Notum is a carboxylesterase that suppresses Wnt signaling through deacylation of an essential palmitoleate group on Wnt proteins. There is a growing understanding of the role Notum plays in human diseases such as colorectal cancer and Alzheimer's disease, supporting the need to discover improved inhibitors, especially for use in models of neurodegeneration. Here, we have described the discovery and profile of 8l (ARUK3001185) as a potent, selective, and brain-penetrant inhibitor of Notum activity suitable for oral dosing in rodent models of disease. Crystallographic fragment screening of the Diamond-SGC Poised Library for binding to Notum, supported by a biochemical enzyme assay to rank inhibition activity, identified 6a and 6b as a pair of outstanding hits. Fragment development of 6 delivered 8l that restored Wnt signaling in the presence of Notum in a cell-based reporter assay. Assessment in pharmacology screens showed 8l to be selective against serine hydrolases, kinases, and drug targets.
Notum is a carboxylesterase that suppresses Wnt signaling through deacylation of an essential palmitoleate group on Wnt proteins. There is a growing understanding of the role Notum plays in human diseases such as colorectal cancer and Alzheimer's disease, supporting the need to discover improved inhibitors, especially for use in models of neurodegeneration. Here, we have described the discovery and profile of 8l (ARUK3001185) as a potent, selective, and brain-penetrant inhibitor of Notum activity suitable for oral dosing in rodent models of disease. Crystallographic fragment screening of the Diamond-SGC Poised Library for binding to Notum, supported by a biochemical enzyme assay to rank inhibition activity, identified 6a and 6b as a pair of outstanding hits. Fragment development of 6 delivered 8l that restored Wnt signaling in the presence of Notum in a cell-based reporter assay. Assessment in pharmacology screens showed 8l to be selective against serine hydrolases, kinases, and drug targets.
The
Wnt signaling pathway is a complex, evolutionarily conserved
system from Drosophila and Caenorhabditis elegans through to mammals, which
plays key roles in both embryonic development and the adult animal.
These roles include cell fate determination and maintenance, and cell
division in many different tissues.[1] The
“canonical Wnt signaling pathway” is characterized by
the secreted activator (or agonist) Wnt proteins, which initiate an
intracellular signaling cascade by binding to the cell surface receptor
(a member of the Frizzled family of cell surface proteins, complexed
with the coreceptor LDL-receptor-related protein family 5/6).[1] The intracellular signaling cascade utilizes
β-catenin and GSK3β to activate gene transcription via
T-cell factor (TCF)/lymphoid enhancing factor (LEF) transcription
factors. This signaling system is tightly regulated by a complex network
of modulators and feedback inhibitory proteins, including the family
of dickkopf (Dkk) proteins[2] and the carboxylesterase
Notum.[3,4] Given the pleiotropic and essential function
of Wnt signaling, it is unsurprising that aberrant Wnt signaling can
contribute to disease in humans, including certain cancers, diabetes,
and osteoporosis.[1,5] In the mammalian brain, Wnt signaling
performs several important roles, including the maintenance of synapse
function,[6] modulating microglial signaling
and survival,[7] neurogenesis,[8] and maintaining the integrity and function of
the blood–brain barrier (BBB).[9,10] In Alzheimer’s
disease (AD), when the Wnt signaling tone is disrupted by an increase
in the inhibitory Dkk proteins, this leads to synapse loss, which
may contribute to cognitive impairment.[11] In stroke, loss of Wnt signaling leads to BBB breakdown, contributing
to the pathology.[12,13] In a model of multiple sclerosis,
reactivation of Wnt signaling at the BBB partially restores functional
BBB integrity and limits immune cell infiltration into the CNS.[14] Therefore, augmenting Wnt signaling in these
disorders of the central nervous system (CNS) has therapeutic potential.Notum is a negative regulator of the Wnt signaling pathway. It
is a secreted protein that functions as a carboxylesterase, removing
a palmitoleate ester from a conserved serine residue in Wnt proteins
that is essential for its binding and activation of the cell surface
coreceptors.[3] While first identified in Drosophila, its function in mammals, in both healthy
and diseased, is being increasingly explored. The key tools to enable
this progress are fit-for-purpose small-molecule inhibitors of Notum
carboxylesterase activity.[15,16]First-generation
inhibitors of Notum activity were LP-922056 (1)[17] and the irreversible inhibitor
ABC99 (2)[18] that have proved
to be valuable research tools in exploring the link between pharmacological
inhibition of Notum activity and disease association in rodent models
(Figure ). LP-922056
is a potent inhibitor of Notum suitable for chronic, oral dosing in
rodents discovered through the optimization of a high-throughput screening
(HTS) hit.[17] LP-922056 was used to show
that inhibition of Notum activity is a potential novel anabolic therapy
for strengthening the cortical bone and preventing non-vertebral fractures.[19] Notum has recently been identified as a key
mediator for people at high risk of developing colorectal cancer.[20,21] Pharmacological inhibition of Notum with LP-922056 abolished the
ability of Apc-mutant cells to expand and form intestinal
adenomas.[21] ABC99 is a potent and selective
covalent inhibitor of Notum activity developed by competitive activity-based
protein profiling (ABPP) from a library of activated carbamates.[18] Inhibition of Notum produced by Paneth cells
in mice with ABC99 restored the regeneration of aged intestinal epithelium.[22] In the brain, Notum regulates neurogenesis in
the subventricular zone (SVZ) by modulating Wnt signaling, and inhibition
of Notum with ABC99 leads to an activation of Wnt signaling and increased
proliferation in the SVZ.[8]
Figure 1
First-generation Notum
inhibitors used in disease association studies.
First-generation Notum
inhibitors used in disease association studies.A number of structurally distinct Notum inhibitors have been reported
from hit-to-lead programs (Figure ).[23−27] However, most of these inhibitors have limited brain penetration
as directly measured in mouse pharmacokinetic (PK) studies;[24,26−28] this restricts their use to peripheral (non-CNS)
indications. Only ABC99 has been reported to reach the brain and activate
the Wnt pathway as assessed by qRT-PCR of Axin2 in
the ventricular-SVZ tissue following an intraperitoneal (i.p.) injection
to Nestin-CFP mice.[8]
Figure 2
Representative
chemical structures of Notum inhibitors.
Representative
chemical structures of Notum inhibitors.Our objective was to identify a screening hit that could be optimized
to a potent, selective, and brain-penetrant inhibitor of Notum activity
suitable for chronic, oral delivery. Such an inhibitor would complement
LP-922056 and ABC99 as it could be administered in rodent models of
neurodegenerative disease.Fragment-based drug discovery (FBDD)
is an established method for
hit finding of biologically active compounds and is now supported
by multiple complementary screening technologies.[29,30] The concept of a “fragment” has essentially remained
consistent with an emphasis on small, low-molecular-weight organic
molecules with high aqueous solubility as described by Congreve et
al.[31] In contrast, there is an increase
in the application of FBDD to more challenging, non-traditional targets
such as GPCRs and ion channels, and to phenotypical screens including
parasites.[29] The creation of fragment libraries
with a clearly defined purpose beyond chemical diversity has also
received considerable attention. Examples of such libraries include
reactive covalent fragments,[32] 3D fragments,[33] natural product-like compounds,[34] very small fragments such as FragLites and Minifrags,[35] halogen-enriched fragments,[36] and synthetically poised fragment libraries.[37]The original Diamond-SGC Poised Library
(DSPL) was created by Cox
et al. as a 768-membered fragment library that was designed to allow
rapid follow-up synthesis to provide quick structure–activity
relationships (SARs) around fragment screening hits. Each member of
the collection contained at least one functional group that could
be created with reliable chemical reactions, that is, the fragment
library was created to be structurally diverse and synthetically enabled.[37,38]Herein, we describe the discovery of crystallographic fragment
screening hits 6a and 6b from the DSPL and
their optimization to identify 1-phenyl-1,2,3-triazoles as a new Notum
inhibitor chemotype. We present the profile of lead compound 8l that supports it to be a potent, selective, and brain-penetrant
inhibitor of Notum activity.
Results and Discussion
In order
to find hit compounds for the development of new Notum
inhibitors, we chose to conduct a crystallographic fragment screen.
The structure of Notum has a clearly defined lipophilic pocket adjacent
to the catalytic triad that accommodates a C16-lipid palmitoleate
group of Wnt.[3,39] The size and topography of this
pocket gave encouragement that suitable fragments could be found to
bind as the pocket has been assessed to be classically druggable.[40]An X-ray crystallography-based fragment
screen was performed at
the XChem platform of Diamond Light Source (Oxford, U.K.). The highly
automated XChem platform, in combination with synchrotron beamline
I04-1, enabled us to efficiently screen 768 compounds from the DSPL
fragment library for binding to Notum. Compounds were individually
soaked into crystals of Notum and high-resolution structures were
determined for each of these putative Notum–fragment complexes.
The electron density maps provided evidence of compound binding associated
with the active site of Notum for 59 of the crystal structures (hit
rate: 7.7%).[23,26,41] The complete output of the hits from this crystallographic screen
with the DPSL will be published separately.[42,43] Hit validation was then performed by demonstration that these binders
were able to inhibit Notum carboxylesterase activity in a biochemical
assay (up to 1 mM).A pair of outstanding hits were 4-(hydroxymethyl)triazoles 6a (IC50 12 μM)[26] and 6b (IC50 0.5 μM), which also highlighted
some early beneficial SAR (Figure ). A 3,4-Cl2 disubstituted phenyl ring conferred
a substantial improvement in Notum inhibition when compared to a single
4-Cl (23-fold). Both the Notum-6a and -6b X-ray structures showed that the ligand formed a H-bond between
N3 of the triazole with the backbone of Trp128. The increase in potency
of 6b over 6a can be attributed to the improved
occupancy of the palmitoleate pocket by the 3,4-Cl2 disubstituted
phenyl ring (Figures and S1). Specific interactions in the
pocket include the potential stacking of the ligand with Trp128, Tyr129,
and Phe268 as well as interactions by the phenyl ring substituents
with Phe320.
Figure 3
Preferred hits 6a and 6b from
the crystallographic
screen of the DSPL fragment library.
