Discovery and Characterization of Clinical Candidate LXE408 as a Kinetoplastid-Selective Proteasome Inhibitor for the Treatment of Leishmaniases

Article abstract of DOI:10.1021/acs.jmedchem.0c00499

Visceral leishmaniasis is responsible for up to 30,000 deaths every year. Current treatments have shortcomings that include toxicity and variable efficacy across endemic regions. Previously, we reported the discovery of GNF6702, a selective inhibitor of t

Full text of DOI:10.1021/acs.jmedchem.0c00499

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Drug Annotation
Discovery and Characterization of Clinical Candidate
LXE408 as a Kinetoplastid-Selective Proteasome
Inhibitor for the Treatment of Leishmaniases
Advait Nagle, Agnes Biggart, Céline Be, Honnappa Srinivas, Andreas Hein, Diana Caridha, Richard J
Sciotti, Brandon S Pybus, Mara Kreishman-Deitrick, Badry Bursulaya, Yin Lai, Mu-Yun Gao, Fang Liang,
Casey J. N. Mathison, Xiaodong Liu, Vince Yeh, Jeffrey Smith, Isabelle Lerario, Yongping Xie, Donatella
Chianelli, Michael Gibney, Ashley Berman, Yen-Liang Chen, Jan Jiricek, Lauren Davis, Xianzhong Liu,
Jaime Ballard, Shilpi Khare, Fabian Kurt Eggimann, Alexandre Luneau, Todd Groessl, Michael Shapiro,
Wendy Richmond, Kevin Johnson, Patrick Rudewicz, Srinivasa P S Rao, Christopher Thompson, Tove
Tuntland, Glen Spraggon, Richard Glynne, Frantisek Supek, Christian Weismann, and Valentina Molteni
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c00499 • Publication Date (Web): 15 Jul 2020
Downloaded from pubs.acs.org on July 15, 2020
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Richmond, Wendy; Genomics Institute of the Novartis Research
Foundation
Johnson, Kevin; Genomics Institute of the Novartis Research Foundation
Rudewicz, Patrick; Novartis Institutes for BioMedical Research Emeryville
Rao, Srinivasa; Novartis Institute for Tropical Diseases, Drug Discovery
Unit
Thompson, Christopher; Novartis Institutes for BioMedical Research Inc
Tuntland, Tove; Genomics Institute of the Novartis Research Foundation
Spraggon, Glen; Genomics Institute of the Novartis Research
Foundation, Protein Science
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Glynne, Richard; Genomics Institute of the Novartis Research Foundation
Supek, Frantisek; Genomics Institute of the Novartis Research
Foundation,
Weismann, Christian; Novartis Institutes for BioMedical Research Basel,
Expertise Protease Platform
Molteni, Valentina; Genomics Institute of the Novartis Research
Foundation,
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TITLE PAGE
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Discovery and Characterization of Clinical
Candidate LXE408 as a Kinetoplastid-Selective
Proteasome Inhibitor for the Treatment of
Leishmaniases
Advait Nagle,*,# Agnes Biggart,# Celine Be,† Honnappa Srinivas,† Andreas Hein,† Diana
Caridha,֍ Richard J. Sciotti,֍,Ω Brandon Pybus,֍ Mara Kreishman-Deitrick,֍ Badry
Bursulaya,# Yin H. Lai,# Mu-Yun Gao,# Fang Liang,# Casey J. N. Mathison,# Xiaodong Liu,#
Vince Yeh,#,± Jeffrey Smith,# Isabelle Lerario,#,§ Yongping Xie,#,∥ Donatella Chianelli,# Michael
Gibney,#, ± Ashley Berman,#,Ϭ Yen-Liang Chen,‡ Jan Jiricek,‡ Lauren C. Davis,#,⊥ Xianzhong
Liu,# Jaime Ballard,# Shilpi Khare,#,€ Fabian Kurt Eggimann,† Alexandre Luneau,† Todd
Groessl,# Michael Shapiro,# Wendy Richmond,# Kevin Johnson,#¤ Patrick J. Rudewicz,‡
Srinivasa P. S. Rao,‡ Christopher Thompson,֍ Tove Tuntland,#,Δ Glen Spraggon,# Richard J.
