A Molecular Hybridization Approach for the Design of Potent, Highly Selective, and Brain-Penetrant N-Myristoyltransferase Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.8b00884

Crystallography has guided the hybridization of two series of Trypanosoma brucei N-myristoyltransferase (NMT) inhibitors, leading to a novel highly selective series. The effect of combining the selectivity enhancing elements from two pharmacophores is shown to be additive and has led to compounds that have greater than 1000-fold selectivity for TbNMT vs HsNMT. Further optimization of the hybrid series has identified compounds with significant trypanocidal activity capable of crossing the blood-brain barrier. By using CF-1 mdr1a deficient mice, we were able to demonstrate full cures in vivo in a mouse model of stage 2 African sleeping sickness. This and previous work provides very strong validation for NMT as a drug target for human African trypanosomiasis in both the peripheral and central nervous system stages of disease.

Full text of DOI:10.1021/acs.jmedchem.8b00884

Article
pubs.acs.org/jmc
Cite This: J. Med. Chem. XXXX, XXX, XXX−XXX
A Molecular Hybridization Approach for the Design of Potent,
Highly Selective, and Brain-Penetrant N‑Myristoyltransferase
Inhibitors
Justin R. Harrison, Stephen Brand, Victoria Smith, David A. Robinson, Stephen Thompson,
Alasdair Smith, Kenneth Davies, Ngai Mok, Leah S. Torrie, Iain Collie, Irene Hallyburton,
Suzanne Norval, Frederick R. C. Simeons, Laste Stojanovski, Julie A. Frearson, Ruth Brenk,
Paul G. Wyatt, Ian H. Gilbert,* and Kevin D. Read*
Drug Discovery Unit, Wellcome Centre for Anti-Infectives Research, Division of Biological Chemistry and Drug Discovery, School
of Life Sciences, University of Dundee, Dundee, DD1 5EH, United Kingdom
S
* Supporting Information
ABSTRACT: Crystallography has guided the hybridization of two series of Trypanosoma brucei N-myristoyltransferase (NMT)
inhibitors, leading to a novel highly selective series. The effect of combining the selectivity enhancing elements from two
pharmacophores is shown to be additive and has led to compounds that have greater than 1000-fold selectivity for TbNMT vs
HsNMT. Further optimization of the hybrid series has identified compounds with significant trypanocidal activity capable of
crossing the blood−brain barrier. By using CF-1 mdr1a deficient mice, we were able to demonstrate full cures in vivo in a mouse
model of stage 2 African sleeping sickness. This and previous work provides very strong validation for NMT as a drug target for
human African trypanosomiasis in both the peripheral and central nervous system stages of disease.
the successful chemical validation of TbNMT using the
prototype NMT inhibitor DDD85646 (2) derived from
pyrazole sulfonamide screening hit DDD64558 (1) (Scheme
1), which is highly effective at curing mouse models of
hemolymphatic (peripheral) infection.4,5 Unfortunately, we
were not able to obtain conclusive evidence for TbNMT
validation in stage 2 disease as the blood−brain barrier
penetrant compounds we developed did not have a sufficient
therapeutic window.6
Several research groups have also interrogated NMT as a
potential target for the treatment of leishmaniasis,7,8 malaria,9
fungal infections, and cancer.10−21 In most/all of these
inhibitors, there is a basic center in the compound that
maintains a key interaction with the C-terminal carboxylic acid,
found in the peptide binding site of NMT.
INTRODUCTION
■
Human African trypanosomiasis (HAT), or sleeping sickness,
is caused by two different subspecies of the protozoan parasite
Trypanosoma brucei: T.b. gambiense and T.b. rhodensiense. The
disease, which is fatal unless treated, is transmitted by the bite
of an infected tsetse fly. It has two stages: an initial peripheral
infection during which the parasites are found in the
bloodstream, giving rise to nonspecific symptoms, and a
second stage when the parasites enter the central nervous
system, causing the classic symptoms of HAT, eventually
leading to coma and death. Currently, there are five treatments
available, although none of them are satisfactory due to
toxicity, treatment failures, or the requirement for parenteral
administration, which is inappropriate in a rural African
setting.1
N-Myristoyltransferase (NMT) represents a potential drug
target for HAT because in T. brucei RNAi knockdown of NMT
has been shown to cause cidality in cell culture and to abrogate
infectivity in several animal models.2,3 We have also reported
Received: June 4, 2018
© XXXX American Chemical Society
A
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Scheme 1. Pyrazole Sulfonamide NMT Inhibitors (“S” = HsNMT IC50/T. brucei EC50): DDD64558 (1),5 DDD85646 (2),5 and
DDD100097 (3)6
Figure 1. Structure of DDD85646 (2) bound to LmNMT (PDB ID: 2WSA).4 (A) DDD85646 (C atoms gold) bound in the peptide binding cleft.
(B) H-bond interactions between the ligand and protein.
NMT catalyzes the cotranslational transfer of myristate from
myristoyl-CoA to the N-terminal glycine of ∼60 proteins; a
modification that is implicated in localization and/or activation
of the substrate−proteins.22 The enzyme operates via a Bi−Bi
mechanism in which it first binds myristoyl-CoA, causing a
conformational rearrangement that subsequently reveals the
peptide binding site.2 There is a significant deficit in our
understanding of the identity of downstream targets of NMT
in T. brucei; however, the ribosylation factors Arf and Arl have
been shown to be NMT substrates important for survival of
the parasite in cell culture.23
closest homologue of TbNMT for which a crystal structure has
been elucidated: the primary sequence id was 74% identical
overall and 94% identical within the ligand binding cavities.
Structural analysis was carried out on selected pyrazole
sulfonamide ligands and has been previously described in
detail.4,5 The ligands occupy the substrate binding groove of
LmNMT with the pyrazole occupying a pocket that binds
residues 4 and 5 of the peptide substrate. The pyrazole
nitrogen lone pair forms a key hydrogen bond with the side
chain hydroxyl group of Ser330 (Figure 1B). The pyrazole N-
methyl group interacts with the side chains of Val81 and the
aromatic ring of Phe90, and consequently, larger substituents
than methyl were not tolerated (Figure 1B). There was a
degree of flexibility in the position and orientation of the
sulfonamide group. For most ligands within the series, the
Attempts to crystallize TbNMT have been unsuccessful to
date. However, we were able to obtain a structure for
Leishmania major NMT (LmNMT), which we have used as
a surrogate model for TbNMT (Figure 1A). LmNMT is the
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Scheme 2. Methyl Ester Hit and Close Analogues
sulfonamide moiety hydrogen bonded through a highly
coordinated water molecule to the side chain of His219 and
the backbone amides of Asp396 and Gly397. In other ligands
(described elsewhere5), the water-bridged interaction with
His219 was replaced by a direct hydrogen bond between the
sulfonamide oxygen and the side chain of Asn376. The aryl
rings of DDD85646 (2) did not form directional interactions
with the protein, though they stacked above the side chain of
Tyr217, which lines the floor of the peptide binding site.
