Appl. WO 2013182472 A1, 2013.
(
24) Drewry, D. H.; Hunter, R. N., III; Jung, D. K.; Linn, J. A.; Sehon,
C.; Stavenger, R. A. Benzamide rho-kinase (ROCK-1) inhibitors; N-(2-
phenylethyl)-5-(4-pyridinyl)-2-thiophenecarboxamide. US
2008293716, 2008.
(
25) Moebius, D. C.; Kingsbury, J. S. Catalytic homologation of
cycloalkanones with substituted diazomethanes. Mild and efficient
single-step access to α-tertiary and α-quaternary carbonyl compounds. J.
Am. Chem. Soc. 2009, 131, 878−879.
(
26) Javed, M. I.; Brewer, M. Diazo preparation via dehydrogenation of
hydrazones with “activated” DMSO. Org. Lett. 2007, 9, 1789−1792.
27) Junek, H.; Aigner, H. Synthesen mit nitrilen, XXXV. Reaktionen
von Tetracyanathylen mit Heterocyclen. Chem. Ber. 1973, 106, 914−
21.
28) Staab, H. A.; Luking, M.; Durr, F. H. Darstellung von
Imidazoliden. Synthese von Amiden, Hydraziden und Hydroxamsauren
nach der Imidazolidmethode. Chem. Ber. 1962, 95, 1275−1283.
29) Chitnumsub, P.; Ittarat, W.; Jaruwat, A.; Noytanom, K.;
(
̈
9
(
̈
̈
̈
(
Amornwatcharapong, W.; Pornthanakasem, W.; Chaiyen, P.;
Yuthavong, Y.; Leartsakulpanich, U. The structure of Plasmodium
falciparum serine hydroxymethyltransferase reveals a novel redox switch
that regulates its activities. Acta Crystallogr., Sect. D: Biol. Crystallogr.
2
(
014, 70, 1517−1527.
30) Pinthong, C.; Maenpuen, S.; Amornwatcharapong, W.;
Yuthavong, Y.; Leartsakulpanich, U.; Chaiyen, P. Distinct biochemical
properties of human serine hydroxymethyltransferase compared with
the Plasmodium enzyme: implications for selective inhibition. FEBS J.
2
(
014, 281, 2570−2583.
31) Chitnumsub, P.; Jaruwat, A.; Riangrungroj, P.; Ittarat, W.;
Noytanom, K.; Oonanant, W.; Vanichthanankul, J.; Chuankhayan, P.;
Maenpuen, S.; Chen, C.-J.; Chaiyen, P.; Yuthavong, Y.;
Leartsakulpanich, U. Structures of Plasmodium vivax serine hydrox-
ymethyltransferase: implications for ligand-binding specificity and
functional control. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2014, 70,
3
(
177−3186.
32) Derbyshire, E. R.; Prudencio, M.; Mota, M. M.; Clardy, J. Liver-
̂
stage malaria parasites vulnerable to diverse chemical scaffolds. Proc.
Natl. Acad. Sci. U.S.A. 2012, 109, 8511−8516.
(
33) Dorn, A.; Stoffel, R.; Matile, H.; Bubendorf, A.; Ridley, R. G.
Malarial haemozoin/β-haematin supports haem polymerization in the
absence of protein. Nature 1995, 374, 269−271.
(
34) Trager, W.; Jensen, J. B. Human malaria parasites in continuous
culture. Science 1976, 193, 673−675.
(
35) Huber, W.; Koella, J. C. A comparison of three methods of
estimating EC50 in studies of drug resistance of malaria parasites. Acta
Trop. 1993, 55, 257−261.
(
36) Franke-Fayard, B.; Trueman, H.; Ramesar, J.; Mendoza, J.; van der
Keur, M.; van der Linden, R.; Sinden, R. E.; Waters, A. P.; Janse, C. J. A
Plasmodium berghei reference line that constitutively expresses GFP at a
high level throughout the complete life cycle. Mol. Biochem. Parasitol.
2
(
004, 137, 23−33.
37) Jimenez-Diaz, M. B.; Mulet, T.; Viera, S.; Gomez, V.; Garuti, H.;
Ibanez, J.; Alvarez-Doval, A.; Shultz, L. D.; Martinez, A.; Gargallo-Viola,
D.; Angulo-Barturen, I. Improved murine model of malaria using
Plasmodium falciparum competent strains and non-myelodepleted
null
NOD- scid IL2Rγ
mice engrafted with human erythrocytes.
Antimicrob. Agents Chemother. 2009, 53, 4533−4536.
(
38) Brunner, R.; Aissaoui, H.; Boss, C.; Bozdech, Z.; Brun, R.;
Corminboeuf, O.; Delahaye, S.; Fischli, C.; Heidmann, B.; Kaiser, M.;
Kamber, J.; Meyer, S.; Papastogiannidis, P.; Siegrist, R.; Voss, T.;
Welford, R.; Wittlin, S.; Binkert, C. Identification of a new chemical class
of antimalarials. J. Infect. Dis. 2012, 206, 735−743.
N
DOI: 10.1021/jm501987h
J. Med. Chem. XXXX, XXX, XXX−XXX