Glynne,#,£ Frantisek Supek,# Christian Wiesmann,*,† Valentina Molteni.*,#
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#Genomics Institute of the Novartis Research Foundation (GNF), San Diego, California 92121, United States
†Novartis Institutes for Biomedical Research, CH-4056 Basel, Switzerland
֍ Experimental Therapeutics, Walter Reed Army Institute of Research, Silver Spring, Maryland 20910, United
States
֍ Novartis Institutes for Biomedical Research, Cambridge, Massachusetts 02139, United States
‡Novartis Institute of Tropical Diseases, Emeryville, California 94608, United States
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KEYWORDS: Kinetoplastid • Leishmania • Proteasome • LXE408 • Bortezomib
ABSTRACT: Visceral leishmaniasis is responsible for up to 30,000 deaths every year. Current
treatments have shortcomings that include toxicity and variable efficacy across endemic
regions. Previously, we reported the discovery of GNF6702, a selective inhibitor of the
kinetoplastid proteasome, which cleared parasites in murine models of leishmaniasis, Chagas
disease and human African trypanosomiasis. Here, we describe the discovery and
characterization of LXE408, a structurally related kinetoplastid-selective proteasome inhibitor
currently in Phase 1 human clinical trials. Furthermore, we present high-resolution cryo-EM
structures of the Leishmania tarentolae proteasome in complex with LXE408, which provides a
compelling explanation for the non-competitive mode of binding of this novel class of inhibitors
of the kinetoplastid proteasome.
INTRODUCTION
Visceral leishmaniasis (VL) is an infectious disease causing 50,000-90,000 new cases and
30,000 deaths every year.1 In the Indian subcontinent the first-line treatment for this infection is
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a single infusion of AmBisome® (liposomal amphotericin B). However, liposomal amphotericin B
has limited efficacy in East Africa both as a monotherapy and in combination with other drugs,2
therefore, in this endemic area the first line of treatment is a 17-day therapy with antimony
stibogluconate and paromomycin combination.3, 4 Overall, existing parenteral treatments are
difficult to administer, require hospitalization and have dose-limiting toxicities.5 New drugs that
are orally bioavailable, safe and can effect rapid cure are necessary to address needs of VL
patients in developing economies.
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Previously, we reported the identification of a novel pan-kinetoplastid growth inhibitor series,
exemplified by GNF6702, and demonstrated that this triazolopyrimidine scaffold exerts its
antiparasitic activity through selective inhibition of kinetoplastid proteasome chymotrypsin
activity.6 Subsequently, a GSK/University of Dundee group reported the discovery of a
structurally related scaffold, as well as its unique binding mode to Leishmania tarentolae
proteasome, explaining the scaffold’s exceptional selectivity for the kinetoplastid compared to
the mammalian proteasome.7 Here, we describe the optimization of GNF6702 that led to the
selection of 1 (LXE408), a compound with remarkable efficacy in murine models of visceral and
cutaneous leishmaniasis that is currently in Phase 1 human clinical trials. We also disclose the
high-resolution cryo-EM co-structure of 1 with the Leishmania tarentolae proteasome in the
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presence of the prototypical competitive proteasome inhibitor bortezomib which clarifies the
non-competitive nature of proteasome inhibition for this novel class of anti-leishmanial agents.6
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RESULTS and DISCUSSION
The progression of GNF6702 (2) into clinical testing was hampered by solubility-limited oral
absorption. While this limitation could have potentially been addressed by a specialized
formulation (e.g.; nanosuspension, solid dispersion), such an approach would likely increase
the cost of treatment and complicate its use in developing economies. As a result, we set out to
perform medicinal chemistry optimization of GNF6702 in order to identify a development
candidate with improved solubility and improved oral exposure across preclinical species. Initial
efforts, focusing on reducing logP and increasing the sp3 fraction to increase solubility, led to
molecules with less potency or high clearance. A solubility analysis pointed to the high crystal
packing energy of GNF6702 (melting point = 224 °C) as the key solubility-limiting issue.8 As the
molecule was quite planar, we decided to introduce small substitutions at the pyridine 3-position
(R) to force the pyridine out of plane with respect to the bicyclic core in an effort to reduce the
crystal packing energy, and thus improve solubility (Table 1).9
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Table 1. In vitro data for key analogues
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7
R
F
8
9
N
N
N
N
O
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N
HN
O
N
Compound
No.
R
L.donovani L.donovani HT sol.
proteasome EC50 (M)b pH 6.8.
ERd mouse/rat/ dog/
human
Melting point
group
temperature (°C)
IC50 (M)a
0.04
(M)c
17
1 (LXE408)
Me
H
0.04
0.02
<0.21 /<0.18/ < 0.24/ 0.27
<0.21/<0.27/0.46/ 0.3
139
224
2
0.035
10
(GNF6702)
3
4
5
6
CHF2
Cl
0.06
0.1
0.11
0.05
0.07
0.5
21
7
0.51/ <0.18/ 0.57/ 0.55
<0.21/n.t /<0.21 /0.21
0.4/ n.t/ 0.56/< 0.21
n.t./ n.t/ n.t./n.t.
n.t.
229
n.t.
n.t.
OMe
n.t
7
CH2OH
0.07
n.t. e
a) Half-maximum inhibition of chymotrypsin-like activity of purified L. donovani proteasome; b)
Half-maximum inhibition of L. donovani amastigote proliferation within primary mouse
macrophages; c) Solubility determined in a high-throughput assay format; d) Microsomal
extraction ratio. The assay measures amount of parent compound remaining after 1 hour
incubation in microsomes; e) n.t. = not tested. IC50 and EC50 values correspond to means (n = 4
technical replicates).