Figure 4
(A) Structure
of 6a (PDB: 6ZUV). N3 of the triazole forms a hydrogen
bond with the backbone of Trp128 (dashed line, 2.36Å) (ref (26)). (B) Structure of 6b (PDB: 7B89). The compound forms a hydrogen bond with the backbone of Trp128
(dashed line, 2.21 Å). Select binding site residues are shown.
Water molecules have been removed for clarity. The surface of the
Notum binding pocket is outlined (gray).
Preferred hits 6a and 6b from
the crystallographic
screen of the DSPL fragment library.(A) Structure
of 6a (PDB: 6ZUV). N3 of the triazole forms a hydrogen
bond with the backbone of Trp128 (dashed line, 2.36Å) (ref (26)). (B) Structure of 6b (PDB: 7B89). The compound forms a hydrogen bond with the backbone of Trp128
(dashed line, 2.21 Å). Select binding site residues are shown.
Water molecules have been removed for clarity. The surface of the
Notum binding pocket is outlined (gray).Triazole 6b has molecular and physicochemical properties
consistent with lead-like chemical space[44] (MW 2444; clogP, 2.2; LogD7.4, 2.4;
TPSA 51 Å2) and scored high when assessed by design
metrics (LE 0.59; LLE 4.1),[45,46] including a favorable
prediction of brain penetration (CNS MPO 5.6/6.0; BBB Score 4.9/6.0).[47,48] Triazole 6b was further evaluated in standard in vitro
ADME assays and showed good aqueous solubility, moderate stability
in liver microsomes, and excellent cell permeability (Table ).
Table 1
Notum Inhibition
SAR of Substituted
(1-Phenyl-1H-1,2,3-triazol-4-yl)methanols 6 and 7
All values are
mean ± s.d.
of n = 2–13 experiments quoted to 2 s.f.
40–50% I@10 μM.
40–50% I@100 μM.
In vitro ADME data for 6b: LogD7.4 2.4; aqueous solubility, 100 μg/mL;
mouse
liver microsomes (MLM), Cli 88 μL/min/mg protein;
human liver microsomes (HLM), Cli 12 μL/min/mg protein;
MDCK-MDR1, AB/BA Papp 57/59 × 10–6 cm s–1; efflux ratio (ER), 1.0.
In vitro ADME data for 7y: aqueous solubility, 210 μg/mL; MLM, Cli 6.7 μL/min/mg
protein; mouse hepatocytes, Cli 48 μL/min/106 cells; MDCK-MDR1, AB/BA Papp 46/28
× 10–6 cm s–1; ER, 0.61.
All values are
mean ± s.d.
of n = 2–13 experiments quoted to 2 s.f.40–50% I@10 μM.40–50% I@100 μM.In vitro ADME data for 6b: LogD7.4 2.4; aqueous solubility, 100 μg/mL;
mouse
liver microsomes (MLM), Cli 88 μL/min/mg protein;
human liver microsomes (HLM), Cli 12 μL/min/mg protein;
MDCK-MDR1, AB/BA Papp 57/59 × 10–6 cm s–1; efflux ratio (ER), 1.0.In vitro ADME data for 7y: aqueous solubility, 210 μg/mL; MLM, Cli 6.7 μL/min/mg
protein; mouse hepatocytes, Cli 48 μL/min/106 cells; MDCK-MDR1, AB/BA Papp 46/28
× 10–6 cm s–1; ER, 0.61.Hits 6 were an
attractive starting point to improve
Notum activity and develop PK properties for oral dosing to achieve
good plasma exposure and brain penetration.The optimization
of 6 to deliver this profile was
directed at three principle areas of these hit structures: (1) aryl
ring 7 (Table ); (2) the C4 substituent on triazole 8 (Table ); and (3) further
fine-tuning of the triazole 9 (Table ). Inhibition of Notum activity was routinely
measured in a biochemical assay with human Notum (81–451 Cys330Ser)
as the enzyme and trisodium 8-octanoyloxypyrene-1,3,6-trisulfonate
(OPTS) as the substrate.[23−26] This assay was used to establish SAR and to identify
preferred leads for additional studies. As Notum activity was developed,
representative examples were screened in standard in vitro ADME assays
to assess their suitability for progression to mouse PK studies.
Table 2
Notum Inhibition SAR of Substituted
1-(2,4-Dichloro-3-(trifluoromethyl)phenyl)-1H-1,2,3-triazoles 8
All values are
mean ± s.d.
of n = 2–6 experiments quoted to 2 s.f.
n > 20.
Table 3
Notum Inhibition,
Aqueous Solubility,
Microsomal Stability, and Cell Permeability of 7y, 8l, and 9
compound
7y
8l
9a
9b
9c
d2-8l
Notum IC50 (nM)a
9.5 ± 0.2
6.7 ± 1.6
1300 ± 210
140 ± 20
7.5 ± 1.6
4.6 ± 1.4
aq. solubility (μg/mL)/(μM)
210/680
68/240
NDb
ND
400/1400
44/155
MLM/HLM Cli (μL/min/mg protein)
6.7/1.7
3.2/1.5
ND
ND
4.9/ND
5.4/ND
MDCK-MDR1
AB/BA Papp (×10–6 cm s–1)
46/28
31/39
ND
ND
2/5
48/35
ER
0.61
1.25
2.5
0.73
All values are mean ± s.d.
of n = 3–6 experiments quoted to 2 s.f.
ND, not determined.
All values are
mean ± s.d.
of n = 2–6 experiments quoted to 2 s.f.n > 20.All values are mean ± s.d.
of n = 3–6 experiments quoted to 2 s.f.ND, not determined.As before, trusted design metrics
were calculated to aid compound
design and assess progress, supported by structure-based drug design
(SBDD) through inhibitor docking and X-ray structures. However, a
significant change in our strategy was to discount any weakly acidic
functional groups or heterocycles as these had failed to give sufficient
brain penetration in mouse PK experiments (e.g., 4 and 5).[26,27] Instead, lead compounds would
need to be non-ionized, or weakly basic, at physiologically relevant
pH to be advanced through the screening sequence.General synthetic
methods to prepare inhibitors 7–9 (Tables –3) are shown in Schemes –5. Our initial efforts
focused on exploration of the aryl ring SAR, thus a reliable synthesis
of substituted (1-aryl-1H-1,2,3-triazol-4-yl)methanols 7 was required (Scheme ). Target compounds 7 were readily prepared in
two steps from commercially available starting materials. Either the
prerequisite boronic acid 10 or aniline 11 was converted to the required aryl azide 12 and then
subsequent copper(I)-catalyzed azide–alkyne cycloaddition (CuAAC)
with propargyl alcohol afforded the desired triazoles 7.
Scheme 1
Preparation of (1-Phenyl-1H-1,2,3-triazol-4-yl)methanols 7
Representative reagents and conditions:
(a) NaN3 (2.0 equiv), CuSO4·5H2O (0.2 equiv), MeOH, 40 °C, 5 h; (b) (i) NaNO2 (1.2
equiv), CF3CO2H, 0 °C → RT, 1.5
h; (ii) H2O, RT → 0 °C, (iii) NaN3 (1.1 equiv), 0 °C → RT, 1 h; (c) HC≡CCH2OH (1.0 equiv), sodium l-ascorbate (0.4 equiv), CuSO4·5H2O (0.2 equiv), BuOH-H2O, 50 °C, 2 h.
Scheme 5
Preparation of (1,2,3-Triazol-1-yl)pyridine 9c
Reagents and conditions: (a)
(i) NH2NH2·H2O (1.5 equiv),
EtOH, 85 °C, 4 h; (ii) HCl, RT → 0 °C, NaNO2 (2.0 equiv), 30 min; (iii) NaOH, 0 °C → RT; (b) sodium l-ascorbate (1.0 equiv), CuSO4·5H2O (0.1 equiv), HC≡CSiMe3 (1.0 equiv), BuOH-H2O, 40 °C, 16 h, 2%.
Preparation of (1-Phenyl-1H-1,2,3-triazol-4-yl)methanols 7
Representative reagents and conditions:
(a) NaN3 (2.0 equiv), CuSO4·5H2O (0.2 equiv), MeOH, 40 °C, 5 h; (b) (i) NaNO2 (1.2
equiv), CF3CO2H, 0 °C → RT, 1.5
h; (ii) H2O, RT → 0 °C, (iii) NaN3 (1.1 equiv), 0 °C → RT, 1 h; (c) HC≡CCH2OH (1.0 equiv), sodium l-ascorbate (0.4 equiv), CuSO4·5H2O (0.2 equiv), BuOH-H2O, 50 °C, 2 h.
Preparation of 1,2,3-Triazoles 8a–e, 8j
Representative reagents and conditions:
(a) (i) NaNO2 (2.0 equiv), aq. HCl, MeCN, RT, 1 h; (ii)
RT → 0 °C, NaN3 (2.0 equiv), 0 °C, 1 h;
or (i) NaNO2 (1.2 equiv), CF3CO2H,
0 °C → RT, 1.5 h; (ii) RT → 0 °C, H2O, NaN3 (1.1 equiv), 0 °C → RT, 1 h; (b) RC≡CH
(13) (1.0 equiv), sodium l-ascorbate (0.4 equiv),
CuSO4·5H2O (0.2 equiv), MeOH-BuOH-H2O, 40 °C, 16 h.