Hydrophobic packing was increased further by the two
chlorine atoms of compound 2, explaining their beneficial
effect on potency.
Significant gains in potency were obtained by appending a
basic functionality, which formed an ionic interaction with the
carboxylate of the C-terminal residue (Leu421) and a series of
water-mediated hydrogen bonds. In the case of 2, this was
obtained with a constrained amine, but it was subsequently
shown that similar gains in potency could be obtained with a
more flexible analogue such as DDD100097 (3) (Scheme 1).6
Lead optimization of the pyrazole sulfonamides subse-
quently led to the identification of potently trypanocidal brain-
penetrant inhibitors, which were evaluated for efficacy in a
stage 2 mouse model of HAT (e.g., DDD100097 (3)).6
Unfortunately, a fully curative dose regimen was not achieved
due to dose-limiting toxicity. This toxicity is likely to be
associated with the simultaneous blockade of host NMT
(Mouse NMT is 98% identical to Human NMT (HsNMT)),
although it is conceivable that some as of yet unidentified
action of the compound is causing the dose-limiting toxicity).
Nonselective NMT inhibitors like compound 2 can be well
tolerated at an efficacious dose to treat the first stage of HAT, a
consequence of the high dependency of T. brucei on NMT.
However, the higher doses often required to also effectively
treat the second CNS stage of the disease have been poorly
tolerated, as was the case for compound 3, a blood−brain
barrier penetrant compound. We therefore deliberately sought
compounds with maximal selectivity to reduce the possibility
of target-driven toxicity and provide a suitable therapeutic
window to achieve a fully curative dose regimen for stage 2
HAT. In the case of the pyrazole sulfonamide series, we
identified the subpocket around the Leu421 as an area in
which we could obtain selectivity. Leu421 is the C-terminal
residue of TbNMT that interacts with the amine. We
attempted to obtain an increase in selectivity by optimization
of the amine but were only able to obtain a modest increase.6
We were unable to rationalize how to build in selectivity, and
this was achieved empirically. At the time of carrying out this
work, there was no human NMT structure available.
Comparison of multiple parasitic and human NMT structures
showed the active sites to be highly conserved with minimal
evidence of conformational change; hence, there was no design
rational to be drawn to generate ligands with higher degrees of
selectivity.
AIMS AND OBJECTIVES
■
The overall aim of the research was to develop lead
compounds with a profile suitable for delivering a fully
curative, well-tolerated dose regimen in the stage 2 mouse
model of HAT. In this article, we discuss the identification of a
novel scaffold with selectivity for TbNMT over HsNMT.
Structural information and our existing knowledge of NMT
inhibitors was then used to increase potency in two new
subseries. Further, we were also able to combine the separate
selectivity-enhancing elements of our previously developed
pyrazole sulfonamide series with those of this new series to
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Figure 2. Crystal structure of 4 bound to LmNMT:MCoA showing key interactions. (A) Ester carbonyl forms H-bond to Ser330; sulfonamide
oxygen forms H-bond to Asp396, and the lower phenyl ring stacks with Tyr217. (B) Binding mode of 4 (C atoms gold) compared to pyrazole
sulfonamide 1 (C atoms purple) showing retention of the interaction with Ser330. (C) Conformational change in residues 231−236 (gray ribbon)
induced by azepane ring of 4 (C atoms gold) compared to LmNMT:MCoA (green ribbon, PDB ID: 3H5Z). The side chain of Phe232 from each
structure is shown in stick representation.
enhance the overall selectivity. We were also able to derive
compounds with blood−brain barrier permeability.
high throughput screening, was at least 10-fold more selective
for TbNMT over HsNMT compared to the pyrazole
sulfonamide hit 1, making it a potentially attractive start
point for optimization. From previous work, a ratio of
inhibition of the HsNMT potency compared to activity in a
parasite growth assay (selectivity “S” = HsNMT IC50/T. brucei
EC50) was found to give a better indication of selectivity rather
than direct comparison of potency at either enzyme.6 “S” for 4
was also significantly improved (>8.3 compared to 1.0 for 1).
We therefore sought to optimize 4 through exploration of its
structure−activity relationships and through hybridization with
key elements from the pyrazole sulfonamides with the aim of
maximizing selectivity.
RESULTS AND DISCUSSION
Identification of a TbNMT Selective Hit: Methyl Ester
Series. Methyl ester 4 (Scheme 2), identified as a singleton by
■
Scheme 3
Initial work on SAR generated some interesting results, but
it proved difficult to rationalize these until we had structural
information. Although replacement of the azepane moiety of 4
with a pyrrolidine gave an equipotent compound 5, removal of
the azepane group entirely resulted in loss of activity (7).
Unexpectedly, piperidine analogue 9 was also inactive.
Modifications to substituents on the phenylsulfonamide ring
were tolerated (cf. 8). However, carboxylic acid 6 proved
inactive (Scheme 2). This was particularly important because
the methyl ester undergoes rapid hydrolysis in plasma, which
would result in a complete loss of activity. The development of
Figure 3. Crystal structure of LmNMT:MCoA:10 showing water-mediated interactions in the C-terminal region. (A) Conformation of 10 (C
atoms gold) bound to LmNMT; H-bonds are shown as dashed black lines. (B) Binding mode comparison of 10 with the pyrazole sulfonamide
ligand DDD85646 (1; C atoms purple).
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Scheme 4. Design of Isosteres of the Methyl Ester
Scheme 5. Biaryl Hydrogen Bond Acceptors
efficacious compounds therefore required the identification of
stable ester bioisosteres capable of maintaining activity against
NMT in vivo.
protein able to change conformation, whereas the HsNMT
protein, being less flexible, suffers a steric clash preventing
binding of the ligand. Furthermore, the azepane ring was
observed to adopt a folded conformation not favorable for a
piperidine ring, which might explain the lack of activity of the
piperidine analogue (9). The pyrrolidine ring of 18 (a more
decorated version of compound 5) can also adopt a similar
folded conformation to the azepane of compound 4 in
agreement with our selectivity hypothesis.
Use of Existing Knowledge of NMT Inhibitors to
Increase Potency in the Methyl Ester Series. From our
previous work,5,6 appending a basic moiety to DDD64558 (1)
to reach the C-terminal carboxylic acid gave an ∼1000-fold
increase in potency (cf. compounds 2 and 3).5,6 Using the
crystallographic information, an amine-containing substituent
was appended to the arylsulfonamide core of 4 to give 10
(Scheme 3) to place a basic nitrogen adjacent to the C-
terminal carboxylic acid (Leu421). This substitution gave a
marked increase in potency versus TbNMT and also
maintained selectivity over HsNMT. A crystal structure of 10
bound to LmNMT:MCoA confirmed that the ligand retained
the interactions of 4 with the protein, including the azepane-
generated conformational shift. Additional water-mediated
interactions with the C-terminal carboxylic acid of the protein
accounted for the 150-fold gain in potency (Figure 3). Overlay
with the crystal structure of 2 showed that the two ligands
Use of Structural Information to Find the Binding
Mode of the Methyl Ester Series. We were unable to obtain
TbNMT crystal structures; however a structure was obtained
for 4 bound to LmNMT:MCoA (Figure 2A) as a surrogate
showing the ligand could bind in the peptide binding groove.