In general, the compounds synthesized with different R groups maintained an inhibitory
activity on purified L. donovani proteasome similar to the unsubstituted compound 2.
Compounds 1 and 4 emerged as frontrunners, having good potency in the more relevant
intracellular L. donovani growth inhibition assay and favorable microsomal stability across
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species. Finally, using the melting point temperature as an indicator of crystal packing
energy, we selected compound 1 over compound 4 for further characterization due to its
lower melting point temperature (melting point = 139 °C). A salt and form selection screen
was conducted on compound 1 (22 different counter ions and co-crystal formers in 4
solvents). The fumarate (co-crystal) was selected as the preferred candidate exhibiting
appropriate properties in terms of stability, solubility, and excipient compatibility. The
melting point of the thermodynamically stable polymorph fumarate co-crystal was 224 °C.
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40
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0
GNF6702
LXE408
0
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Time (min)
Figure 1. Time course of dissolution of 1 (LXE408, fumarate co-crystal) vs 2 (GNF6702)
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In a dissolution study, the fumarate co-crystal of 1 showed a significantly higher
dissolution rate than the free base of 2. Furthermore, previous studies have shown that
compounds with higher super-saturation stability in fasted state simulated intestinal fluid
(FASSIF) display better absorption in the intestine.10 When compared to free base of
compound 2, fumarate co-crystal of compound 1 was found to maintain a 5-fold higher
super-saturation in FASSIF achieving a concentration of 73 M at 16 minutes.11 It should
be noted that no suitable salt or co-crystal form of compound 2 with the right properties
could be identified upon screening with various counter ions. Overall, the data presented
supported our decision to select compound 1 (LXE408) for further characterization.
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To determine efficacy in vivo, compound 1 was administered for 8 days at three dose
levels in a murine model of VL. In mice infected with L. donovani,12 oral dosing (PO) with
1 effected a more pronounced reduction in liver parasite burden than miltefosine, the only
oral anti-leishmanial drug available in clinical practice (Fig. 2a). Dosing compound 1 at 1
mg/kg b.i.d. led to a 95% reduction of parasite burden in the liver, an efficacy equivalent
to that effected by a 12 mg/kg q.d. regimen of miltefosine. This miltefosine regimen
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yielded plasma exposures in mice similar to the miltefosine clinical regimen. The in vivo
efficacy of compound 1 plateaued at the 3 mg/kg b.i.d. dose with no further reduction of
liver parasite burden at the 10 mg/kg b.i.d. dose. The plasma exposure of 1 increased
dose-proportionally, and the free plasma concentration of 1 (mouse plasma protein
binding = 94.4%) at the 3 and 10 mg/kg b.i.d. doses was above the EC50 value of 1 on
intramacrophage L. donovani amastigotes throughout the duration of treatment (Fig. 2b).
This observation is consistent with our previously published PK-PD model for this class
of compounds, which indicated that the proteasome inhibitor efficacy is driven by free
AUC and/ or by time above Cmin.6 Such a PK-PD profile suggests that sustained inhibition
of the parasite proteasome, which results in intracellular accumulation of ubiquitylated
proteins, is required for rapid clearance of L. donovani in vivo.
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Figure 2. a) Compound 1 post-treatment L. donovani liver burdens in murine model of VL
as assessed by qPCR. Circles: burdens in individual mice; thick horizontal lines: means
of the treatment groups; red dotted line: limit of L. donovani detection by qPCR; circles
below the red dotted line: mice with no detectable L. donovani parasites; RU: relative
units. Data points below the limit of detection are ‘jittered’ to show number of animals in
group. b) Time profiles of mean free plasma concentration of compound 1; free compound
1 concentration values were predicted from measured total plasma concentration values
collected on day 8 of treatment. Dashed red line corresponds to intramacrophage L.
donovani EC50 of 40 ± 8.1 nM. Circles: means ± s.d.; n=5 mice; fraction unbound in mouse
plasma=0.056. For data points lacking error bars, standard deviations are smaller than
circles the representing means.
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Next, we tested the in vivo efficacy of 1 in a murine model of cutaneous leishmaniasis
(CL). In BALB/c mice infected with L. major (a model of Old World leishmaniasis), oral
dosing with 1 effected robust healing of parasite-induced skin lesions at the base of the
tail. Administration of 1 for 10 days at a dose of 20 mg/kg b.i.d. produced a therapeutic
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effect comparable to that of liposomal amphotericin B, the most potent anti-leishmanial
drug available today for CL therapy (Fig. 3). Previously, we showed that compound 2
had superior anti-parasitic activity in BALB/c footpad model of L. major infection when
compared to miltefosine.6 Data presented here further extend these findings and
establish 1 as a promising oral agent for treatment of cutaneous leishmaniasis.