Preparation of 1,2,3-Triazoles 8f–i, 8k–l
Reagents and conditions: (a)
(i) NaNO2 (2.0 equiv), aq. HCl, MeCN, RT, 1 h; (ii) RT
→ 0 °C, NaN3 (2.0 equiv), 0 °C, 1 h; (iii)
HC≡CCO2Me (1.0 equiv), sodium l-ascorbate
(0.4 equiv), CuSO4·5H2O (0.2 equiv), MeOH-BuOH-H2O, 40 °C, 16 h, 71%;
(b) (i) aq. NaOH (2.0 equiv), MeOH, RT, 1 h; (ii) aq HCl, 95%; (c)
NH3, MeOH, 70 °C, 3 h, 46%; (d) MeNH2,
MeOH, 70 °C, 3 h, 64%; (e) NaNO2 (1.2 equiv), CF3CO2H, 0 °C → RT, 1.5 h; (ii) RT →
0 °C, aq. NaN3 (1.1 equiv), 0 °C → RT,
1 h; (iii) aq. NaOH; (iv) HC≡C-CH2NHBoc (1.0 equiv),
sodium l-ascorbate (0.4 equiv), CuSO4·5H2O (0.2 equiv), BuOH-H2O, 50 °C, 16 h, 8%; (f) HCl (excess), dioxane, 50 °C, 16
h, quant.; (g) (i) NaNO2 (1.2 equiv), CF3CO2H, 0 °C → RT, 1.5 h; (ii) RT → 0 °C,
H2O, NaN3 (1.1 equiv), 0 °C → RT,
1 h; (iii) sodium l-ascorbate (0.4 equiv), CuSO4·5H2O (0.2 equiv), HC≡CCH2SiMe3 (1.0 equiv), BuOH-H2O, 50 °C, 16 h; (h) (i) MeOH, K2CO3 (10
equiv), reflux, 2%; (ii) TBAF (2.0 equiv), THF, RT, 16 h; (j) (i)
NaNO2 (4.7 equiv), CF3CO2H, 0 °C
→ RT, 1 h; (ii) RT → 0 °C, NaN3 (4.7
equiv), 0 °C → RT, 16 h; (iii) RT → 0 °C,
aq. NaOH; (iv) HC≡CSiMe3 (2.0 equiv), sodium l-ascorbate (0.9 equiv), CuSO4·5H2O (0.5 equiv), MeOH-BuOH-H2O, RT, 16 h; (k) MeOH, K2CO3 (10 equiv), RT,
24 h, 67%; (l) MeOH-d4, D20,
K2CO3 (3.0 equiv), RT, 3 days, 67%.
Preparation of 1,2,4-Triazole 9a and
1,2,3,4-Tetrazole 9b
Reagents and conditions:
(a)
ClSiMe3 (15 equiv), 1,2-diformylhydrazine (3.0 equiv),
Et3N, pyridine, 0 °C → 130 °C, 20 h, 50%;
(b) HC(OMe)3 (3.0 equiv), NaN3 (3.1 equiv),
AcOH, 100 °C, 1.5 h, 10%.
Preparation of (1,2,3-Triazol-1-yl)pyridine 9c
Reagents and conditions: (a)
(i) NH2NH2·H2O (1.5 equiv),
EtOH, 85 °C, 4 h; (ii) HCl, RT → 0 °C, NaNO2 (2.0 equiv), 30 min; (iii) NaOH, 0 °C → RT; (b) sodium l-ascorbate (1.0 equiv), CuSO4·5H2O (0.1 equiv), HC≡CSiMe3 (1.0 equiv), BuOH-H2O, 40 °C, 16 h, 2%.This approach (11 → 12 → 7) proved to be versatile and reliable and
was applied to
the synthesis of a number of inhibitors. 2,4-Dichloro-3-(trifluoromethyl)aniline
(11a) was converted to the corresponding azide 12a, and reaction with the appropriate 1-alkyne 13 under CuAAC conditions gave a variety of 4-substituted 1,2,3-triazoles
(8, 14–17) (Schemes and 3). A number
of inhibitors were prepared by functional group conversion and/or
protecting group strategies. Methyl ester 14 was converted
to the acid 8f by base hydrolysis, and to amides 8g and 8h by amination with NH3 and
MeNH2, respectively. Acid deprotection of the Boc group
from 15 gave methyl amine 8i, and K2CO3/TBAF deprotection deprotection of 16 furnished 4-methyl triazole 8k. CuAAC-promoted [2 +
3] cyclization of 12a with Me3SiC≡CH
gave the corresponding 4-TMS triazole 17, and desilylation
with K2CO3/MeOH afforded 8l. Desilylation
of 17 with CD3OD and D2O as the
solvents promoted deuteration at the C4 and C5 positions of the triazole
and afforded d2-8l with high-isotope
incorporation.
Scheme 2
Preparation of 1,2,3-Triazoles 8a–e, 8j
Representative reagents and conditions:
(a) (i) NaNO2 (2.0 equiv), aq. HCl, MeCN, RT, 1 h; (ii)
RT → 0 °C, NaN3 (2.0 equiv), 0 °C, 1 h;
or (i) NaNO2 (1.2 equiv), CF3CO2H,
0 °C → RT, 1.5 h; (ii) RT → 0 °C, H2O, NaN3 (1.1 equiv), 0 °C → RT, 1 h; (b) RC≡CH
(13) (1.0 equiv), sodium l-ascorbate (0.4 equiv),
CuSO4·5H2O (0.2 equiv), MeOH-BuOH-H2O, 40 °C, 16 h.
Scheme 3
Preparation of 1,2,3-Triazoles 8f–i, 8k–l
Reagents and conditions: (a)
(i) NaNO2 (2.0 equiv), aq. HCl, MeCN, RT, 1 h; (ii) RT
→ 0 °C, NaN3 (2.0 equiv), 0 °C, 1 h; (iii)
HC≡CCO2Me (1.0 equiv), sodium l-ascorbate
(0.4 equiv), CuSO4·5H2O (0.2 equiv), MeOH-BuOH-H2O, 40 °C, 16 h, 71%;
(b) (i) aq. NaOH (2.0 equiv), MeOH, RT, 1 h; (ii) aq HCl, 95%; (c)
NH3, MeOH, 70 °C, 3 h, 46%; (d) MeNH2,
MeOH, 70 °C, 3 h, 64%; (e) NaNO2 (1.2 equiv), CF3CO2H, 0 °C → RT, 1.5 h; (ii) RT →
0 °C, aq. NaN3 (1.1 equiv), 0 °C → RT,
1 h; (iii) aq. NaOH; (iv) HC≡C-CH2NHBoc (1.0 equiv),
sodium l-ascorbate (0.4 equiv), CuSO4·5H2O (0.2 equiv), BuOH-H2O, 50 °C, 16 h, 8%; (f) HCl (excess), dioxane, 50 °C, 16
h, quant.; (g) (i) NaNO2 (1.2 equiv), CF3CO2H, 0 °C → RT, 1.5 h; (ii) RT → 0 °C,
H2O, NaN3 (1.1 equiv), 0 °C → RT,
1 h; (iii) sodium l-ascorbate (0.4 equiv), CuSO4·5H2O (0.2 equiv), HC≡CCH2SiMe3 (1.0 equiv), BuOH-H2O, 50 °C, 16 h; (h) (i) MeOH, K2CO3 (10
equiv), reflux, 2%; (ii) TBAF (2.0 equiv), THF, RT, 16 h; (j) (i)
NaNO2 (4.7 equiv), CF3CO2H, 0 °C
→ RT, 1 h; (ii) RT → 0 °C, NaN3 (4.7
equiv), 0 °C → RT, 16 h; (iii) RT → 0 °C,
aq. NaOH; (iv) HC≡CSiMe3 (2.0 equiv), sodium l-ascorbate (0.9 equiv), CuSO4·5H2O (0.5 equiv), MeOH-BuOH-H2O, RT, 16 h; (k) MeOH, K2CO3 (10 equiv), RT,
24 h, 67%; (l) MeOH-d4, D20,
K2CO3 (3.0 equiv), RT, 3 days, 67%.
Alternative heterocycles were prepared by standard
methods (Scheme ).
Condensation of
aniline 11a with 1,2-diformylhydrazine gave 1,2,4-triazole 9a, and the reaction of 11a with trimethyl orthoformate
and NaN3 gave 1,2,3,4-tetrazole 9b. 2-Chloropyridine 18 was sequentially treated with hydrazine and NaNO2/HCl to give 19, and then CuAAC with HC≡CSiMe3 afforded 2-triazolepyridine 9c (Scheme ). Tetrazolo[1,5-a]pyridine 19 acts as a synthon for the 2-azidopyridine
in this reaction.
Scheme 4
Preparation of 1,2,4-Triazole 9a and
1,2,3,4-Tetrazole 9b
Reagents and conditions:
(a)
ClSiMe3 (15 equiv), 1,2-diformylhydrazine (3.0 equiv),
Et3N, pyridine, 0 °C → 130 °C, 20 h, 50%;
(b) HC(OMe)3 (3.0 equiv), NaN3 (3.1 equiv),
AcOH, 100 °C, 1.5 h, 10%.
We first explored the SAR of the aryl ring
with a variety of single
substituents at the ortho-, meta-, and para-positions. The unsubstituted
phenyl 7a (IC50 110 μM) was prepared
and screened as a benchmark to assess these changes, along with the
original hits 6a and 6b. Substitutions were
biased toward small, lipophilic groups, based upon the available structural
information, and building upon previous Notum SAR from phenyl-azole
scaffolds.[25,26] The tactic of walking single
Cl, Me, and CF3 groups around the phenyl ring proved to
be fruitful, with the 3-Me (7e), 3-Cl (7f), 3-CF3 (7g), 4-Me (7k), and
4-CF3 (7l) analogues affording the largest
improvements in potency over 7a (ca. 10 to 48-fold).
