1,2,4-Triazolo-[1,5-a]pyridine HIF Prolylhydroxylase Domain-1 (PHD-1) Inhibitors with a Novel Monodentate Binding Interaction

Article abstract of DOI:10.1021/acs.jmedchem.7b00352

Herein we describe the identification of 4-{[1,2,4]triazolo[1,5-a]pyridin-5-yl}benzonitrile-based inhibitors of the hypoxia-inducible factor prolylhydroxylase domain-1 (PHD-1) enzyme. These inhibitors were shown to possess a novel binding mode by X-ray crystallography, in which the triazolo N1 atom coordinates in a hitherto unreported monodentate interaction with the active site Fe²⁺ ion, while the benzonitrile group accepts a hydrogen-bonding interaction from the side chain residue of Asn315. Further optimization led to potent PHD-1 inhibitors with good physicochemical and pharmacokinetic properties.

Full text of DOI:10.1021/acs.jmedchem.7b00352

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Article
1,2,4-Triazolo-[1,5-a]pyridine HIF Prolylhydroxylase Domain-1
(PHD-1) Inhibitors With a Novel Monodentate Binding Interaction
Saleh Ahmed, Andrew Ayscough, Greg R Barker, Hannah E. Canning, Richard Davenport, Robert
Downham, David Harrison, Kerry Jenkins, Natasha Kinsella, David G. Livermore, Susanne Wright,
Anthony David Ivetac, Robert Skene, Steven J. Wilkens, Natalie A. Webster, and Alan G. Hendrick
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 08 Jun 2017
Downloaded from http://pubs.acs.org on June 9, 2017
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Journal of Medicinal Chemistry
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1,2,4ꢀTriazoloꢀ[1,5ꢀa]pyridine HIF Prolylhydroxylase
Domainꢀ1 (PHDꢀ1) Inhibitors With a Novel
Monodentate Binding Interaction
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Saleh Ahmed,† Andrew Ayscough, † Greg R. Barker, † Hannah E. Canning, † Richard Davenport,†
Robert Downham, † David Harrison, † Kerry Jenkins,*, † Natasha Kinsella, † David G. Livermore,†
Susanne Wright, † Anthony D. Ivetac, ǂ Robert Skene, ǂ Steven J. Wilkens, ǂ Natalie A. Webster ¥ and
Alan G. Hendrick. ¥
† Department of Medicinal Chemistry, Takeda Cambridge Ltd., 418 Cambridge Science Park, Milton
Road, Cambridge, CB4 0PZ, United Kingdom
ǂ Department of Computational Sciences & Crystallography, Takeda California Inc., 10410 Science
Center Dr, San Diego, CA 92121, United States
¥ Department of in vitro Pharmacology, Takeda Cambridge Ltd., 418 Cambridge Science Park, Milton
Road, Cambridge, CB4 0PZ, United Kingdom
ABSTRACT: Herein we describe the identification of 4ꢀ{[1,2,4]triazolo[1,5ꢀa]pyridinꢀ5ꢀyl}benzonitrile
based inhibitors of the hypoxiaꢀinducible factor prolylhydroxylase domainꢀ1 (PHDꢀ1) enzyme. These
inhibitors were shown to possess a novel binding mode by Xꢀray crystallography, in which the triazolo
N1 atom coordinates in a hitherto unreported monodentate interaction with the active site Fe2+ ion,
whilst the benzonitrile group accepts a hydrogen bonding interaction from the side chain residue of
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Asn315. Further optimization led to potent PHDꢀ1 inhibitors with good physicochemical and
pharmacokinetic properties.