The main interactions were hydrogen bonds between the ester
carbonyl and Ser330 and between a sulfonamide oxygen and
Asp396 together with a stacking interaction between the lower
phenyl ring and Tyr217. Figure 2B shows the overlay of 4 with
pyrazole hit 1 showing that both molecules adopted twisted
conformations due to their common sulfonamide motif and
formed a similar key hydrogen-bonding interaction with
Ser330.
A significant difference was observed, however, in the
conformation of the protein when 4 was bound (Figure 2C):
there was a conformational change in residues 231−236, part
of the DE loop, to alleviate a steric clash between the azepane
ring and the side chain of Phe232. This loop is highly flexible
as judged by crystallographic B-factors, allowing a 5 Å shift in
the position of the Phe232 side chain. The flexibility is
facilitated by the Gly234 residue, which is not present in the
HsNMT protein. Hence, this could explain the high selectivity
observed for this ligand with the LmNMT (and TbNMT)
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Scheme 6. Activities of Fused Bicyclic Hydrogen Bond Acceptors against TbNMT
adopted similar conformations with an amine group occupying
a very similar position relative to the C-terminus.
and indazole systems (Scheme 7). The lactone analogue of the
methyl ester (15) was inactive.
Quinoline Series. Appending an amine tail to 16 to reach
the C-terminal carboxylic acid of NMT gave 18, resulting in
the expected gain in potency while maintaining the selectivity
observed with the methyl ester series. A crystal structure of 18
bound to LmNMT:MCoA (Figure 4) showed that the binding
mode of the ester series was retained with the quinoline
nitrogen of 18 forming a similar hydrogen bonding interaction
to Ser330 as the ester carbonyl of 4. The pyrrolidine ring of 18
occupies similar space to the azepane of 4, possibly explaining
the retention of high selectivity.
Head Group Optimization. Compounds with an amine in
the quinoline 6-position such as 16 were found to be
chemically unstable with mass spectrometry suggesting that
oxidation of the pyrrolidine ring under air was occurring.
Hence, an array of 6-alkyl-substituted quinolines was prepared
(Scheme 8, Table 1). Synthesis was achieved by Negishi
coupling with bromide 19 to install the 6-alkyl group (20)
followed by nitro reduction and coupling with 4-bromophe-
nylsulfonyl chloride to form sulfonamide 21.
Removal of an electron-donating nitrogen by replacement of
the 6-pyrrolidine with a 6-cyclopentyl group (24) resulted in a
chemically stable compound with similar potency. However,
substitution with a cyclohexyl or phenyl ring (25, 26) resulted
in loss of activity. This is consistent with the observed loss of
activity for a piperidine substituent in the methyl ester series
(9, Scheme 2). Benzyl and 3-pentyl groups were tolerated (29,
30), but the best potency was obtained with isobutyl and
cyclohexylmethyl groups (27, 28). Of these, the isobutyl
substituent (27) showed good potency and selectivity with the
minimum of addition of molecular weight and lipophilicity, i.e.,
equal ligand efficiency (LE)24 but improved ligand-lipophilicity
Compound 10 was also highly potent against the T. brucei
parasite. However, the ester was rapidly hydrolyzed to the
inactive acid (cf. 6) in mouse plasma (t1/2 < 15 min; method in
Supporting Information). Two approaches were therefore
adopted to find an alternative stable framework, which is
isosteric with the ester, to retain the hydrogen bond donor
functionality important for binding to Ser330 (Scheme 4).
Biaryl Isostere Strategy to Increase Metabolic
Stability. The first approach was to replace the ester with a
pendent heterocyclic hydrogen bond acceptor to maintain the
H-bond acceptor interaction with Ser330 (Scheme 5). A wide
range of heterocycles (pyrazoles, imidazoles, thiazoles,
oxazoles, etc.) were investigated. However, all compounds
proved inactive in our biochemical assay except 3-pyridyl 11,
which showed weak activity, ∼50-fold lower than that of the
methyl ester analogue (cf. 8, Scheme 2).
Bicyclic Isosteres to Improve Metabolic Stability. A
second approach was to explore the incorporation of a
hydrogen bond acceptor into a fused bicyclic system (Scheme
6). It was noted that removal of the azepane or pyrrolidine
groups from the methyl ester series (7, Scheme 2) resulted in
loss of activity. However, to aid ease of synthesis, compounds
without an azepane or pyrrolidine substituent were originally
prepared. Of these, quinoline 12, indazole 13, and
benzothiazole 14 showed weak activity against TbNMT. The
quinoline and indazole hits were selected for further studies.
Reinstatement of the azepane group into the quinoline
scaffold (22, Table 1) resulted in a compound with similar
potency and selectivity to hit compound 4. Similarly, addition
of a pyrrolidine group improved the potency of both quinoline
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Table 1. Quinazoline Head Group Optimization
a
b
c
LE = ligand efficiency (ΔG/number of non-hydrogen atoms). LLE = ligand-lipophilicity efficiency (pIC50 − clogP).25 “S” = HsNMT IC50/T.
brucei EC50.
Scheme 7. Fused Bicycles with an Added Amino Group
efficiency (LLE),25 and was thus selected for further
optimization.
Strategy to Increase Selectivity in the Quinoline
Series by Tail Group Optimization. It was known from our
previous work in the pyrazole sulfonamide series that
selectivity could be improved by optimization of the amine-
containing “tail group” moiety.6 Hence, combining the
selectivity conferred by the quinoline isobutyl substituent
with that conferred by different terminal amines was
attempted. An array of alternative pendant amines was then
prepared in the search for more selective compounds (Table
2). The amine moieties were appended by first hydroboration
of an appropriate alkene precursor with 9-BBN followed by
Appending an alkyl chain-linked piperidine “tail” (31 and
32, see Table 2) to the 6-isobutylquinoline system gave the
expected improvement in potency in analogous fashion to that
observed in the pyrazole and methyl ester series (cf.
compounds 3 and 10). The crystal structure of 32 bound to
LmNMT:MCoA showed that the overall binding mode of the
series was retained with the 6-isobutyl group sufficient to
induce the conformational change in residues 231−236 that is
proposed to confer selectivity over HsNMT (Figure 5).