A 2-Cl group (7c) afforded a modest increase in potency
showing the potential benefit of substitution at this position. Inhibitors 7f, 7g, and 7l were superior to
hit 6a.Additive effects from multiple substituents
were then investigated
by preparing a set of dichlorophenyl triazole-matched molecular pairs
(7q–t). This set of inhibitors was prioritized
due to their synthetic accessibility from available commercial reagents
and the precedent from 6b. The original hit 6b was preferred over all other isomers with activity following the
order: 3,4-Cl2 (6b, IC50 0.5 μM)
> 3,5-Cl2 (7t, IC50 2.4 μM)
> 2,3-Cl2 (7q, IC50 5.1 μM)
≈ 2,4-Cl2 (7r, IC50 5.6
μM) > 2,5-Cl2 (7s, IC50 23
μM). Improvements could be achieved with alternative 3,4-disubstitutions
when combinations of preferred substituents (Me, Cl, and CF3) were used with a clear preference for 3-CF3-4-Cl (7w, IC50 0.065 μM). Addition of a 2-Cl as
a third substituent onto 7w as 2-Cl-3-CF3-4-Cl
(7y) gave a further boost in potency with 7y (IC50 0.0095 μM) being 50-fold more active than
hit 6b and 10,000-fold more active than the unsubstituted
phenyl ring 7a.At this point in the program, mouse
PK data for 7y was generated in vivo to determine the
extent of plasma and brain
exposure and to verify the correlation of the in vitro ADME data with
in vivo outcomes in this series. Triazole 7y was selected
as it combined excellent Notum activity (IC50 9.5 nM) with
an attractive ADME profile as it possessed good aqueous solubility,
microsomal stability, and cell permeability (Table and Supporting Information). Following a single oral dose of 10 mg/kg, plasma exposure for 7y was low (Cmax 300 ng/mL; AUC(0→inf) 150 ng h/mL), which we attributed to higher
clearance than predicted by MLM and highlighted the need to further
improve metabolic stability (Figure S2).
Our hypothesis for this disconnection between MLM stability and plasma
clearance of 7y was due to phase 2 metabolism/conjugation
of the OH group, and this was subsequently supported by the higher
clearance of 7y in mouse hepatocytes (Cli 48
μL/min/106 cells). Hence, we designed inhibitors
that explored direct modification and/or replacement of the −CH2OH (Table ). Despite the poor plasma exposures, 7y displayed the
highest brain-to-plasma ratio [Kp 1.2,
based on AUC0–inf (total drug)] observed to date
and was far superior to previous leads 1 (Kp ca. 0),[28]3 (Kp 0.29),[24]4 (Kp 0.16),[26] and 5 (Kp ca.
0)[27] where comparable studies were performed.
This was viewed to be a significant advantage for this series.SAR of the C4-substituent of the triazole was studied with retention
of the preferred aryl ring of 7y as this group conferred
significant activity to the series and was superior to all other substitution
patterns explored so far (Table ). Substitution at the C5 position was not investigated
as previous studies with related 4-Cl-phenyl azoles showed this to
be unfavorable.[26]Modification of
the −CH2OH through addition of
the α-Me group (8a), elongation of the carbon chain
(8b–8c), or a combination of both these approaches
(8d–8e) leads to a dramatic reduction in potency
(IC50 > 1 μM). Oxidation to the carboxylic acid 8f was tolerated with a modest reduction in potency (7y vs 8f), whereas activity was significantly
diminished upon conversion to the corresponding primary 8g and secondary amides 8h. Primary amine 8i also had a significant reduction in potency as did “capping”
the −CH2OH as the −CH2OCH3 ether 8j. Deletion of the −OH from 7y to give the 4-Me triazole 8k had a more modest
effect upon the reduction in potency (12-fold) that was consistent
with the loss of one H-bonding group from the ligand. So far, all
changes to the C4 substituent had been unfavorable, then, somewhat
unexpected, potent inhibition of Notum activity was entirely recovered
by removal of the substituent as unsubstituted triazole 8l (IC50 6.7 nM) was the most potent inhibitor prepared
in this series. Finally, revisiting the aryl ring substitution with
2,3,4-Cl38m, as a simpler and direct analogue
of 8l, was slightly inferior.The Notum-8l X-ray structure is consistent with the
ligand retaining the H-bond between N3 of the triazole and the backbone
of Trp128. In addition, 8l has near complete occupancy
of the palmitoleate pocket through the 2-Cl-3-CF3-4-Cl
substituents on the aryl ring. The near optimal filling of the pocket
is achieved while still positioning the triazole in a favorable position
for a hydrogen bond with the backbone of Trp128 (Figure ). This combination of features
is likely why such high potency is observed.
Figure 5
(A) Side view of structure
of 8l (PDB: 7PK3). (B) Top view of
the structure of 8l (PDB: 7PK3). The compound forms a hydrogen bond
with the backbone of Trp128 (dashed line, 2.47 Å) and displays
excellent occupancy of the lipophilic pocket. Select binding site
residues shown within 4 Å of the compound. Water molecules have
been removed for clarity. The surface of the Notum binding pocket
outlined (gray). (C) Illustration of space occupied by 8l (green mesh, PDB: 7PK3) and the outline of the pocket (gray mesh, PDB: 7PK3).
(A) Side view of structure
of 8l (PDB: 7PK3). (B) Top view of
the structure of 8l (PDB: 7PK3). The compound forms a hydrogen bond
with the backbone of Trp128 (dashed line, 2.47 Å) and displays
excellent occupancy of the lipophilic pocket. Select binding site
residues shown within 4 Å of the compound. Water molecules have
been removed for clarity. The surface of the Notum binding pocket
outlined (gray). (C) Illustration of space occupied by 8l (green mesh, PDB: 7PK3) and the outline of the pocket (gray mesh, PDB: 7PK3).1,2,3-Triazole 8l was superior in potency to
the isomeric
1,3,4-triazole 9a or the 1,2,3,4-tetrazole 9b (Table ). These
effects seem to be quite subtle as most key inhibitor–Notum
interactions are available with these alternative 1-aryl azoles and
may be due to a more optimal electronic interaction of 8l with Trp128 or simply that an N4 atom is detrimental.[49] Modification of the phenyl ring of 8l to the corresponding pyridine 9c was equipotent and
demonstrated that some modest polarity could be accommodated in the
palmitoleate pocket. Dual deuteration of 8l at the C4
and C5 positions of the triazole gave d2-8l as a tool to investigate the metabolic fate of the
parent molecule.Triazole 8l has physicochemical
and molecular properties
consistent with drug-like space.[50] It was
noteworthy that 8l had a much lower measured distribution
coefficient (LogD7.4 1.3 ± 0.05) than had been predicted
by the calculated partition coefficient (clogP 3.9) despite being non-ionized at physiologically relevant
pH. This disconnection could be attributed to the close proximity
of the 2-Cl, 3-CF3, and 4-Cl substituents on the aromatic
ring that results in a reduction in overall hydrophobic surface area
when compared to summing their individual lipophilicity fragmental
constants (π). These results highlight the importance of generating
empirical data to correlate with calculated values. Evaluation of 8l against standard design metrics using Notum inhibition
from the biochemical assay (IC50 6.7 nM, pIC50 8.2) and measured lipophilicity (LogD7.4 1.3) calculates 8l to have high LE (0.67), LLE (6.9), and a favorable prediction
of brain penetration (CNS MPO 5.4/6.0; BBB Score 5.6/6.0).Triazole 8l displayed satisfactory aqueous solubility
in phosphate-buffered saline (PBS), good metabolic stability in both
MLM and HLM, and high permeability in the MDCK-MDR1 cell transit assay
with minimal evidence for recognition and efflux by the P-gp transporter
(Table and Supporting Information). In contrast, pyridine 9c performed poorly in the permeability assay although these
results were confounded by low compound recovery. Comparison of MLM
stability of 8l with d2-8l (Cli 3.2 vs 5.4 μL/min/mg protein) showed
deuteration of the triazole afforded no improvement suggesting that
CYP-mediated metabolism of the triazole C–H bonds was not a
major route of clearance in this system.Based on these data, 8l was selected as our nominated
lead from this series for further profiling as it had the best combination
of Notum activity and in vitro ADME properties including lipophilicity.Lead 8l was then evaluated in additional in vitro
pharmacology and ADME screens (Table ). Triazole 8l restored
Wnt signaling in the presence of Notum (EC50 110 nM; n = 4) in a in a cell-based TCF/LEF (Luciferase) reporter
assay[23,26,27] and gave standard
S-shaped concentration–response curves up to 10 μM (Figure S9). Performing these experiments in the
absence of Notum showed a maximal Wnt response at all concentrations
tested (up to 10 μM; n = 4). The activation
of Wnt signaling was due to direct on-target inhibition of Notum by 8l and not by assay interference or cell toxicity (up to 10
μM).
Table 5
Extended
Profile of 8la
Non-standard abbreviations:
m, mouse;
r, rat; d, dog; h, human; mid., midazolam; and test., testosterone.
Values are mean; n = 3 per time point; terminal
blood and brain levels were measured
up to 24 h. All animals were normal throughout the study period.Male C57BL6 mice.Sprague–Dawley male rats.Formulation A: dimethylsulfoxide
(DMSO) (2% v/v) + 10% v/v Solutol in 1× PBS (98% v/v).Formulation B: 0.1% Tween80 in water.Kp was
calculated by the ratio of brain concentration–time profile
(AUC(0–inf),brain) to that of plasma concentration–time
profile (AUC(0–inf),plasma). Kp,uu was calculated by multiplying Kp with the ratio of free fraction in brain homogenate to plasma (fu,brain/fu,plasma).Non-standard abbreviations:
m, mouse;
r, rat; d, dog; h, human; mid., midazolam; and test., testosterone.The aqueous solubility of 8l could be significantly
enhanced to 1 mg/mL with cosolvents bringing added flexibility to
study design. Triazole 8l has good stability in liver
microsomes and hepatocytes across mouse, rat, dog, and human, which
predicts for low clearance across these species. Screening in HLM
including eight standard substrates of cytochrome P450 (CYP450) enzymes
showed moderate/weak inhibition of 1A2 (IC50 0.96 μM),
2B6 (IC50 5.2 μM), 2C9 (IC50 15 μM),
and 2C19 (IC50 8.1 μM) but not 2C8, 2D6, or 3A4 (IC50 > 50 μM). Furthermore, there was no evidence for
CYP
time-dependent inactivation (TDI) as assessed by the preincubation
of HLM with 8l in the presence and absence of NADPH,
followed by the incubation with the discrete marker substrates (IC50 shift). There was good cell permeability across a Caco-2
monolayer consistent with good absorption from the gut. The hepatocyte
data for 8l was encouraging as this deletion of the −CH2OH group from 7y seems to have removed the liability
of this metabolic soft spot. Hence, 8l was advanced to
rodent PK studies.PK data for 8l was generated
in vivo in mouse, and
subsequently in rat, to evaluate plasma and brain exposure (Table and Supporting Information). Following single intravenous administration
of 8l to mouse, plasma clearance was low compared to
liver blood flow and volume of distribution was moderate resulting
in an elimination half-life of 2.4 h (Figure S3, Table S11). Following a single oral dose to mouse, 8l was rapidly absorbed and showed good oral bioavailability (66%)
(Figure S4, Table S12). Good brain penetration
was confirmed with drug levels in the brain similar to drug levels
in the plasma (Kp 1.1). The brain-to-plasma
ratio was 0.77 (Kp,uu) when free drug
concentrations were calculated. This oral dose of 10 mg/kg achieved
a concentration in the brain of Cmax ≈
300 nM (free drug) that exceeded the Notum EC50 from the
cell-based TCF/LEF assay, and brain levels were maintained above 100
nM for ∼4 h. Although encouraging, this dose, and the resulting
level of brain exposure, may be insufficient to promote a sustained
pharmacodynamic (PD) response, where Notum activity was reduced for
an extended period of time. This will need to be determined empirically.