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Figure 3. Efficacy of 1 in the BALB/c mouse model of Old World leishmaniasis. Data in
the graph show progression of lesion sizes at the base of the tail of BALB/c mice infected
with 1x107 L. major parasites after 10 days of treatment. Experimental time points
represent the mean lesion sizes ± SEM (n=6). Single-factor ANOVA (Dunnet's test) was
used to determine the statistical significance of differences in lesion sizes between vehicle
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and compound-treated groups (P < 0.05). Starting from day 4 post-treatment, lesion sizes
in the vehicle control group were bigger, and statistically differed from lesion sizes in
compound-treated groups except for the compound 1 at 1 mg/kg b.i.d. dose.
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Compound 1 was profiled in vitro for off-target activity on a panel of receptors including
GPCRs, nuclear receptors and kinases. For VMAT2, 61% inhibition was observed at 10
M concentration, however compound 1 had a low propensity to cross the blood brain
barrier (brain/plasma AUC ratio = 0.03 in mice) suggesting minimal potential modulation
of VMAT2 receptor in the brain. Overall, compound 1 was not considered to pose a risk
of off-target activity at clinically relevant concentrations (see Supporting Information). In
addition, the compound also showed no inhibition of the hERG channel (IC50 > 30 M) in
a manual patch clamp assay indicating a low risk for QT prolongation. Finally, in vitro
AMES and in vivo micronucleus (MNT) studies in rat did not indicate a risk of mutagenicity
or clastogenicity, respectively.
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Pharmacokinetic studies in selected preclinical species (mouse, dog, rat and cynomolgus
monkey) using the fumarate salt (Table 2) revealed that 1 had low clearance (CL, 2-17%
of hepatic blood flow) and low steady-state volume of distribution values (Vss, smaller or
comparable to the volume of total body water) in mouse, rat and monkey following
intravenous (IV) compound administration. However, the CL and VSS values in dog were
in the moderate range (CL: 37% of hepatic blood flow and Vss larger than the total volume
of body water). The plasma terminal half-life of 1 was in the range of 3-10 hours across
species following IV administration. Oral bioavailability was 27-67% across the tested
species when using conventional suspension formulations.
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Table 2. Pharmacokinetic profile of compound 1 in selected preclinical species
PK parameters
CL (mL/min/kg)
Vss (L/kg)
Mousea Ratb Dogc Monkeyd
2.3
0.63
3.3
4.6
7.5
0.8
2.1 11.6
0.53 2.1
0.9
0.5
9.7
9.7
12
T1/2 IV (h)
3.8
4.3
12
3.8
3.3
1.5
0.3
MRT IV(h)
DN AUC PO (hM)
DN Cmax PO (M)
1.0
1.0
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F (%)
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27
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a) Balb/C mice, 5 mg/kg IV and 20 mg/kg PO doses; b) Male Sprague Dawley rat, 3 mg/kg
IV and 10 mg/kg PO doses; c) Male beagle dog, 0.3 mg/kg IV and 1.0 mg/kg PO doses; d)
Male cynomolgus monkey, 0.3 mg/kg IV and 10 mg/kg PO doses. For IV dosing, 1 was
formulated in 75% PEG300/25% D5W vehicle; for PO dosing, 1 was formulated in 0.5%
methylcellulose, 0.2 % Tween 20 vehicle; CL: plasma clearance after administration of a
single IV bolus dose; Vss: volume of distribution at steady-state; DN AUC PO: dose-
normalized area under the plasma concentration-time curve after administration of a
single oral dose; DN Cmax PO: dose-normalized maximum plasma concentration after
administration of a single oral dose; F(%): oral bioavailability after administration of a
single oral dose.
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Figure 4. Compound 1 main oxidative metabolites
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OH
F
F
F
N
N
N
N
N
N
N
N
N
N
N
N
O
O
O
N
N
N
HN
HO
HN
HN
O
O
O
OH
N
N
N
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In order to select the preclinical species for toxicology studies that would reflect most
closely the metabolism of 1 in human, we identified and quantified the most abundant
metabolites in in vitro hepatocytes and in vivo. In vitro, 1 was mainly metabolized to mono-
hydroxylated metabolites 6-8. Metabolite 6 showed a modest activity of 0.5 M on
parasites, whereas the other two metabolites had an activity of greater than 5 M. No
unique metabolites were detected in human hepatocytes. While the metabolism profile of
1 was qualitatively similar across the tested species, we observed species differences in
the extent of formation of individual metabolites. The predominant metabolite in dog
hepatocytes was 8 with minor formation of the other two metabolites (6 and 7). In contrast,
in mouse, rat, monkey, and human hepatocytes, the predominant metabolite was 7 with
minor formation of the other two metabolites (6 and 8).