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selectivity “S” with the longer butyl linker giving better
selectivity (cf. 31 vs 32, 36 vs 37). The stereochemistry of the
ligand could also affect selectivity (cf. 41 vs 42). Adding bulk
to the ligand could also improve selectivity (cf. 39 vs 40).
However, by far the most selective compound was the
pseudotropine-derived compound 41, which was nearly 4000-
fold selective (enzyme selectivity 3900; “S” = 325).
Interestingly, the analogous tropine-derived compound (42)
was much less selective (albeit more potent). The pseudo-
tropine group was known to confer modest selectivity in the
pyrazole series (cf. DDD101140; Figure 6).6 Therefore, we
had established, at least in this case, that a synergistic
combination of selective “head” (the isobutylquinoline) and
“tail” (the pseudotropine) groups resulted in a compound with
extremely high selectivity. The origin of the contribution to
selectivity, which the pseudotropine group makes, is unknown.
A crystal structure of this ligand bound to LmNMT could not
be obtained. The region of the active site around the C-
terminus is highly conserved between species with no obvious
differences that could explain differences in selectivity. It is
possible that the particular size and shape of the pseudotropine
ligand can be better accommodated in the T. brucei enzyme
due to differences in protein flexibility or the stability of the
water molecules that form the hydrogen bonding network.
In general, these compounds had lower potency against the
T. brucei parasite than the pyrazole series (e.g., compound 3).
This is in keeping with lower potency against the TbNMT
enzyme (2−16 nM for the quinoline series against typically <2
nM for the pyrazole series).
Figure 4. Crystal structure of 18 bound to LmNMT:MCoA. (A)
Binding mode of 18 (C atoms green) highlighting the H-bond
between the quinolone nitrogen and the side chain of Ser330. (B)
Comparison of the binding mode of 18 with 4 (C atoms gold). The
quinolone N atom mimics the ester carbonyl of 4.
Indazole Series Headgroup Optimization. A 5-
pyrrolidinylindazole, 17 (Scheme 7), had also shown some
activity against TbNMT, and a series of indazoles were also
evaluated as a quinoline surrogate. A limited array of 5-
alkylindazoles was prepared based around the most successful
compounds in the quinoline series (Table 3) using a similar
chemical route (Scheme 10). An isobutyl group (51) again
resulted in good potency and selectivity, as did a methyl-
cyclohexyl group (52). In this series, these two compounds
showed at least 10-fold better potency than those compounds
with a cyclic group appended directly to the indazole.
a
Scheme 8. Compounds 18−21
Tail Group Optimization to Increase Selectivity in the
Indazole Series. The tail group amine was then optimized in
analogous fashion to the quinoline series (Table 4). Synthesis
was performed using the same hydroboration-Suzuki coupling
protocol utilized for the quinoline series (Scheme 11). Groups
that gave good selectivity in the quinoline series (Table 2) also
performed well in the indazole series, often with improved
selectivity over the analogous quinoline. Six compounds were
found to have greater than 500-fold selectivity for TbNMT
over HsNMT (55, 57, 58, 60, 61, and 62) with the
pseudotropine-derived compound 60 again showing the
greatest selectivity (“S” = 318). A crystal structure of the 5-
isobutyl indazole ligand 54 bound to LmNMT:MCoA showed
the binding mode to be comparable to the 6-isobutyl quinoline
with the indazole N1 lone pair forming the key H-bond to
Ser330 (Figure 7). The binding mode of the remainder of the
molecule is highly conserved with all key interactions described
previously retained. The indazole series showed better potency
against T. brucei than the quinoline series in keeping with its
higher potency against the TbNMT enzyme.
a
(a) (R = alkyl, phenyl) RZnBr, Pd(dppf)Cl2, CuI, DMF, THF, 80
°C; (b) (R = amino) azepane or pyrrolidine, toluene, microwave 110
°C; (c) H2, Pd/C, EtOAc or Zn, NH4Cl, THF, MeOH, H2O; (d) 4-
BrPhSO2Cl, pyridine, CH2Cl2 (see Table 1 for the different “R”
groups: compounds 22−30).
Suzuki coupling of the crude borane with bromide 27 (Scheme
9).26,27 In some cases, a t-Boc-protected amine precursor was
used. These t-Boc-protected products were converted into
secondary amines by cleavage of the t-Boc group with TFA or
into N-methyl amines by cleavage of the t-Boc group followed
by reductive amination with paraformaldehyde or directly by
reduction of the t-Boc group with lithium aluminum hydride.
Over 100-fold selectivities for TbNMT over HsNMT were
achieved for a range of amine substituents. Piperidine (32, 43),
piperazine (39, 40), homopiperazine (37), fused-piperazine
(34, 35), and ether-linked morpholino groups (44, 45) all gave
highly selective compounds. Generally, piperazines were more
selective than piperidines (but less potent, cf. 31 vs 38, 33 vs
39), as noted previously.5,6 Secondary amines and their N-
methyl-capped analogues tended to show similar activities (cf.
32 vs 33, 44 vs 45). The length of the linker chain could affect
DMPK and in Vivo Efficacy. DMPK data for key
compounds are shown in Table 5. Compounds demonstrated
a range of mouse microsomal intrinsic clearance with examples
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a
Table 2. Tail Group Optimization
a
See Supporting Information for full table. “S” = HsNMT IC50/T. brucei EC50
from both indazole and quinoline series having acceptable
metabolic stability (i.e., Cli <5 mL min−1 g−1). Metabolite
identification studies on one of the more metabolically
unstable quinoline series compounds, 32, following incubation
with mouse liver microsomes, suggested that oxidation of the
central phenyl ring, or of the alkyl chain linker, predominated
rather than N-demethylation or isobutyl group metabolism.
Both series also demonstrated moderate to low plasma protein
binding. One major concern was hERG inhibition with both
series demonstrating low micromolar inhibition, potentially
driven by the basic, terminal amine group. Standard techniques
to reduce hERG inhibition are to lower lipophilicity and
basicity.28,29 To this end, a series of compounds bearing a
morpholine group were prepared, as this gave compounds with
the desired lower logD and pKa (See Figure 8). No significant
improvement in hERG activity was observed, and a substantial
penalty was paid in unacceptably high metabolic instability.
Because of its markedly superior EC50 against the T. brucei
parasite and acceptable metabolic stability (Cli), compound 54
was selected for progression toward a stage I mouse efficacy
model of HAT. When dosed orally at 50 mg/kg, 54
demonstrated good exposure (AUC0−8 = 2,900,000 ng-min/
mL; Cmax = 11,000 ng/mL; Tmax = 1 h) and in keeping with its
high “S” value (47), 54 was well-tolerated (maximum tolerated
dose >300 mg/kg when dosed twice a day orally for 3 days). In
the stage 1 HAT mouse model, with female NMRI mice
infected with T. brucei s427, compound 54 was fully curative at
10 mg/kg when dosed twice a day orally for 4 days, starting
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kg) would only give a cardiovascular window of ∼10-fold,
highlighting a significant issue for progression for this series
unless hERG liability could be reduced while maintaining high
potency.