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Introduction. Hypoxia-inducible factors (HIFs) are heterodimeric transcription factors comprising of
an alpha subunit (HIFꢀ1α, HIFꢀ2α or HIFꢀ3α) and a beta subunit (HIFꢀ1β, HIFꢀ2β or HIFꢀ3β). HIFs,
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and in particular HIFꢀ1α, have been shown to be master regulators of oxygen homeostasis in response to
limited oxygen environments.1 Multiple target genes have been identified with the main downstream
effects being to increase anaerobic respiration via stimulation of the glycolytic pathways,2 to improve
tissue oxygenation by enhancing vascularisation (angiogenesis),3 and increasing red blood cell
production (erythropoiesis).4 In normoxic environments HIFꢀ1 is negatively regulated by hydroxylation
of the αꢀsubunit and subsequent proteosomal degradation via the von HippelꢀLindau (VHL) ꢀ E3
ubiquitin ligase system.5 The hydroxylation of HIFs are mediated by prolylhydroxylase domain
enzymes (PHDs) which are nonꢀheme iron(II) dependent enzymes of which there are three catalytically
competent isoforms (PHDꢀ1, 2 & 3).6 The PHD enzymes utilise 2ꢀoxoglutarate (2ꢀOG) and molecular
oxygen as substrates and hence in oxygen depleted environments (i.e. hypoxia) their ability to mediate
HIF turnover is diminished resulting in HIF stabilisation, binding to hypoxia response elements (HRE)
and gene activation. Despite the active site across the PHD isoforms being highly conserved, the
different isoforms appear to perform some distinct as well as overlapping roles. This can be attributed in
part to differential expression and localisation but also due to subtle differences in substrate
specificities.6f
There is increasing interest in exploring pharmacological intervention of the HIFꢀPHD axis in a variety
of disease settings due to its key role in ischemia and inflammation, such as in inflammatory bowel
disorders (e.g. Ulcerative Colitis, Crohn’s disease),7 ischemic reperfusion injuries (cardiac,8
cerebrovascular,9 liver 10 and renal 11) and neurodegenerative processes. 12 Other potential clinical
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Journal of Medicinal Chemistry
settings include anemia,13 wound healing and organ transplantation.14 There have been extensive efforts
towards the development of small molecule inhibitors of PHD enzymes.15 Direct pharmacological
inhibitors of PHDs fall into four categories: (1) Fe2+ substitutes (e.g. Co2+, Ni2+ and Cu2+); (2) Fe2+
sequesters (e.g. desferrioxamine, Lꢀmimosine etc.); (3) 2ꢀOG mimetics (e.g. dimethyloxalylglycine and
NꢀoxalylꢀDꢀphenylalanine); and (4) targeted activeꢀsite inhibitors (e.g. Roxadustat (formerly FGꢀ
4592),16 Nꢀ{[5ꢀ(3ꢀchlorophenyl)ꢀ3ꢀhydroxyꢀ2ꢀpyridinyl]carbonyl}glycine (Vadadustat, formerly AKBꢀ
6548),17 2ꢀ[6ꢀ(4ꢀmorpholinyl)ꢀ4ꢀpyrimidinyl]ꢀ4ꢀ(1Hꢀ1,2,3ꢀtriazolꢀ1ꢀyl)ꢀ1,2ꢀdihydroꢀ3Hꢀpyrazolꢀ3ꢀone
(Molidustat, formerly BAY 85ꢀ3934)18 etc.). Clearly the scope for development and use of iron
substitutes, sequesters and 2ꢀOG mimetics for clinical use are severely hampered by the lack of
specificity over a multitude of offꢀtarget metabolic systems. Targeting the PHD active site gives far
more scope for specific and selective modulation of PHDs/HIFs, however it is a notable facet of the
majority of inhibitors reported to date that chelation binding to the activeꢀsite ferrous ion plays a
significant role in driving ligand potency. This has nonꢀtrivial consequences from a molecular properties
standpoint that may limit the potential for some of these compounds as in vivo tool compounds and as
potential drug candidates.
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Results and Discussion. A fragment screening campaign identified triazolopyrimidine 1 as a
moderately potent inhibitor of PHDꢀ1 (7.1μM) with a reasonably good ligand efficiency19 of 0.30
(Figure 1.). We were intrigued by the relative simplicity of the architecture of this hit and by its
structural divergence from other PHD inhibitor motifs seen internally and in the literature.15 Initially, we
sought to investigate the key requirements for binding through systematic deletion of the heterocyclic
core nitrogen atoms. Removal of one of the 5ꢀmembered rings’ nonꢀbridgehead nitrogen atoms
(compound 2) resulted in a 3ꢀfold drop in potency, suggesting this nitrogen might be making a modest
binding interaction. Deletion of the other nonꢀbridgehead nitrogen from the 5ꢀmembered ring
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(compound 3) abolished potency demonstrating that this atom has a key role in binding. Conversely,
removal of the nonꢀbridgehead nitrogen from the 6ꢀmembered ring (compound 4) actually improved
potency ~10ꢀfold to 0.67μM (L.E. 0.36). In view of the promising leadꢀlike potential of this compound
we decided to continue optimization on the 1,2,4ꢀtriazoloꢀ[1,5ꢀa]pyridine scaffold.