Hence, we subsequently explored alternative dosing regimes to provide
some flexibility in the determination of the required efficacious
concentrations (Ceff) in rodent models
of disease, and its relationship to Notum pharmacology (EC50), that is, establish the PK–PD relationship (vide infra).
Table 4
Rodent Plasma and Brain PK Parameters
of 8la
mouseb
ratc
Intravenous
(i.v.)
dose (mg/kg)
1.0d
1.0d
CL (mL/min/kg)
10.4
7.5
Vdss (L/kg)
2.0
2.0
t1/2 (h)
2.4
3.3
Values are mean; n = 3 per time point; terminal
blood and brain levels were measured
up to 24 h. All animals were normal throughout the study period.
Kp was
calculated by the ratio of brain concentration–time profile
(AUC(0–inf),brain) to that of plasma concentration–time
profile (AUC(0–inf),plasma). Kp,uu was calculated by multiplying Kp with the ratio of free fraction in brain homogenate to plasma (fu,brain/fu,plasma).
PK evaluation of 8l in rat also showed low plasma
clearance and moderate volume of distribution with a half-life of
3.3 h (Table ). Oral
administration to rat showed good absorption, drug levels, and brain
penetration (Kp 1.4). The oral bioavailability
was supramaximal with Fo 140%. The plasma
(and brain) concentration–time plots clearly show double Cmax peaks (Figure S7), which could indicate some contribution from excreted drug being
reabsorbed from the gastrointestinal tract and/or an effect on gastric
emptying times. On balance, these results established that 8l has PK properties compatible with evaluation in rodent models of
disease.Triazole 8l emerged as a promising lead
with potent
inhibition of Notum enzymic activity in both biochemical and cellular
assays, along with excellent brain penetration in rodent. The wider
pharmacology activity of 8l was then assessed against
serine hydrolases, kinases, and representative drug safety targets
to identify any off-target activity and to establish its selectivity.Selectivity against serine hydrolases was assessed by unbiased
proteomic analysis in a human colorectal cancer cell line SW620 that
has been characterized to have high Notum expression.[51] Quantitative ABPP was performed using FP-biotin to label
active serine hydrolases in concentrated conditioned media and cell
lysates, combined with tandem mass tagging technology (TMT) for relative
quantification.[52] Triazole 8l showed concentration-dependent inhibition of Notum activity (IC50 170 nM in conditioned media) and demonstrated high selectivity
in this system as there was no cross reactivity against any of the
other 39 serine hydrolases detected (Figures , S11 and S12).[18,52b]
Figure 6
Concentration–response
curves from proteomic analysis of 8l in SW620 cells.
The response for Notum is shown in dark
blue circles (•) and all other serine hydrolases in gray. (A)
Concentrated conditioned media, Notum IC50 170 nM. (B)
Cell lysates. Each sample set was preincubated with an eight-point
concentration–response curve of 8l or DMSO, then
labeled for 20 min with FP-Biotin (2 μM) to capture active serine
hydrolases. Labeled proteins were enriched on NeutrAvidin agarose
and then digested using LysC and trypsin. The subsequent peptides
were isobarically labeled with eight channels of a TMT10plex and combined
for final LC–MS/MS analysis. The MS proteomics data have been
deposited to the ProteomeXchange Consortium via the PRIDE partner
repository with the dataset identifier PXD031338.
Concentration–response
curves from proteomic analysis of 8l in SW620 cells.
The response for Notum is shown in dark
blue circles (•) and all other serine hydrolases in gray. (A)
Concentrated conditioned media, Notum IC50 170 nM. (B)
Cell lysates. Each sample set was preincubated with an eight-point
concentration–response curve of 8l or DMSO, then
labeled for 20 min with FP-Biotin (2 μM) to capture active serine
hydrolases. Labeled proteins were enriched on NeutrAvidin agarose
and then digested using LysC and trypsin. The subsequent peptides
were isobarically labeled with eight channels of a TMT10plex and combined
for final LC–MS/MS analysis. The MS proteomics data have been
deposited to the ProteomeXchange Consortium via the PRIDE partner
repository with the dataset identifier PXD031338.Compound 8l showed excellent selectivity for Notum
over a panel of 485 distinct kinases with <50% inhibition at 10
μM for all these kinases including casein kinase 1 (CK1) and
GSK3 (Table ). This
was of critical importance to studies wishing to assess the function
of Notum with 8l, as CK1 and GSK3 phosphorylate β-catenin
leading to its degradation and thus reducing Wnt signaling.[53] For some context, 8l showed the
highest activity in this kinase panel at CDK14/cyclin Y with 43% inhibition
at 10 μM (n = 2) (Table S20).Compound 8l was screened for off-target
pharmacology
across multiple drug target classes in the DiscoverX Safety47 panel.
This suite of 78 functional assays assesses pharmacological modulation
of 47 human off-target liabilities that have been highlighted for
the initial safety assessment of lead compounds.[54] There was minimal activity at 75/78 targets having IC50/EC50 > 30 μM with only modest activity
at HTR3A (IC50 4.5 μM), nAChR(a4/b2) (IC50 10 μM), and MAOA (IC50 28 μM) (Table ). Liability through direct
interaction with cardiac ion channels was low as 8l had
no significant effect on either the hERG or the cardiac sodium channels
(IC50 > 30 μM) (Table S21).Having established 8l as a potent and selective
inhibitor
of Notum carboxylesterase activity, our efforts shifted to developing
a multigram synthesis of 8l and identification of a suitable
dosing regimen to investigate the role of Notum in mouse models of
neurodegenerative disease.The multigram synthesis of 8l essentially followed
the discovery route but with additional precautions to minimize the
risk of high energy intermediates on scale such as the azide 12a (Scheme ). Differential scanning calorimetry (DSC) analysis of azide 12a determined an exotherm onset temperature at 125.8 and
so 42 °C was established as the maximum safe handling temperature
(Figure S15). Reactions were performed
in smaller batches and then pooled for purification to give a single,
homogenous product.
Reagents
and conditions: (a)
(i) NaNO2 (4.7 equiv), CF3CO2H, 0
°C, 0.5 h → RT, 1.5 h; (ii) NaN3 (4.7 equiv),
H2O, 0 °C → RT, 16 h; (iii) NaOH, 0 °C;
(b) HC≡CSiMe3 (2.0 equiv), sodium l-ascorbate
(0.9 equiv), CuSO4·5H2O (0.5 equiv), MeOH-BuOH-H2O, RT, 16 h; (c) K2CO3 (10 equiv), MeOH, RT, 24 h; overall yield,
67%.Increasing the oral dose of 8l to mouse from 10 mg/kg
to twice daily dosing of 30 mg/kg (2 × 30 mg/kg, p.o. at T = 0 and 9 h, formulation A) achieved higher and prolonged
drug concentrations in the plasma and brain (Figure S5, Table S13). The sustained free drug levels of 8l in the brain now exceeded the Notum EC50 for ca. 20 h.
This twice daily oral dosing of 8l was well tolerated
in both wild-type and aged APP/PS1 mice for 7 days and was therefore
deemed suitable for the in vivo pathfinder experiments. These studies
are ongoing and will be reported in due course.
Conclusions
Crystallographic
fragment screening of the DSPL for binding to
Notum, supported by a biochemical enzyme assay to rank inhibition
activity, proved to be successful in identifying chemically enabled
hits. This screening strategy identified 58 hits that were shown to
bind to the active site of Notum with 20 of these having IC50 < 100 μM. A pair of outstanding hits were 4-(hydroxymethyl)triazoles 6a (IC50 12 μM) and 6b (IC50 0.5 μM), and these were selected for fragment development
to improve Notum activity and develop PK properties for oral dosing
to achieve good plasma exposure and brain penetration.Exploration
of the substituents on the aryl ring that binds in
the palmitoleate pocket of Notum gave potent early lead 7y (IC50 9.5 nM) and further optimization of the triazole
group delivered 8l (IC50 6.7 nM). It is noteworthy
that advanced lead 8l can be characterized as a fragment-sized
molecule (MW 282; HAC 17) with high LE and LLE, and that inhibition
of Notum activity was increased by over 15,000-fold by optimization
of the substituents on the aryl ring (8l vs 7a). These findings highlight the benefits of FBDD when aligned with
a suitable drug target.The Notum-8l X-ray structure
clearly showed that 8l has near complete occupancy of
the palmitoleate pocket
through the 2-Cl-3-CF3-4-Cl substituents on the aryl ring.
Triazole 8l restored Wnt signaling in the presence of
Notum (EC50 110 nM) in a cell-based TCF/LEF reporter assay
confirming functional activity. Assessment in broader off-target and
safety pharmacology screens showed 8l to be selective
against serine hydrolases, kinases, and representative drug targets.
PK studies with 8l in mouse and rat showed good plasma
exposure and brain penetration, and was well tolerated.In summary,
triazole 8l (ARUK3001185) is a potent
and selective inhibitor of Notum activity with good brain penetration
suitable for oral dosing in rodent models of disease.
Experimental Section
Notum Protein Expression and Purification
Methods have
been described in detail elsewhere.[25]
Notum OPTS Biochemical Assay
Methods have been described
in detail elsewhere.[23−26] Representative concentration–response curves are provided
in the Supporting Information.