The ability to cross the blood−brain barrier is essential for
treatment of stage 2 HAT, when the parasites are present in
the central nervous system. Compounds in the quinoline series
were brain penetrant but unfortunately lacked the right balance
of potency and metabolic stability for progression. Further-
more, the hERG inhibition was marginally higher within this
series. In contrast, the indazole series was very poorly brain
penetrant, and for 54, the most profiled exemplar from the
series, the observed very poor brain penetration was
demonstrated to be due to the fact it was a strong P-
glycoprotein substrate. Following coadministration in rat with
the Pgp inhibitor, GF120918, the brain:blood ratio for 54
increased to 2.2 (0.09 without GF120918 coadministration).
Validation of NMT as a Drug Target for Stage 2 HAT.
In previous studies, we had validated NMT as a drug target in
stage 1 HAT.4−6 However, these molecules were not brain
penetrant or did not offer sufficient tolerability in rodents to be
able to deliver a dosing regimen to conclusively validate NMT
as a viable drug target in stage 2 HAT. Owing to its high
potency against the T. brucei parasite, good oral pharmacoki-
netics and excellent tolerability, 54 appeared to be a good tool
molecule to validate NMT for stage 2 HAT. For the purposes
of the stage 2 HAT model, the poor CNS penetration of 54
could be overcome by using CF-1 mdr1a deficient mice.30
Using this model, a completely curative dosing regimen was
obtained for 54 (Figure 9) at 50 mg/kg orally twice daily for 5
days, validating NMT as a suitable drug target for stage 2 HAT.
Using female NMRI mice, not mdr1a deficient, compound 54
relapsed at all dose levels (50, 100, and 200 mg/kg orally twice
daily for 5 days) in the stage 2 model.
Figure 5. Crystal structure of the 6-isobutyl derivative 32 bound to
LmNMT:MCoA. Structure of 32 (C atoms gold) bound to LmMNT
(gold ribbon). The main chain of LmNMT bound to the pyrolidine
derivative 18 is overlaid (gray ribbon).
a
Scheme 9. Compound 27
We have previously shown that poor CNS penetration could
be overcome by application of a metabolically stable
difluoromethyl “cap” to the sulfonamide.6 Pleasingly, when
this modification was made to indazole 54 (i.e., 66, see Scheme
12) the brain:blood ratio was significantly improved (1.9 vs <
0.1), whereas other properties were largely left unaffected,
including trypanocidal activity, metabolic stability (mouse Cli
= 2.3 mL/min/g), selectivity “S” and also unfortunately hERG
(IC50 = 6 μM). Consequently, because of the likely
unacceptable cardiovascular safety window (this was only
∼3-fold at 50 mg/kg, delivering stage 2 efficacy for 54), 66 was
not progressed further.
a
(a) 9-BBN, THF, reflux; (b) Pd(dppf)Cl2, K3PO4, DMF, H2O (see
Table 2 for the different “R” groups: compounds 31−45; in some
cases, an additional synthetic step was required, see the Supporting
Information for details).
CONCLUSIONS
■
We show here that for the first time it is possible to obtain
inhibitors of TbNMT that are highly potent, selective, and
CNS penetrant (i.e., 41 and 66). The substantial gains in
selectivity achieved relative to earlier series has resulted from
the additive effect of combining selectivity-enhancing pendent
amines from a previous series with new highly selective head
groups, which translates into improved tolerability in vivo.
High-throughput screening identified 4, an inhibitor of
TbNMT, with selectivity over HsNMT. A chemistry campaign
based around crystal structures of the ligand bound to
LmNMT led to the discovery of two novel alternative
scaffolds: one based on a quinoline and one based on an
indazole. Both scaffolds showed good potency and selectivity
for the parasite enzyme. From crystallographic studies of our
previous pyrazole series, we were able to hybridize these
Figure 6. Modest selectivity afforded by a pseudotropine substituent
(published previously: compound 71 in Supporting Information6).
day 3 postinfection. All animals relapsed at dose levels of 0.3, 1,
or 3 mg/kg orally twice daily for 4 days. Assuming linearity of
exposure, the free Cmax at the minimal efficacious dose (10 mg/
J
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Table 3. Indazole Head Group Optimization
a
Scheme 10. Compounds 46−48
HAT. It remains to be seen whether this series offers the
potential to deliver a lead with all the properties required for
further development toward a treatment for both stages of
HAT, particularly with regard to a molecule potent against the
parasite, having good pharmacokinetics and brain penetration
and an acceptable cardiovascular safety window.
EXPERIMENTAL SECTION
■
General Experimental Information. Chemicals and solvents
were purchased from the Aldrich Chemical Co., Fluka, ABCR, VWR,
Acros, Fisher Chemicals, and Alfa Aesar and were used as received
unless otherwise stated. Air- and moisture-sensitive reactions were
carried out under an inert atmosphere of argon in oven-dried
glassware. Analytical thin-layer chromatography (TLC) was per-
formed on precoated TLC plates (layer 0.20 mm silica gel 60 with
fluorescent indicator UV254 from Merck). Developed plates were air-
dried and analyzed under a UV lamp (UV254/365 nm). Flash column
chromatography was performed using prepacked silica gel cartridges
(230−400 mesh, 40−63 μm, from SiliCycle) using a Teledyne ISCO
a
(a) RZnBr, Pd(dppf)Cl2, CuI, DMF, THF, 80 °C (for compounds
49−52); (b) pyrrolidine, toluene, microwave 110 °C (for compound
17); (c) H2, Pd/C; (d) 4-BrPhSO2Cl, pyridine, CH2Cl2 (see Table 3
for the different “R” groups: compounds 49−52).
scaffolds with our previous pyrazole series leading to
compounds with potent activity against TbNMT and greater
than 1000-fold selectivity for TbNMT vs HsNMT and with “S”
values ranging from 10- up to 300-fold. Although some of
1
these compounds had antitrypanocidal activities with EC50
<
Combiflash Companion or Combiflash Retrieve. H NMR spectra
100 nM, in general they were less potent than the pyrazole
series with which we had previously worked. Some of the
quinoline series showed blood−brain barrier penetration, but
none had the right balance of potency against the parasite,
metabolic stability, and selectivity to progress further. In
contrast, one of the indazole compounds, 54, had good
potency against the T. brucei parasite (EC50 = 5 nM), good
microsomal stability (Cli = 2.9 mL/min/g), good selectivity
(“S” = 47), good oral exposure, and good in vivo tolerability. It
was efficacious in a stage 1 mouse model of HAT and could
potentially be further developed as a drug to treat stage 1 of the
disease, except the cardiovascular toxicity window was too low
because of the hERG liability. Although, compound 54 lacked
sufficient brain penetration for treating stage 2 HAT as a
consequence of strong Pgp interaction, a fully curative oral
dose regimen was achieved in the stage 2 GVR35 mouse model
of HAT when using CF1 mdr1a deficient mice for the
infection. This validates NMT as a drug target for stage 2
were recorded on a Bruker Avance II 500 spectrometer (1H at 500.1
MHz) or a Bruker DPX300 spectrometer (1H at 300.1 MHz).