Table 3. Distribution of metabolites in various preclinical species[a]
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Abundancea
Mouse Rat Dog Monkey Human
1
6
7
8
+++ +++ +++
+++
+
+++
+
+
+
+
+
+
+
+
+
+
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n.d
++
+
+
a Semi-quantitative metabolite profiles were obtained after incubation of 10 μM of 1 for 4 hours
and analysis using UV chromatographic data (250 nm). All integrable peaks were normalized to
the percent area of the largest observed peak.
+++ > 75%; ++ ≤ 75%>25%; + ≤ 25%
The in vivo metabolic profiles of 1 in mouse, rat, dog, and monkey plasma after oral
administration were consistent with the in vitro hepatocytes data (Table 3). After a single
oral dose, the exposure of metabolite 8 was higher than that of 1 in dog plasma (120%),
while it was minor (0.2-5%) in plasma of all the other species. Levels of 6 and 7 were in
moderate or low range in dog plasma (≤ 10% of parent). In mouse, rat and monkey
plasma, 6 was less than 10% relative to the equivalent exposure of 1 and 7 was in the
range of 5-17% of parent exposure. It is worth noting that the aniline resulting from amide
hydrolysis was not detected in vivo. Using a panel of dog recombinant CYP enzymes, we
performed P450 phenotyping studies that demonstrated that 1 was metabolized to 8
predominantly by CYP2D15 in dog. Importantly, compound 1 was not a substrate of
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human CYP2D6, the human ortholog of dog CYP2D15. Human recombinant CYP
phenotyping experiments showed that CYP3A4, CYP1A1, 1A2, and 2J2 were the major
enzymes responsible for the formation of metabolite 8. These data suggested that the
metabolism in dog was not predictive of human metabolism and therefore monkey was
selected as the non-rodent species for toxicological studies.
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Table 4. Percentage of metabolite exposure relative to 1 in preclinical species following oral
administration at 10 mg/kg
Species Metabolites (%)a, b
6
7
8
Mouse
Rat
1.5
1.5
9.1
4.5
17
2.3
11
0.2
1.5
120
5.1
Dog
Monkey 1.7
a)
AUC of metabolites was dose-normalized before relative amounts were calculated. b) The
concentrations were analyzed from experiments described in table 2.
To further characterize compound 1 we wanted to establish its mode of binding to the
proteasome.
Previously published mechanism of action studies indicated that 2 is an inhibitor of the
kinetoplastid proteasome that selectively inhibits the chymotrypsin-like activity of the β5
proteasome subunit (PSMB5). In a biochemical assay, 2 inhibited the chymotrypsin-like
activity in a non-competitive manner when the fluorogenic peptide Suc-LLVY-[Rh110]-
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[Dpro] was used as the substrate. This observation, together with the identified resistant
mutations in the β4 proteasome subunit (PSMB4), pointed to a potential binding site for
2 at the interface of the PSMB4 and PSMB5 subunits.6 However, the exact binding site
of 2 and how the compound binding mode relates to its non-competitive mode of inhibition
remained unanswered.7 To this end, we elucidated the atomic structure of 1 in complex
with the 20S proteasome from L. tarentolae using cryo-EM (Figure 6a). The resultant 3.4Å
resolution structure revealed a clearly defined density for compound 1 (Figure 6b) with 1
situated within the active site pocket of PSMB5 partly formed by the side chains of Ile29
and Phe24 of the PSMB4 subunit (Figure 6c).13 There, the dimethyl-oxazole unit of 1
packs tightly with the two hydrophobic residues. The conformation is analogous to the
one recently published by Wyllie et al.7 and is in agreement with binding profiles from
parasite resistant mutations. First, the 5 Y113F mutation of T. brucei proteasome leads
to loss of activity for compound 1 (Supporting Information; 113 refers to the amino acid
position in the mature 5 subunit) and, secondly the F24L mutation of the L. tarentolae
proteasome exhibits a significant up shift in IC50 value in response to the inhibitor (Figure
5). This is in contrast to the binding curve for bortezomib (Figure 5) which shows no
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reduction in activity in response to the resistant mutation. Bortezomib is a prototypical
peptidomimetic inhibitor of the proteasome, which inhibits proteasome chymotrypsin-like
activity competitively with peptide substrates such as Suc-LLVY-[Rh110]-[Dpro]. This lack
of resistance is in complete agreement with a large number of structural and biochemical
studies of bortezomib in homologous proteasomes.14
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Figure 5. F24L mutation in proteasome β4 subunit confers selective resistance to 1.