TCF/LEF Reporter
(Luciferase) Cell-Based Assay
Methods
have been described in detail elsewhere.[23,26,27] Concentration–response curves are
provided in the Supporting Information.
Proteomic Analysis in SW620 Cells
Cell Culture
SW620
cells were grown in Leibovitz’s
L-15 medium supplemented with 10% FBS, Pen/Strep 10 U/mL, and 0.075%
sodium bicarbonate.
Conditioned Medium Generation
SW620
cells were grown
to around 80% confluence, washed three times with PBS, and then placed
in serum-free media for 48 h. The medium was then removed, spun at
300G to remove any solids, and then concentrated in 10k MWCO protein
concentrators (Pierce) by centrifugation.
Cell Lysate Generation
SW620 cells were grown to around
80% confluence, the medium was removed and cells were washed three
times with PBS, and then lysed in the ice cold IP lysis buffer (Pierce).
Lysates were clarified by centrifugation at 15k G for 10 min at 4
°C, then protein quantification was performed by the BCA assay
(Pierce) and lysates were equalized to 1.5 mg/mL.
Proteomics
Protocol
Either lysates or conditioned medium
was treated with the appropriate concentration of each inhibitor or
DMSO across a range of concentrations and then equilibrated at RT
for 45 min. Following this, 2 μM FP-Biotin (Santa Cruz) was
added to each sample for 20 min with agitation. Lysates were then
precipitated using chloroform–methanol, and the protein pellet
was washed two times with MeOH. Protein pellets were dissolved in
50 mM HEPES pH 8.0 with 2% SDS and 10 mM DTT, then the SDS concentration
was adjusted to 0.2% with further 50 mM HEPES pH 8.0. Each solution
was then added to an aliquot of equilibrated NeutrAvidin beads,[55] which had been washed two times with 50 mM HEPES
pH 8.0 with 0.2% SDS, for 16 h at RT with agitation. Beads were then
pelleted by centrifugation, the supernatant was removed and then washed
three times with 50 mM HEPES pH 8.0 with 0.2% SDS, followed by three
washes with 50 mM HEPES pH 8.0. The beads were then incubated with
10 mM DTT in 50 mM HEPES pH 8.0 for 15 min at RT, followed by 20 mM
iodoacetamide, which was incubated for 30 min in the dark. A further
aliquot of 10 mM DTT was added to the beads, then they were pelleted
by centrifugation, the supernatant was removed, the beads were washed
with 50 mM HEPES pH 8.0, and all the supernatants were removed. LysC
(Wako) was dissolved in 50 mM HEPES pH 8.0 to give a 1 μg/mL
solution, of which 50 μL (50 ng of LysC) was added to the beads
and incubated overnight at 37 °C. The beads were pelleted, the
supernatant was removed, and then washed with additional 50 mM HEPES
pH 8.0 to extract as many peptides as possible. 75 ng of trypsin (Pierce)
was added from a 20 ng/μL solution in 50 mM HEPES pH 8.0 and
incubated at 37 °C for 7 h. Each set of conditions were then
labeled using eight channels of a TMT10plex (Thermo) following the
manufacturer’s instructions, then combined. TMT-labeled tryptic
peptides were filtered using 1 mm Empore C18 solid-phase extraction
discs aka “stage tips” (3M) prior to vacuum centrifugation
and resuspension in 0.1% trifluoroacetic acid.On an Ultimate
3000 nanoRSLC HPLC (Thermo Scientific), 1–10 μL of sample
was loaded onto a 20 mm × 75 μm Pepmap C18 trap column
(Thermo Scientific) prior to elution via a 50 cm × 75 μm
EasySpray C18 column into a Lumos Tribrid Orbitrap mass spectrometer
(Thermo Scientific). A 70′ gradient of 6–40% B was used,
followed by washing and re-equilibration (A = 2% MeCN, 0.1% formic
acid; B = 80% MeCN, 0.1% formic acid).The Orbitrap was operated
in “Data Dependent Acquisition”
mode, followed by MS/MS in “TopS” mode, with Orbitrap
accumulation at R = 50,000 of HCD fragmented parent
ions.Raw files were processed using MaxQuant (maxquant.org)
and Perseus
(maxquant.net/perseus) with a recent download of the uniprot homo
sapiens reference proteome database and a common contaminants database.
A decoy database of reversed sequences was used to filter false positives,
at a peptide false detection rate of 1%.Analysis was performed
using Perseus and filtered against contaminants,
reversed, and proteins identified by the site. Protein groups were
then filtered by those which contained valid values in at least six
of the eight conditions. Serine hydrolases were identified by filtering
for those proteins identified as such in these two reference publications[18,52b] with intensity values normalized relative to the DMSO control, and
concentration–response curves and IC50 values were
determined using GraphPad Prism 9.1.0.The identity and quantification
of each serine hydrolase identified
in SW620 cells by FP-biotin ABPP for 8l (and 1) is provided in the Supporting Information. The MS proteomics data have been deposited to the ProteomeXchange
Consortium via the PRIDE partner repository[56] with the dataset identifier PXD031338.
Structural Biology
The methods of protein production,
crystallization, data collection, and structure determination have
been described in detail.[26] Data collection
and refinement statistics are presented in the Supporting Information.To prepare the figures of the
crystal structures, the PDB entry was processed using the Protein
Preparation Wizard in Schrödinger Maestro 12.4 using the default
settings and optimizing the hydrogen bond assignment. No changes were
made to the position of heavy atoms. Final images were prepared using
PyMol version 2.4.
ADME Assays
Molecular properties
(MW, clogP, and TPSA) were calculated
with ChemDraw Professional
v16.0.1.4(77). Distribution coefficients (LogD7.4) were
measured using the shake flask method.Selected compounds were
routinely screened for aqueous solubility in PBS (pH 7.4), transit
performance in MDCK-MDR1 cell lines for permeability, and metabolic
stability in MLM and HLM as a measure of clearance. Lead 8l was also screened for inhibition of representative CYP450 enzymes,
permeability across the Caco-2 cell monolayer, and metabolic stability
in liver microsomes and hepatocytes. Assay protocols and additional
data are presented in the Supporting Information. ADME studies reported in this work were independently performed
by Cyprotex (Macclesfield, U.K), GVK Biosciences (Hyderabad, India),
and WuXi AppTec (Shanghai, China).
PK Studies
In
vivo PK data in mouse and rat was independently
generated at Charles River Laboratories (Groningen, Netherlands),
GVK Biosciences (Hyderabad, India), and WuXi AppTec (Shanghai, China).
PK was supported by the development of suitable formulations for the
route of administration, and the measurement of plasma protein binding
(PPB) and brain tissue binding for the calculation of free drug levels
in these compartments. Study protocols and additional data are presented
in the Supporting Information.
Kinase Selectivity
Panel
Kinase selectivity screening
was performed by Thermo Fisher Scientific (Paisley, U.K.) in their
SelectScreen Biochemical Kinase Profiling Service. Assay information
and data are presented in the Supporting Information.
Safety Pharmacology Studies
Screening for off-target
pharmacology across multiple drug target classes was performed in
the DiscoverX Safety47 panel as the SAFETYscan E/IC50 ELECT service
by Eurofins (San Diego CA, U.S.A.). Assay information and data are
presented in the Supporting Information.
Chemistry
General Information
Methods have
been described in
detail elsewhere.[27a] The purity of compounds 6–9 was evaluated by NMR spectroscopy and LCMS analysis.
All compounds had purity ≥95% unless otherwise stated.
General
Methods
*Caution* Azides are potentially
explosive reagents and therefore all solutions containing azides were
concentrated under reduced pressure behind a blast shield at RT. Furthermore,
sodium azide reacts with acids to generate gaseous HN3,
which is a toxic gas. All reactions were conducted behind a blast
shield under a flow of nitrogen, and exit gases were passed through
an aqueous base prior to their release into a ventilated fume hood.
Preparation of 1-Phenyl-1H-1,2,3-triazoles
General
Method 1.1
*Caution* Step 1: Sodium
azide (260 mg, 4.0 mmol, 2.0 equiv) and copper(II) sulfate pentahydrate
(100 mg, 0.4 mmol, 0.2 equiv) were added to a solution of phenylboronic
acid 10 (2.0 mmol, 1.0 equiv) in MeOH (10 mL). The reaction
was stirred at 40 °C for 5 h, concentrated to ∼50% volume
under reduced pressure, and then diluted with H2O (ca.
10 mL) and EtOAc (ca. 50 mL). The organic phase was separated, diluted
with t-BuOH (1.7 mL), and then cautiously concentrated
under reduced pressure at 25 °C to provide a solution of crude
azide 12 in t-BuOH, which was used without
further purification.Step 2: Copper(II) sulfate pentahydrate
(100 mg, 0.4 mmol, 0.22 equiv), sodium l-ascorbate (56 mg,
0.28 mmol, 0.15 equiv), H2O (1.7 mL), and propargyl alcohol
(0.1 mL, 1.8 mmol, 1.0 equiv) were added to the solution of crude
azide 12 from step 1. The reaction was stirred at 40
°C for 16 h, cooled to RT, diluted with H2O (ca. 10
mL), extracted with EtOAc (ca. 50 mL), and the organic phase was washed
with H2O (ca. 10 mL) and brine (ca. 10 mL). The organic
phase was dried, filtered, and concentrated under reduced pressure.
The crude product was purified by column chromatography (0–5%
MeOH in CH2Cl2) to give the triazoles 6b and 7.