Chemical shifts (δ) are expressed in ppm recorded using the residual
solvent as the internal reference in all cases. Signal splitting patterns
are described as singlet (s), doublet (d), triplet (t), quartet (q),
multiplet (m), broad (b), or a combination thereof. Coupling
constants (J) are quoted to the nearest 0.1 Hz. LC-MS analyses were
performed with either an Agilent HPLC 1100 series connected to a
Bruker Daltonics MicrOTOF or an Agilent Technologies 1200 series
HPLC connected to an Agilent Technologies 6130 quadrupole LC-
MS, where both instruments were connected to an Agilent diode array
detector. All assay compounds had a measured purity of ≥95% as
determined using this analytical LC-MS system (TIC and UV). High-
resolution electrospray measurements were performed on a Bruker
Daltonics MicrOTOF mass spectrometer. Microwave-assisted chem-
istry was performed using a Biotage Initiator Microwave Synthesizer.
Purity (>97%) and molecular mass were confirmed by HPLC and
high-resolution mass spectrometry.
Prototypical Procedure for the Negishi Coupling between
an Aryl Bromide and an Alkylzinc Reagent. 6-Isobutyl-5-
K
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Table 4. Indazole Tail Group Optimization
a
filtered through a plug of SiO2, and concentrated. The products were
purified by flash chromatography on silica (0−100% EtOAc-hexane)
to give the title compound as a brown gum (311 mg, 1.43 mmol,
96%).
Scheme 11. Compound 51
1H NMR (CDCl3, 500 MHz) δ 8.99 (1H, d, J = 4.2 Hz), 8.20 (1H,
d, J = 8.8 Hz), 8.07 (1H, ddd, J = 8.6, 1.4, 0.7 Hz), 7.64 (1H, d, J =
8.8 Hz), 7.56 (1H, dd, J = 8.6, 4.2 Hz), 2.71 (2H, d, J = 7.4 Hz), 2.07
(1H, m), 0.98 (6H, d, J = 6.7 Hz). LCMS (ES+) [M + H]+ m/z 231.1.
Prototypical Procedure for the Hydrogenation of an Aryl
Nitro Group to an Amine. 6-Isobutylquinolin-5-amine. A solution
of 6-isobutyl-5-nitro-quinoline (286 mg, 1.42 mmol) in EtOAc (10
mL) was subjected to hydrogenation conditions with 5%Pd/C (93
mg) under a hydrogen balloon at room temperature for 6 h. The
reaction mixture was filtered through Celite, and the filtrate was
concentrated to give the crude title compound (285 mg, 1.19 mmol,
92%) as a brown solid, which was used without purification. LCMS
(ES+) [M + H]+ m/z 201.1.
a
(a) 9-BBN, THF, reflux; (b) Pd(dppf)Cl2, K3PO4, DMF, H2O. In
some cases, an additional synthetic step was required, see the
Supporting Information for details.
Prototypical Procedure for the Coupling on an Aryl Amine
with a Sulfonyl Chloride. 4-Bromo-N-(6-isobutylquinolin-5-yl)-
benzenesulfonamide (27). 5-Amino-6-isobutylquinoline (220 mg,
1.10 mmol) was taken up in a mixture of pyridine (2 mL) and DCM
(3 mL); 4-bromobenzene-1-sulfonyl chloride (337 mg, 1.32 mmol)
was added, and the reaction mixture was stirred at room temperature
for 3 days. The solvent was removed under reduced pressure; the
residue was partitioned with DCM and saturated aqueous sodium
bicarbonate solution, and the organics were dried (MgSO4) and
concentrated to dryness. Purification by flash chromatography (0−
100% EtOAc-CH2Cl2) gave the title compound (299 mg, 0.71 mmol,
65%) as a cream solid.
nitroquinoline. 6-Bromo-5-nitroquinoline (0.378 g, 1.49 mmol),
Pd(dppf)Cl2·CH2Cl2 (66 mg, 0.08 mmol, 5 mol %), and copper(I)
iodide (48 mg, 0.25 mmol, 17 mol %) were added to a dry 50 mL
flask under argon. The flask was purged three times with a vacuum/
argon cycle, and DMF (12 mL) was added. The mixture was stirred at
room temperature, and the flask was purged a further three times with
a vacuum/argon cycle. The mixture was cooled to 0 °C, and a 0.5 M
solution of the isobutylzinc bromide in THF (4.5 mL, 2.25 mmol, 1.5
equiv) was added dropwise. The mixture was then heated at 80 °C
overnight.
The reaction mixture was then concentrated, treated with EtOAc
(20 mL) and water (20 mL), and stirred vigorously for 30 min. The
mixture was filtered; the phases were separated, and the aqueous
phase was extracted with further EtOAc (2 × 20 mL). The combined
organic extracts were washed with water and brine, dried (Na2SO4),
1H NMR (DMSO-d6, 500 MHz) δ 10.14 (1H, s), 8.79 (1H, dd, J =
4.2, 1.6 Hz), 8.00 (1H, d, J = 8.5 Hz), 7.93 (1H, d, J = 8.8 Hz), 7.76
(2H, d, J = 8.6 Hz), 7.66 (1H, d, J = 8.8 Hz), 7.52 (2H, d, J = 8.6 Hz),
7.34 (1H, dd, J = 8.6, 4.2 Hz), 2.50 (2H, br.s), 1.92 (1H, m), 0.75
L
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Figure 7. Crystal structure of the indazole ligand 54 bound to LmNMT:MCoA. (A) Binding mode of 54 (C atoms light blue) showing H-bond to
Ser300. (B) Binding mode comparison of 54 with quinoline scaffold 32 (C atoms gold).
Table 5. DMPK Data for Selected Compounds
a
TbNMT IC50 HsNMT IC50 T. brucei EC50
enzyme
selectivity
Cli mouse microsomes mL−1
min−1 g−1
mouse
brain:blood ratio
mouse PPB
(fu)
hERG
cmpd
(nM)
(nM)
(nM)
“S”
IC50/μM
Quinoline Series
32
34
37
41
<5
<5
6
250
640
1400
27000
19
40
59
91
>50
>130
59
13
16
24
300
6.2
6.2
1.8
1.5
0.8
0.8
0.8
0.17
0.093
0.18
1.3
1.6
7
3900
Indazole Series
54
58
60
62
63
64
<5
<5
5
7
<5
<5
230
2500
12000
4600
190
5
31
39
220
26
9
>46
>500
2500
660
>38
>26
46
80
320
21
7
2.9
3.7
1.4
20
24
22
<0.1
<0.1
<0.1
0.062
3.7
3.0
3.3
3.1
5.6
6.7
130
14
a
hERG values were determined using IonWorks patch-clamp electrophysiology at Essen Bioscience, UK.