Inhibition of chymotrypsin-like activity of purified wild-type (PSMB4WT) and PSMB4F24L L.
tarentolae proteasome by 1 and Bortezomib; IC50 values of chymotrypsin-like activity
are listed inside the plot. Data shown represents the means from one of the
representative experiments (n = 3 technical replicates). RU (relative units) corresponds
to the percentage of chymotrypsin-like activity relative to the DMSO control.
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However, when the structure of the canonical bortezomib binding mode is superimposed
with the structure of the binary complex with 1, steric clashes indicate that 1 and
bortezomib cannot bind to the PSMB5 pocket simultaneously without a conformational
change in the pocket for one or both of the compounds. To understand this at an atomic
level, a Cryo-EM structure of the ternary complex with L. tarentolae proteasome in the
presence of 1 and bortezomib was solved. The resulting proteasome structure, solved at
3.2 Å resolution, clearly revealed that both 1 and bortezomib can occupy the pocket as a
ternary complex with the proteasome. The binding mode of 1 remained unchanged when
compared to the binary complex between the compound and the proteasome. In contrast,
bortezomib adopted a novel conformation with its phenyl group rotated ~60 degrees
around the dihedral angle connection formed by the phenyl with the bortezomib backbone
when compared to the previously published crystal structures of the human proteasome
in complex with bortezomib (Figure 7a and 7b).13 Overall, these findings provide atomic
level resolution details of the binding of 1 to the kinetoplastid proteasome and explain its
non-competitive mode of chymotrypsin-like activity inhibition.
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Figure 6. Binding of 1 to L. tarentolae proteasome. a) Positions of the two compound 1
molecules in 5 subunit of proteasome, related 5 domains colored cyan, 4 subunits
colored green, Compound 1 carbon atoms colored as yellow spheres, b) Electron density
of the 3.4 Å structure around compound 1 (at 3 sigma deviations above mean. EMDB and
PDB code: EMD-10472, PDB ID 6TCZ. c) Key interactions around compound 1, with
resistance mutants in 4 subunit colored as orange sticks.
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Figure 7. a) Final density map of the ternary complex of compound 1 and bortezomib bound to
the β5 pocket. The map is displayed as a blue isomesh contoured at 3.0 σ above the mean and for
clarity is displayed only around the ligands at a radius of 2.0 Å. The β5 chain is colored green in a
cartoon representation, 1 and bortezomib are represented as sticks with carbons colored as
yellow and beige respectively. b) Rotation of bortezomib when competing with 1,
bortezomib in beige, 1 in yellow, relative to bortezomib. Leishmania tarentolae
proteasome 20S subunit complexed with 1 and bortezomib accession code(s): EMD-
10463, PDB ID: 6TD5.
Chemical Synthesis of Compound 1
Scheme 1 outlines the research synthesis of compound 1 that was achieved in 8 steps with an
overall 8% yield. The synthesis was initiated from commercially available 2-fluoro-5- nitrobenzoic
acid 9, which upon treatment with thionyl chloride was converted to acid chloride. The excess
thionyl chloride was removed under reduced pressure to be used for next step. The crude acid
chloride was re-suspended in toluene and the intermediate was treated with aminoguanidine
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carbonate for 12 h, which yielded 10 as a solid. Amino guanidine intermediate 10 was stirred in
water at 100 °C for 8 h to yield the amino-triazole 11 in 51% yield. Triazole 11 was condensed
with bromomalonaldehyde and the resulting nitro compound was further reduced with Raney
nickel in the presence of zinc iodide to afford the key intermediate 12. The resulting amide from
coupling of aromatic amine 12 with 2,4-dimethyl oxazole carboxylic acid was further
functionalized to boronic ester using a Miyaura Borylation reaction.15 Finally, Suzuki coupling
between the boronic acid and 2-bromo-methylpyridine gave the desired compound 1.
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NO2
NO2
N
O2N
N
NH
NH2
a, b
NH2
H
N
c
N
OH
H
N
F
H
F
O
F
O
9
10
11
F
H2N
Br
N
N
N
N
N
N
N
N
f, g, h
O
d,e
N
N
NH
F
O
12
1
Scheme 1. Synthesis of Compound 1a
aReagents and conditions. (a) SOCl2, 80 °C, 4 h, 98% (b) aminoguanidine carbonate, toluene, room temp.
12 h, 97% (c) H2O, AcOH-carboxylic acid, HATU, DIEA, DMF, 0.5 h, 53% (g) 4,4,4',4',5,5,5',5'-
octamethyl-2,2'-bi (1,3,2-dioxaborolane), Pd(dppf)Cl2, KOAc, dioxane, 100 °C, 16 h, 24% (h) 2-bromo-
3-methylpyridine, Pd(dppf)Cl2, 1M, aq. Na2CO3, 80 °C, 16 h, 39%
CONCLUSIONS
The current target product profile for a new antileishmanial drug is to be safe, effective,
oral with a short course of treatment (< 11 days).16 Optimization of GNF6702 successfully
led to compound 1 (LXE408) which when dosed orally, demonstrates excellent efficacy
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in mouse disease models and has an appropriate safety profile in preclinical species.