General Method 1.2
*Caution* Step 1: Sodium
nitrite (155 mg, 2.25 mmol, 1.2 equiv) was added portionwise to a
solution of aniline 11 (1.87 mmol, 1.0 equiv) in TFA
(2.0 mL) at 0 °C over 30 min. The reaction mixture was then allowed
to warm to RT for 1.5 h before recooling to 0 °C. A solution
of sodium azide (133 mg, 2.05 mmol, 1.1 equiv) in H2O (1.5
mL) was added dropwise over 5 min, stirred for 30 min, and then warmed
to RT over 60 min. The mixture was basified to pH 8–9 by dropwise
addition of aqueous NaOH (5 M), then diluted with t-BuOH (2.0 mL) to provide a solution of crude azide 12, which was used without further purification.Step 2: Copper(II)sulfate
pentahydrate (93 mg, 0.37 mmol, 0.2 equiv), sodium l-ascorbate
(148 mg, 0.75 mmol, 0.4 equiv), and mono-substituted alkyne 13 (1.87 mmol, 1.0 equiv) were added to the solution from
step 1. The reaction mixture was stirred at 50 °C for 16 h, cooled
to RT, diluted with brine (ca. 10 mL), and then extracted with EtOAc
(ca. 50 mL). The organic phase was dried, filtered, and concentrated
under reduced pressure. The crude product was purified by column chromatography
(0–5% MeOH in CH2Cl2) to give triazoles 7 and 8.
General Method 1.3
*Caution* Step 1: Aniline 11 (0.65 mmol,
1.0 equiv) was dissolved in H2O
(0.5 mL) and MeCN (3.0 mL), then aqueous HCl (37%, 7.3 mL) was added
at RT and stirred vigorously for 10 min. Sodium nitrite (90 mg, 1.3
mmol, 2.0 equiv) was added portionwise and the solution was stirred
for 1 h, cooled to 0 °C, and then sodium azide (85 mg, 1.3 mmol,
2.0 equiv) was added portionwise. After 1 h, the solution was diluted
with H2O (ca. 10 mL) and Et2O (ca. 50 mL). The
organic phase was separated, diluted with t-BuOH
(2.0 mL), and then cautiously concentrated under reduced pressure
at 25 °C to provide a solution of crude azide 12 in t-BuOH, which was used without further purification.Step 2: Sodium l-ascorbate (52 mg, 0.26 mmol, 0.4 equiv),
mono-substituted alkyne 13 (0.65 mmol, 1.0 equiv), copper(II)
sulfate pentahydrate (33 mg, 0.13 mmol, 0.2 equiv), MeOH (1.0 mL),
and H2O (2.0 mL) were added to the solution of crude azide 12 from step 1, and the reaction was stirred at 40 °C
for 16 h. The reaction was cooled to RT, diluted with H2O (ca. 10 mL), extracted with EtOAc (ca. 50 mL), and the organic
phase was washed with H2O (ca. 10 mL) and brine (ca. 10
mL). The organic phase was dried, filtered, and concentrated under
reduced pressure. The crude product was purified by column chromatography
(0–5% MeOH in CH2Cl2) to give triazoles 7 and 8.
General Method 1.4
*Caution* Step 1: Sodium
nitrite (72 mg, 1.0 mmol, 1.2 equiv) was added portionwise to a solution
of aniline 11 (0.9 mmol, 1.0 equiv) in TFA (0.5 mL) at
0 °C over 30 min. The reaction mixture was then allowed to warm
to RT for 1.5 h before recooling to 0 °C. H2O (0.1
mL), sodium azide (62 mg, 1.0 mmol, 1.1 equiv) was added portionwise
over 30 min, and then warmed to RT over 1 h. The mixture was basified
to pH 8–9 by dropwise addition of saturated aqueous NaHCO3, then diluted with CH2Cl2 (ca. 50 mL).
The organic phase was separated, diluted with t-BuOH
(0.5 mL), and then cautiously concentrated under reduced pressure
at 25 °C to provide a solution of crude azide 12 in t-BuOH, which was used without further purification.Step 2: Copper(II) sulfate pentahydrate (43 mg, 0.2 mmol, 0.2 equiv),
sodium l-ascorbate (69 mg, 0.4 mmol, 0.4 equiv), H2O (0.5 mL), and mono-substituted alkyne 13 (0.9 mmol,
1.0 equiv) were added to the solution of crude azide 12 from step 1, and the reaction mixture was stirred at 50 °C
for 2–16 h. The reaction was cooled to RT, diluted with H2O (ca. 10 mL), extracted with EtOAc (ca. 50 mL), and the organic
phase was washed with H2O (ca. 10 mL) and brine (ca. 10
mL). The organic phase was dried, filtered, and concentrated under
reduced pressure. The crude product was purified by column chromatography
(0–5% MeOH in CH2Cl2) to give triazoles 7 and 8.
*Caution* Step 1: Sodium nitrite (180 mg, 2.61 mmol, 1.2 equiv) was added
portionwise to a solution of 2,4-dichloro-3-(trifluoromethyl)aniline 11a (500 mg, 2.17 mmol, 1.0 equiv) in TFA (5 mL, 4.35 mmol)
at 0 °C over 30 min. The reaction was then allowed to warm to
RT for 1.5 h, H2O (0.1 mL) was added, and the mixture was
then recooled to 0 °C. Sodium azide (155 mg, 2.39 mmol, 1.1 equiv)
was added portionwise over 30 min, and the reaction mixture was then
allowed to warm to RT over 1 h. The mixture was basified to pH 8–9
by dropwise addition of saturated aqueous NaCO3H, then
diluted with EtOAc (ca. 50 mL). The organic phase was separated, diluted
with t-BuOH (5.0 mL), and then cautiously concentrated
under reduced pressure at 25 °C to provide a solution of crude
azide 12a in t-BuOH, which was used
without further purification.Step 2: MeOH (5 mL), H2O (5 mL), sodium l-ascorbate (172 mg, 0.9 mmol, 0.4 equiv),
trimethyl(propargyl)silane (244 mg, 2.2 mmol, 1.0 equiv), and copper(II)
sulfate pentahydrate (109 mg, 0.43 mmol, 0.2 equiv) were added to
the solution of crude azide 12a from step one, and the
reaction mixture was stirred at 50 °C for 16 h. The reaction
was cooled to RT, diluted with H2O (ca. 10 mL), extracted
with EtOAc (ca. 50 mL), and the organic phase was washed with H2O (ca. 10 mL) and brine (ca. 10 mL). The organic phase was
dried, filtered, and concentrated under reduced pressure to give 16, which was used immediately without further purification.Step 3: K2CO3 (3.05 g, 21.7 mmol, 10.0 equiv)
was added to crude 16 from step 2 in MeOH (15 mL) and
heated to reflux for 16 h. The mixture was taken to pH 7 by addition
of aqueous HCl (1 M), then diluted with EtOAc (ca. 50 mL). The organic
phase was dried, filtered, and concentrated under reduced pressure.
Due to incomplete desilylation, the crude material was suspended in
THF (10 mL) and TBAF hydrate (1.22 g, 4.35 mmol, 2.0 equiv) in H2O (0.5 mL) was added. The reaction was stirred at RT for 16
h, diluted with EtOAc (ca. 50 mL), and then the organic phase was
dried, filtered, and concentrated under reduced pressure. The crude
product was purified by column chromatography, eluting with 0–30%
EtOAc in cyclohexane to give 8k isolated as an off-white
solid (12 mg, 2%).1H NMR (700 MHz, CDCl3): δ 7.67 (d, J = 0.8 Hz, 1H), 7.64 (d, J = 8.6 Hz, 1H),
7.61 (d, J = 8.7 Hz, 1H), 2.47 (d, J = 0.8 Hz, 3H); 13C NMR (176 MHz, CDCl3): δ
143.7, 136.2 (q, J = 1.2 Hz), 136.1, 131.6, 131.0,
130.7, 128.0 (q, J = 31.1 Hz), 123.6, 122.2 (q, J = 277.2 Hz), 10.9; LCMS m/z: 296.0 [M + H]+.
*Caution* Step 1: 2,4-Dichloro-3-(trifluoromethyl)aniline 11a (1.0 g, 4.3 mmol, 1.0 equiv) was dissolved in TFA (20 mL), cooled
to 0 °C, a solution of sodium nitrite (1.4 g, 20.6 mmol, 4.7
equiv) in H2O (2.8 mL) was added dropwise over 10 min,
and stirred for a further 30 min. The ice bath was removed, the reaction
was stirred at RT for 1 h, and then recooled to 0 °C, before
a solution of sodium azide (1.3g, 20.6 mmol, 4.7 equiv) in H2O (5.4 mL, 20.6 mmol) was added dropwise over 30 min. The reaction
was allowed to warm to RT over 16 h, before recooling to 0 °C.
Aqueous NaOH (52 mL, 5 M) was then cautiously added to take the mixture
to pH 8–9 to provide a solution of crude azide 12a, which was used without further purification.Step 2: MeOH
(10 mL), t-BuOH (10 mL), aqueous copper(II) sulfate
pentahydrate (5.1 mL, 100 mg/mL, 2.1 mmol, 0.5 equiv), aqueous sodium l-ascorbate (4.1 mL, 1 M, 4.1 mmol, 0.9 equiv), and trimethylsilylacetylene
(0.94 mL, 8.7 mmol, 2.0 equiv) were added to the solution of crude
azide 12a from step one at RT. The reaction was stirred
for 16 h, diluted with EtOAc (ca. 500 mL), and the organic phase was
cautiously concentrated under reduced pressure at 25 °C to give
crude 17.Step 3: MeOH (15 mL) and K2CO3 (6.0 g, 43.5
mmol) were added to crude 17, and the reaction was stirred
at RT for 24 h, before H2O (ca. 20 mL) was then added until
all the solid carbonate dissolved. MeOH was then removed under reduced
pressure and the resultant aqueous mixture was diluted with EtOAc
(ca. 500 mL). The organic phase was dried, filtered, and concentrated
under reduced pressure. The crude product was purified by column chromatography
(0–30% EtOAc in cyclohexane) to give 8l and isolated
as a white solid (0.83 g, 67%).mp 111–112 °C; IR
νmax (film): 3164,
3141, 3082, 1248, 1198, 1132, 1026, 790 cm–1; 1H NMR (700 MHz, DMSO-d6): δ
8.61 (d, J = 1.2 Hz, 1H), 8.03 (d, J = 1.1 Hz, 1H), 8.01 (apparent dd, J = 8.6, 0.5
Hz, 1H), 7.98 (apparent dd, J = 8.6, 0.5 Hz, 1H); 13C NMR (176 MHz, DMSO-d6): δ
136.0, 134.9, 133.7, 132.9, 132.1, 130.6, 127.7, 125.7 (q, J = 30.2 Hz), 122.1 (q, J = 276.6 Hz);
LCMS: MS m/z: 282.0 [M + H]+; HRMS C9H4Cl2F3N: calcd 281.9807 [M + H]+; found, 281.9807.Spectroscopic
and analytical data for 8l are presented
in the Supporting Information.