Figure 8. Illustration of the strategy to lower logD and pKa by replacement of the piperidine moiety of 54 with a morpholine group.
(6H, d, J = 5.5 Hz). LCMS (ES+) [M + H]+ m/z 418.9/420.9. HRMS
(ES+): found 419.0433 [M + H]+; C19H20BrN2O2S+ [M + H]+,
requires 419.0423.
Prototypical Procedure Coupling of an Aryl Bromide with a
9-BBN-Derived Trialkylborane under B-Alkyl Suzuki−Miyaura
Conditions. N-(6-Isobutylquinolin-5-yl)-4-(4-(1-methylpiperidin-4-
yl)butyl)benzenesulfonamide (32). 4-(But-3-en-1-yl)-1-methylpiper-
idine (61.2 mg, 0.4 mmol) under argon at room temperature was
treated dropwise with 9-BBN (0.5 M in THF, 0.8 mL, 0.4 mmol).
The reaction mixture was heated under reflux for 1 h. The resulting
solution was then transferred into a stirred mixture of 4-bromo-N-(6-
isobutylquinolin-5-yl)benzenesulfonamide (82.4 mg, 0.196 mmol)
and potassium phosphate (53 mg, 0.25 mmol) in DMF (2 mL) and
water (0.6 mL) under argon. After bubbling argon through the
reaction for 5 min at room temperature, Pd(dppf)Cl2·CH2Cl2 (16 mg,
0.02 mmol, 10 mol %) was added, and the reaction vessel was sealed
and heated in a microwave at 90 °C for 1 h. The reaction mixture was
concentrated under reduced pressure, resuspended in CH2Cl2, and
applied to a 2 g SCX-2 column. The column was washed with CH2Cl2
M
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Figure 9. Kaplan−Meier survival graph for 54 in the NMRI and CF-1 mdr1a deficient mouse infected with Trypanosoma b. brucei GVR35. Mice
(five per group) were dosed with drug vehicle (orally, black), Diaminazene diaceturate (Berenil; intraperitoneally, gray), Melarsoprol
(intraperitoneally, aqua), or 54 (orally, blue (200 mg/kg), purple (100 mg/kg), and red (50 mg/kg)) for 5 days starting at day 21 following
infection. Berenil and Melasoprol were administered once daily, and 54 was administered twice daily.
1H NMR (DMSO-d6, 500 MHz): δ 9.38 (1H, br s), 7.54 (2H, d, J
a
Scheme 12. Compounds 65 and 66
= 7.8 Hz), 7.52 (1H, s), 7.39 (1H, d, J = 8.7 Hz), 7.32 (2H, d, J = 7.8
Hz), 7.04 (1H, d, J = 8.7 Hz), 4.01 (3H, s), 2.73 (2H, d, J = 11.0 Hz),
2.66 (2H, t, J = 7.8 Hz), 2.31 (2H, d, J = 7.8 Hz), 2.14 (3H, s), 1.83
(2H, t, J = 7.8 Hz), 1.78 (1H, m), 1.61−1.55 (4H, m), 1.34−1.21
(4H, m), 1.16−1.09 (3H, m), 0.73 (6H, d, J = 6.8 Hz). 13C NMR
(125 MHz, CDCl3): δ 149.1 (C), 148.6 (C), 136.6 (C), 130.0 (C),
128.9 (2 × CH), 127.3 (CH), 124.6 (CH), 124.5 (C), 121.4 (C),
116.7 (CH), 56.0 (CH2), 46.5 (CH3), 40.3 (CH3), 39.2 (CH2), 36.3
(CH2), 35.8 (CH2), 35.1 (CH), 32.4 (CH2), 31.4 (CH2), 29.5 (CH),
26.3 (CH2), 22.3 (CH3). LCMS (ES+) [M + H]+ m/z 497.2. HRMS
(m/z): [M + H]+ calcd for C28H41N4O2S, 497.2945; found 497.2946.
Enzyme Inhibition Assay and Cell Viability Assay. NMT
enzyme inhibition assays were carried out using a scintillation
proximity assay utilizing [3H]myristoyl-coA and a peptide substrate
derived from T. brucei calpain-type protease CAP5.5 as described
a
previously.6 Assays were performed at room temperature (22−23 °C)
(a) Sodium difluoroacetate, K2CO3, MeCN; (b) 4-(but-3-en-1-yl)-1-
methylpiperidine, 9-BBN, THF, reflux; then Pd(dppf)Cl2, K3PO4,
DMF, H2O.
in 384-well white optiplates (PerkinElmer). Each assay was performed
in a 40 μL reaction volume containing 30 mM Tris buffer, pH 7.4, 0.5
mM EDTA, 0.5 mM EGTA, 1.25 mM dithiothreitol (DTT), 0.1% (v/
v) Triton X-100, 0.125 μM [3H]myristoyl-coA (8 Curie (Ci)
mmol−1), 0.5 μM biotinylated CAP5.5, 5 nM NMT, and various
concentrations of the test compound. Test compound (0.4 μL in
DMSO) was transferred to all assay plates using a Cartesian
Hummingbird (Genomics Solution) before 20 μL of enzyme was
added to assay plates. The reaction was initiated with 20 μL of a
substrate mix and stopped after 15 min (HsNMT1) or 50 min
(TbNMT) with 40 μL of a stop solution containing 0.2 M phosphoric
acid, pH 4.0 and 1.5 M MgCl2 and 1 mg ml−1 PVT SPA beads (GE
Healthcare). All reaction mix additions were carried out using a
Thermo Scientific WellMate (Matrix). Plates were sealed and read on
a TopCount NXT Microplate Scintillation and Luminescence
Counter (PerkinElmer).
T. brucei cell viability assays were carried out as described
previously.6 In brief, bloodstream T. b. brucei s427 was cultured at
37 °C in modified HMI9 medium (56 μM 1-thioglycerol was
substituted for 200 μM 2-mercaptoethanol) and quantified using a
hemocytometer. Cell culture plates were stamped with 1 μL of an
appropriate concentration of test compound in DMSO followed by
the addition of 200 μL of trypanosome culture (104 cells ml−1) to
each well except for one column that received media only. Culture
plates of T. brucei cells were incubated at 37 °C in an atmosphere of
5% CO2 for 69 h before the addition of 20 μL of resazurin (final
concentration, 50 μM). After a further 4 h incubation, fluorescence
(10 mL) and 10% MeOH-CH2Cl2 (20 mL) and then eluted with a
5% mixture of 7N ammonia in methanol and CH2Cl2 (10 mL). The
product-containing fractions were concentrated and purified by flash
chromatography with DCM/MeOH/NH3 to give the title compound
as an off-white solid (55 mg, 0.11 mmol, 57%).