Applying the PK-PD principles, we expect that a dose of 85-190 mg per day will effect
cure in VL patients. Currently, phase I studies in healthy human volunteers are ongoing
to determine safety and tolerability. The compound will most likely be dosed in
combination with other agent once the safety and efficacy is established in humans as a
single agent. Cryo-EM efforts led to a detailed understanding of the mechanism of
inhibition of chymotrypsin activity by 1 and elucidated a novel mode of binding. The
discovery of the non-competitive specific 5 proteasome inhibition and further
understanding of the binding mode of 1 could prove to be transformative in the fields of
infectious diseases and oncology where more subunit-selective proteasome inhibitors are
being sought.17, 18
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EXPERIMENTAL SECTION
Materials and methods:
Commercially available starting materials were used as supplied without further purification.
Reactions were carried out by using dry organic solvents (DCM, ACN, DMF, etc.) unless
otherwise noted. Reactions were monitored using thin layer chromatography and an Agilent
Technologies 1200 series 6140 Quadrupole LC-MS with UV detection at 254 nm in electrospray
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ionization (ESI) mode. For LC-MS, all retention times reported are at 254 nm UV channel unless
otherwise noted. All NMR spectra were recorded on a Bruker AVANCE-400 spectrometer
7
8
9
1
13
operating at a frequency of 400.13 MHz for H and 100.61 MHz for C equipped with a 5 mm
QNP cryoprobe with Z-gradient. Chemical shifts for 1H and 13C spectra were referenced to residual
solvent. MS were obtained on an Agilent Technologies 1200 series 6140 quadrupole LC-MS in
electrospray ionization (ESI) mode. HRMS-ESI data were recorded using an Agilent 6520
Accurate-Mass Q-TOF LC-MS system with HPLC-Chip Cube interface and an Agilent 1200
HPLC. All final compounds were isolated analytically pure, >99% purity by HPLC unless
otherwise indicated. More information can be found in the Supporting Information.
10
11
12
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2-(2-fluoro-5-nitrobenzoyl)hydrazine-1-carboximidamide (10)
A solution of 2-fluoro-5-nitrobenzoic acid (50 g, 270 mmol) in thionyl chloride (100 mL) was
heated to 80°C and stirred for 4 hours. The mixture was allowed to cool down to room temperature
and the solvent was removed to give acid chloride intermediate (54 g, 98% yield).
To a solution of aminoguanidine carbonate (36.2 g, 266 mmol) in dry toluene (300 mL) cooled to
0°C was added the above acid chloride (54 g, 266 mmol) over 30 minutes. The mixture was stirred
at room temperature for 12 hours. The formed precipitate was removed by filtration, and the
residue was treated with H2O (400 mL) and made alkaline with sodium carbonate. The solid was
collected and recrystallized from water to obtain compound 10 (62 g, 97% yield). LCMS: [ESI]
m/z = 241.1 [M+H]+. The synthesis of the compound has been described previously and is
commerically available.
5-(2-fluoro-5-nitrophenyl)-4H-1,2,4-triazol-3-amine (11)
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A solution of compound 10 (62 g, 257 mmol) in H2O (800 mL) was stirred for 8 hours at 100°C.
After cooling, the solid obtained was filtered, and the cake was washed with H2O (100 mL), THF
(100 mL), and dried to give 11 (34 g, 51% yield). 1H NMR (400 MHz, DMSO) 12.42 (s, 1H), 8.74
(dd, J = 6.27 Hz, 3.0 Hz, 1H), 8.26 (dt, J = 7.0 Hz, 3.4 Hz, 1H), 7.57 (t, J = 9.5 Hz, 1H), 6.29 (s,
10
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13
2H). C NMR (101 MHz, DMSO) δ 164.34, 161.71, 157.72, 144.42, 144.39, 125.76, 125.66,
125.25, 125.20, 118.85, 118.61. HRMS found [M+H]+ = 224.0578, C8H7O2N5F predicted [M+H]+
= 224.0578.
3-(6-bromo-[1,2,4]triazolo[1,5-a]pyrimidin-2-yl)-4-fluoroaniline (12)
To a solution of compound 11 (10 g, 44.8 mmol) in acetic acid (50 mL) was added 2-
bromomalonaldehyde (8.12 g, 53.8 mmol) at room temperature. The mixture was heated
to 100 °C and stirred for 4 hours. The mixture was allowed to cool to room temperature
before adding water (100 mL) and filtering. The filter cake was then washed with THF,
dried under vacuum to give compound 6-bromo-2-(2-fluoro-5-nitrophenyl)-
[1,2,4]triazolo[1,5-a]pyrimidine (6.5 g, 43% yield). 1H NMR (400 MHz, DMSO) 10.04 (s, 1H),
13
9.08 (s, 1H), 8.96-9.04 (m, 1H), 8.47 (dt, J = 9.0 Hz, 3.4 Hz, 1H), 7.76 (t, J = 9.5 Hz, 1H). C
NMR (101 MHz, DMSO) δ 165.01, 162.36, 160.38, 157.47, 154.11, 144.68, 138.27, 138.09,
128.47, 128.36, 126.51, 126.46, 119.54, 119.36, 119.30, 107.08. HRMS found [M+H]+ =
337.9688, C11H6O2N5BrF predicted [M+H]+ = 337.9683.