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Figure 1. Initial fragment lead and exploratory core SAR.
Keeping the core triazolo[1,5ꢀa]pyridine constant, we sought to investigate the SAR of the phenyl ‘top
ring’ by conducting a positional scan of simple substituents (Table 1.). The SAR showed a preference
for electron withdrawing substituents in the 4ꢀposition. In particular, the paraꢀnitrile compound, 17,
displayed a significant increase in potency and binding efficiency (0.034μM, L.E. 0.44).
Table 1. SAR of ‘Top Ring’
R
PHDꢀ1 IC50 (ꢁM)
H
30
5
6
7
4ꢀCl
3ꢀCl
0.55
26
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2ꢀCl
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8
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3
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9
4ꢀF
1.3
28
9
3ꢀF
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2ꢀF
24
4ꢀMe
3ꢀMe
4ꢀOMe
3ꢀOMe
2ꢀOMe
4ꢀCN
3ꢀCN
5.6
>100
26
55
>100
0.034
61
XꢀRay Crystallography: Crystal structures of ligands bound to PHDꢀ2 are prevalent in the literature, but
until now, no PHDꢀ1 crystal structures have been reported. During the course if our investigation we
obtained a 2.5Å resolution Xꢀray crystal structure of 17 bound into PHDꢀ1 using a coꢀcrystallization
method (Figure. 2).
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Figure 2. Xꢀray crystal structure of 17 bound in the active site of PHDꢀ1 (PDB accession code 5V1B).
This showed a number of interesting features (Figure 2 and 3), most notably a monodentate
coordination of the triazolopyridine N1ꢀatom with the active site Fe2+ ion. To date, virtually all reported
competitive PHD inhibitors are shown (or are postulated) to make a bidentate chelation interaction to
iron. It has been proposed through computational docking that the PHDꢀ2 inhibitor 6ꢀaminoꢀ1,3ꢀ
dimethylꢀ5ꢀ[(2ꢀpyridinylsulfanyl)acetyl]ꢀ2,4(1H,3H)ꢀpyrimidinedione (TM6089) coordinates in a nonꢀ
chelation fashion although this has not been substantiated by experimental methods.9a To the best of our
knowledge this is the first verified example of a monodentateꢀiron binding PHD inhibitor. The
significance of this is that many chelation binders invoke highly polar or ionisable moieties, such as
amides, phenols and hydroxyl groups. The physicochemical properties of such groups (e.g. pKa, PSA)
often runs counter to desirable drugꢀlike chemical space, especially for CNS indications where crossing
the bloodꢀbrain barrier presents a significant hurdle to achieving efficacious drug concentrations.20
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Other noteworthy features include the benzonitrile moiety that projects into the protein inducing the
formation of an ‘Arg367ꢀout’ pocket with the nitrile group accepting a hydrogen bond interaction from
the side chain of Asn315. Many reported PHD inhibitors target Arg367 (Arg383 in PHDꢀ2 structures)
via a carboxylate or similarly acidic residue – again, moieties that are often not ideal for oral exposure.
Additionally, the phenyl ring is positioned appropriately to make a πꢀstacking interaction with Tyr287
and the triazolopyridine N3 atom is potentially making a waterꢀmediated interaction to Tyr313.
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H2O
3
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1
H2O
H2O
Figure 3. Ligand interaction diagram of 17 bound in the active site of PHDꢀ1.
The crystal structure suggests that there is very limited scope for extending substituents from all but the
7ꢀposition of the triazolopyridine bicyclic scaffold and that substitution of the benzonitrile ‘top ring’
may be restricted to small substituents. These assertions were in agreement with the observed SAR
(Table 2). Although the focus of our research led us to optimize our series against the PHDꢀ1 isoform
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we also generated a crystal structure of 17 bound into PHDꢀ2 (PDB accession code 5V18, not shown)
which showed essentially the same key binding features. This is unsurprising given the identity of the
active site region between PHDꢀ1 and PHDꢀ2, and consistent with the observation that PHDꢀ1 activity
broadly tracked PHDꢀ2 activity for these compounds (data not shown).