*Caution* Step
1: Sodium nitrite (360 mg, 5.22 mmol, 1.2 equiv) was added portionwise
to a solution of 2,4-dichloro-3-(trifluoromethyl)aniline 11a (1.0 g, 4.35 mmol, 1.0 equiv) in TFA (10 mL) at 0 °C over 30
min. The reaction mixture was then allowed to warm to RT over 90 min.
H2O (1.0 mL) was added and then the mixture was recooled
to 0 °C. Sodium azide (310 mg, 4.78 mmol, 1.1 equiv) was added
portionwise over 30 min, then the reaction was allowed to warm to
RT over 1 h. The mixture was basified to pH 8–9 by dropwise
addition of saturated aqueous NaHCO3 and then extracted
with CH2Cl2 (ca. 250 mL). The organic phase
was separated, diluted with t-BuOH (10.0 mL), and
then cautiously concentrated under reduced pressure at 25 °C
to provide a solution of crude azide 12a in t-BuOH, which was used without further purification.Step 2:
Copper(II) sulfate pentahydrate (217 mg, 0.87 mmol, 0.2 equiv), sodium l-ascorbate (345 mg, 1.74 mmol, 0.4 equiv), H2O (10
mL), and then trimethylsilylacetylene (0.6 mL, 4.35 mmol, 1.0 equiv)
were added to the solution of crude azide 12a from step
1. The reaction mixture was stirred at RT for 16 h, diluted with H2O (20 mL), extracted with EtOAc (ca. 100 mL), and the organic
phase was washed with H2O (ca. 20 mL) and brine (ca. 20
mL). The organic phase was dried, filtered, and concentrated under
reduced pressure. The crude product was purified by column chromatography
(0–30% EtOAc in cyclohexane) to give 17 (390 mg,
1 .1 mmol, 25%) as an oily solid.1H NMR (400 MHz,
DMSO-d6): δ 8.62 (s, 1 H), 8.01
(d, J = 8.8 Hz, 1
H), 7.96 (d, J = 8.8 Hz, 1 H), 0.33 (s, 9H).Step 3: 17 (100 mg, 0.28 mmol, 1.0 equiv) and K2CO3 (117 mg, 0.85 mmol, 3.0 equiv) were dried for
2 h under vacuum (0.5 mbar, RT). >99% methanol-d4 (5.0 mL) was added under an argon atmosphere and stirred
at RT for 3 days, before addition of >99% D2O (5.0 mL).
The vessel was quickly transferred to a rotary evaporator and the
organics were removed under reduced pressure, then the vessel was
quickly resealed. Alumina-treated anhydrous CH2Cl2 (ca. 10 mL) was added, stirred for 15 min, and then the organic
layer was removed via a syringe and concentrated under reduced pressure.The crude product was purified by column chromatography (0–30%
EtOAc in cyclohexane) to give d2-8l (>90% d2-isotope) and isolated
as a white solid (60 mg, 67%).1H NMR (700 MHz, DMSO-d6): δ 7.98 (apparent dd, J = 8.7, 0.5 Hz, 1H),
7.72 (apparent dd, J = 8.7, 0.6 Hz, 1H); 13C NMR (176 MHz, DMSO-d6): δ 153.3,
134.14–133.53 (m), 133.0 (q, J = 1.6 Hz),
131.6, 131.3, 128.3, 127.06–126.53 (m), 122.4 (q, J = 275.7 Hz), 121.8 (q, J = 29.5 Hz); LCMS m/z: 284.2 [M + H]+.
1-(2,3,4-Trichlorophenyl)-1H-1,2,3-triazole
(8m)
Steps 1 and 2: azide 12 (R2 = R3 = R4 = Cl) was prepared by General
Method 1.4 from 2,3,4-trichloroaniline (200 mg, 1.0 mmol, 1.0 equiv)
and trimethyl(propargyl)silane and was used directly in step 3.Step 3: Na2CO3 (5.0 g, 47.2 mmol, 47.2 equiv)
was added to azide 12 in MeOH (5.0 mL) and stirred vigorously
at RT for 16 h. Aqueous HCl (2 M, ca. 60 mL) was cautiously added;
once gas evolution had ceased, the organics were removed under reduced
pressure and the mixture was extracted with CH2Cl2 (ca. 60 mL). The organic phase was dried, filtered, and concentrated
under reduced pressure. The crude product was purified by column chromatography
(0–30% EtOAc in cyclohexane) to give 8m, which
was isolated as an off-white solid (181 mg, 72%).1H NMR (600 MHz, DMSO-d6): δ 8.62
(d, J = 0.9 Hz, 1H), 8.02 (d, J =
1.0 Hz, 1H), 7.93 (d, J = 8.7 Hz, 1H),
7.75 (d, J = 8.7 Hz, 1H); 13C NMR (151
MHz, DMSO-d6): δ 135.1, 134.6, 133.7,
131.7, 129.8, 129.5, 127.6, 127.4; LCMS: MS 248.0 m/z: [M + H]+.
Chlorotrimethylsilane
(1.9 mL, 15.0 mmol, 15.0 equiv) was added dropwise to a solution of
2,4-dichloro-3-(trifluoromethyl)aniline 11a (230 mg,
1.0 mmol, 1.0 equiv), 1,2-diformylhydrazine (264 mg, 3.0 mmol, 3.0
equiv), and NEt3 (1.0 mL, 7.0 mmol, 7.0 equiv) in pyridine
(9.0 mL) at 0 °C. The reaction was then heated to 115 °C
for 2.5 h and then to 130 °C for 20 h. The reaction was cooled
to RT, H2O (2.0 mL) was added, and then the mixture was
concentrated under reduced pressure. The crude product was purified
by reversed-phase chromatography (0–100% MeCN in H2O, 0.1% NH4OH modifier) to give 9a, which
was isolated as a tan solid (140 mg, 50%).1H NMR
(700 MHz, DMSO-d6): δ 8.86 (s, 1H),
8.00 (d, J = 8.7, 1H), 7.96 (d, J = 8.7 Hz, 1H); 13C NMR (176 MHz, DMSO-d6): δ 143.3, 134.2, 133.4, 133.1, 132.0, 130.9,
125.5 (q, J = 30.2 Hz), 122.1 (q, J = 276.6 Hz); LCMS m/z: 282.1 [M
+ H]+.
*Caution* Step 1: Hydrazine monohydrate (60 μL, 1.2 mmol, 1.5 equiv)
was added to a solution of 2,3,5-trichloro-4-trifluoromethyl pyridine 18 (200 mg, 0.80 mmol, 1.0 equiv) in EtOH (0.5 mL). The reaction
was slowly warmed to 85 °C behind a blast shield over 4 h, concentrated
under reduced pressure, and the resultant solid was suspended in aqueous
HCl (1 M, 10 mL) at RT, then cooled to 0 °C. A solution of sodium
nitrite (110 mg, 1 .6 mmol) in H2O (0.5 mL) was added over
30 min.Step 2: The mixture was stirred for a further 30 min
before cautious addition of aqueous NaOH (5 M) to adjust the pH to
8–9. H2O (2.0 mL), t-BuOH (2.0
mL), sodium l-ascorbate (159 mg, 0.8 mmol, 1.0 equiv), copper(II)
sulfate pentahydrate (20 mg, 0.08 mmol, 0.1 equiv), and trimethylsilylacetylene
(110 μL, 0.8 mmol, 1.0 equiv) were added and the reaction was
heated to 40 °C for 16 h. The mixture was cooled to RT, diluted
with EtOAc (ca. 50 mL), the organic phase was dried, filtered, and
concentrated under reduced pressure. The crude product was purified
by reversed-phase chromatography (0–100% MeCN in H2O, 0.1% formic acid modifier) to give 9c, which was
isolated as a white solid (5 mg, 2%).1H NMR (700
MHz, CDCl3): δ 8.66 (d, J = 0.6
Hz, 1H), 8.08 (d, J = 1.2 Hz, 1H),
7.90 (d, J = 1.2 Hz, 1H); 13C NMR (176
MHz, CDCl3): δ 149.2, 147.1, 136.7 (q, J = 32.1 Hz), 133.8, 132.0, 126.7, 125.1, 121.2 (q, J = 278.6 Hz); LCMS: MS m/z: 283.0 [M + H]+.
Authors: U Scherf; D T Ross; M Waltham; L H Smith; J K Lee; L Tanabe; K W Kohn; W C Reinhold; T G Myers; D T Andrews; D A Scudiero; M B Eisen; E A Sausville; Y Pommier; D Botstein; P O Brown; J N Weinstein Journal: Nat Genet Date: 2000-03 Impact factor: 38.330
Authors: Satoshi Kakugawa; Paul F Langton; Matthias Zebisch; Steve Howell; Tao-Hsin Chang; Yan Liu; Ten Feizi; Ganka Bineva; Nicola O'Reilly; Ambrosius P Snijders; E Yvonne Jones; Jean-Paul Vincent Journal: Nature Date: 2015-02-25 Impact factor: 49.962
Authors: Yuguang Zhao; William Mahy; Nicky J Willis; Hannah L Woodward; David Steadman; Elliott D Bayle; Benjamin N Atkinson; James Sipthorp; Luca Vecchia; Reinis R Ruza; Karl Harlos; Fiona Jeganathan; Stefan Constantinou; Artur Costa; Svend Kjær; Magda Bictash; Patricia C Salinas; Paul Whiting; Jean-Paul Vincent; Paul V Fish; E Yvonne Jones Journal: ACS Chem Neurosci Date: 2022-06-22 Impact factor: 5.780
Figure 1
Figure 2
Figure 3
Figure 4
Scheme 1
Scheme 5
Scheme 2
Scheme 3
Scheme 4
Figure 5
Figure 6
Scheme 6