1H NMR (CDCl3, 500 MHz) δ 8.86 (1H, dd, J = 4.2, 1.6 Hz), 8.41
(1H, ddd, J = 8.6, 1.5, 0.8 Hz), 8.03 (1H, d, J = 8.7 Hz), 7.53 (2H, d, J
= 8.4 Hz), 7.50 (1H, d, J = 8.7 Hz), 7.33 (1H, dd, J = 8.6, 4.2 Hz),
7.23 (2H, d, J = 8.4 Hz), 2.85 (2H, d, J = 11.7 Hz), 2.68 (2H, t, J =
7.7 Hz), 2.28 (3H, s), 2.20 (2H, d, J = 7.4 Hz), 1.90 (2H, t, J = 6.6
Hz), 1.82 (1H, m), 1.67 (2H, d, J = 11.5 Hz), 1.61 (2H, quintet, J =
7.5 Hz), 1.37−1.19 (7H, m), 0.80 (6H, d, J = 6.6 Hz). LCMS (ES+)
[M + H]+ m/z 494.3. HRMS (ES+): found 494.2832 [M + H]+;
C29H40N3O2S+ [M + H]+, requires 494.2836.
N-(5-Isobutyl-2-methyl-2H-indazol-4-yl)-4-(4-(1-methylpiperi-
din-4-yl)butyl)benzenesulfonamide (54). According to the proce-
dure above, the trialkylborane was preformed with 4-but-3-enyl-1-
methylpiperidine (1.02 g, 6.63 mmol) followed by Suzuki reaction
with 4-bromo-N-(5-isobutyl-2-methyl-2H-indazol-4-yl)-
benzenesulfonamide (51) (2.0 g, 4.74 mmol) at 100 °C for 90 min
on a heating block. The free base was recrystallized from EtOAc to
give the title compound as an off-white powder (2.06 g, 4.14 mmol,
87%).
N
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was measured (excitation 528 nm; emission 590 nm) using a BioTek
flx800 plate reader.
ABBREVIATIONS USED
■
NMT, N-myristoyltransferase; T. brucei, Trpanosoma brucei; T.
br. brucei, T. brucei. brucei; T. b. gambiense, T. brucei. gambiense;
T. b. rhodensiense, T. brucei. rhodensiense; HAT, human African
trypanosomiasis or sleeping sickness; CoA, Coenzyme A;
MCoA, myristoyl-coenzyme A; CNS, central nervous system;
TbNMT, T. brucei N-myristoyltransferase; HsNMT, human
NMT; LmNMT, Leishmania major NMT; MRC-5, Medical
Research Council cell strain 5; LE, ligand efficiency; LLE,
Ligand-Lipophilicity Efficiency; “S”, HsNMT IC50/T. brucei
EC50; PSA, polar surface area; HBA, hydrogen bond acceptor;
9-BBN, 9-borabicyclo(3.3.1)nonane; t-Boc, tert-butoxycarbon-
yl; TFA, trifluoroacetic acid; THF, tetrahydrofuran; DMF,
N,N-dimethylformamide; BBB, blood−brain barrier; Cli,
microsomal intrinsic clearance; PPB, plasma protein binding;
fu, fraction unbound; Pgp, P-glycoprotein; hERG, human
Protein Crystallography. LmNMT protein ligand complexes
were determined using methods described previously.4,5 Diffraction
data were measured at ESRF (beamlines ID14-1, ID41-4, and ID-29)
and Diamond beamline I03. Data were integrated and scaled using
either the HKL suite31 (LmNMT:4 and LmNMT:10) or XDS32 and
SCALA33 (LmNMT:18, LmNMT:32, and LmNMT:54). All
structures were solved by molecular replacement as implemented in
MOLREP34 using the binary LmNMT:MCoA complex (PDB ID:
3H5Z) as a search model. Refinement and model building was carried
out using REFMAC535 and COOT.36 Ligand coordinate files and
restraints were generated using PRODRG.37 Coordinates for
LmNMT:ligand complexes and associated diffraction data have
been deposited in the Protein Data Bank (PDB) with accession
codes 6GNH, 6GNS, 6GNT, 6GNU, and 6GNV for compounds 4,
10, 18, 32, and 54, respectively.
ASSOCIATED CONTENT
* Supporting Information
ether-a-go-go related gene; cLogP, calculated LogP; Cmax
,
■
maximum blood concentration; Tmax, time when maximum
blood concentration achieved; AUC, area under the blood
concentration−time curve; SAR, structure−activity relation-
ship; TLC, thin-layer chromatography; UV, ultraviolet light;
TIC, total ion chromatogram; HPLC, high-performance liquid
chromatography; LC-MS, liquid chromatography−mass spec-
trometry; ES, electrospray; HRMS, high-resolution mass
spectrometry; eq, equivalents; EtOAc, ethyl acetate; DCM,
dichloromethane; EDTA, ethylenediaminetetraacetic acid;
EGTA, ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-
tetraacetic acid; DMSO, dimethyl sulfoxide; DTT, dithio-
threitol; UPLCMSMS, ultraperformance liquid-chromatogra-
phy tandem mass spectrometry
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmed-
chem.8b00884.
Supplementary data table, synthetic details for com-
pounds 4, 10, 12, 13, 16, 17, 22−46, and 49−64, X-ray
data collection and refinement statistics, details of
DMPK experiments including intrinsic clearance (Cli)
experiments, plasma protein binding experiments,
plasma stability experiments, in vivo mouse studies
including mouse pharmacokinetics, mouse brain pene-
tration, rat brain penetration with and without Pgp
inhibitor, HAT stage 1 mouse model of efficacy, and
HAT stage 2 mouse model of efficacy(PDF)
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AUTHOR INFORMATION
Corresponding Authors
*(K.D.R.) Phone: +44 1382 388 688; E-mail: k.read@dundee.
ac.uk.
*(I.H.G.) Phone: +44 1382 386 240; E-mail: i.h.gilbert@
dundee.ac.uk.
■
ORCID
David A. Robinson: 0000-0003-1979-5918
Ngai Mok: 0000-0002-2827-3735
Paul G. Wyatt: 0000-0002-0397-245X
Ian H. Gilbert: 0000-0002-5238-1314
Kevin D. Read: 0000-0002-8536-0130
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Funding for this work was provided by the Wellcome Trust
(Grants WT077705 and WT094090 and Strategic Award
WT083481). We thank Gina McKay for performing HRMS
analyses, Lorna Campbell and Sandra O’Neill for performing
parasite assays, and Daniel James for data management. We
thank the staff at the European Synchrotron Radiation Facility
(ESRF) and Diamond Light Source (DLS) for beam time and
support.
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DOI: 10.1021/acs.jmedchem.8b00884
J. Med. Chem. XXXX, XXX, XXX−XXX

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