To a solution of compound 6-bromo-2-(2-fluoro-5-nitrophenyl)-[1,2,4]triazolo[1,5-
a]pyrimidine (6g, 17.7 mmol) in THF (150 mL) was added Raney Nickel (7 g) and ZnI2
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(2.26 g, 7.1 mmol), the suspension was degassed under vacuum and the mixture was
stirred under H2 (50 psi) at 25°C for 2 hours. The mixture was filtered and the solvents
removed to give the crude product. The crude product was washed with MeOH (50 mL x
2) and dried under vacuum to give compound 12 (4.2 g, 77% yield).1H NMR (400 MHz,
DMSO): 9.93 (d, J = 2.3 Hz, 1H), 8.98 (d, J = 2.0 Hz, 1H), 7.39-7.45 (m, 1H), 6.99-7.10
10
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13
(m, 1H), 6.67-6.76 (m, 1H), 5.76 (s, 1H), 5.25 (brs, 2H). C NMR (101 MHz, DMSO) δ
163.08, 156.52, 154.05, 153.74, 151.32, 145.84, 137.87, 118.03, 117.91, 117.80, 117.73,
117.56, 117.33, 114.86, 106.16. HRMS found [M+H]+ = 307.9941, C11H8O2N5BrF
predicted [M+H]+ = 307.9942.
N-(4-fluoro-3-(6-(3-methylpyridin-2-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-2-yl)phenyl)-2,4-
dimethyloxazole-5-carboxamide (1)
Step F: To a solution of 2,4-dimethyloxazole-5-carboxylic acid (1.92 g, 13.6 mmol) in DMF
(50 mL) was added HATU (6.2 g, 16.32 mmol) and DIEA (3.5 g, 27.2 mmol) at room
temperature. The mixture was stirred for 30 mins, and the compound 12 (4.2 g, 13.6
mmol) was added. The mixture was stirred for 3 hours, water (100 mL) was added. The
mixture was filtered, the filter cake was washed with H2O (50 mL x 2), THF (50 mL x 2)
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and dried to give N-(3-(6-bromo-[1,2,4]triazolo[1,5-a]pyrimidin-2-yl)-4-fluorophenyl)-2,4-
dimethyloxazole-5-carboxamide (3.1 g, 53%). 1H NMR (400 MHz, DMSO) 10.41 (s, 1 H),
9.97 (s, 1H), 9.02 (brs, 1H), 8.75 (s, 1H), 7.93 (s, 1H), 7.40 (t, J = 9.66 Hz, 1H), 2.50 (s,
3H), 2.39 (s, 3H). 13C NMR (101 MHz, DMSO) δ 162.29, 161.49, 156.86, 156.78, 154.16,
143.51, 139.17, 138.05, 135.52, 124.95, 122.80, 117.67, 117.44, 106.48, 14.29, 13.26.
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HRMS found [M+H]+ = 431.0259, C17H13O2N6BrF predicted [M+H]+= 431.0262.
Step G: To a solution of compound N-(3-(6-bromo-[1,2,4]triazolo[1,5-a]pyrimidin-2-yl)-4-
fluorophenyl)-2,4-dimethyloxazole-5-carboxamide (3.0 g, 7.0 mmol) in dioxane (60 mL)
was added 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bi (1,3,2-dioxaborolane) (7.1 g, 28 mmol),
[1,1′-Bis(diphenylphosphino)ferrocene]
dichloropalladium(II),
complex
with
dichloromethane (0.5 g, 0.7 mmol) and KOAc (2.0 g, 21 mmol) under N2. This mixture
°
was heated to 100 C for 16 hours. The solvent was removed and the compound re-
dissolved in THF and filtered through silica gel. The filtrate was concentrated and
triturated with MTBE to give compound (2-(5-(2,4-dimethyloxazole-5-carboxamido)-2-
1
fluorophenyl)-[1,2,4]triazolo[1,5-a]pyrimidin-6-yl)boronic acid (0.67 g, yield, 24%). H NMR:
DMSO 400MHz : 2.40 (s, 3 H), 2.51 (s, 3 H), 7.38 - 7.43 (m, 1 H), 7.92 - 7.96 (m, 1 H), 8.72 -
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