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Core Bicyclic SAR: Introduction of a methyl group at C2 resulted in >15ꢀfold drop in potency (19).
Similarly, an amino substituent at C2 lost 10ꢀfold potency relative to 17 and further Nꢀmethylation or Nꢀ
acylation substantially diminished potency. Incorporation of a methyl substituent at C8 was barely
tolerated and even a fluorine substituent resulted in >250ꢀfold drop off in potency. The former is
consistent with the steric constraints conferred by the binding of N1 to iron and the latter we speculate is
due to the electronic effects of fluorine making N1 a weaker coordinating atom. In the case of the
original triazolopyrimidine hit (1) it is probable that both Nꢀatoms are chelating to iron but in doing so
are presenting the ‘top ring’ in a subꢀoptimal orientation for additional beneficial interactions.
The C6 position could only accommodate small substituents (F, Me, NH2) with 10 to 20ꢀfold drop off
in potency, anything larger abolished potency. As predicted by the crystal structure, C7 was tolerant of a
variety of substituents with similar or improved potencies relative to 17. The vector from this position
leads out of the protein into solvent space, and towards a flexible loop region. This suggested that
opportunities for significant new binding interactions may be limited. Nonetheless, this position became
our main focus for tuning potency and optimizing the physicochemical properties of the series (vide
infra).
Top Ring SAR: The position ortho to the nitrile group supported fluorine substituents with good levels
of potency but anything larger abruptly diminished potency. The meta positions were slightly more
accommodating, with fluorine, methyl and amino retaining potency and chlorine and methoxy showing
only a small dropꢀoff, within 4ꢀfold of 17. Interestingly, CF3 was not tolerated suggesting steric
considerations alone do not explain the SAR and that electronics may also play an important role.
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Combinations of fluorine atoms (o and m positions) and methyl groups (m positions) were generally
well tolerated although diꢀmeta substituted compounds were generally less active than monoꢀsubstituted
analogues.
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In addition to exploring substituents on the top ring we sought to investigate heterocyclic replacements
with a view to altering properties such as lowering lipophilicity, increasing solubility and increasing
fractional sp3 character (Table 3). We found that introducing nitrogen atoms ortho to the nitrile resulted
in modest drops in potency relative to the benzonitrile analogue (55, 5ꢀfold and 56, 2.5ꢀfold). However,
heteroaromatics containing nitrogen atoms in the meta position relative to the nitrile had more
pronounced dropꢀoffs in potency (57, 8ꢀfold, 58, 38ꢀfold and 59, 40ꢀfold), especially when combined
with an ortho nitrogen. None of the 5ꢀmembered heteroaromatic top ring replacements showed any
appreciable inhibition against PHDꢀ1. This is unsurprising given the directional requirements of the
nitrile to making an efficient hydrogen bond with Asn315 whilst maintaining the N1 interaction with
iron. We did however observe some potency with saturated 6ꢀmembered heterocyclic replacements. The
cyano piperidine 64, showed 0.28μM potency whilst piperazine cyanamide was 1.8μM. Molecular
modeling shows that these ligands are able to adopt conformations that allow good binding interactions
to both Asn315 and iron, but that either the increased volume due to the sp3 hybridisation, or the loss of
the π-π interaction is detrimental to binding. The saturated cyano pyrrolidino and 2-
azaspiro[3.3]heptanes examples were not significantly active.
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Table 2. SAR of Core Bicycle and Top Ring.
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R2
R8
R7
R6
A
B
C
D
PHDꢀ1
IC50
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9
(ꢁM)
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Me
0.57
0.24
18
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20
21
22
23
24
25
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27
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29
30
31
32
NH2
NMe2
NHAc
2.5
F
9.6
Me
80
Me
0.039
0.013
0.025
0.008
0.46
0.46
0.77
>10
NH2
NHEt
NHAc
F
Me
NH2
NMe2
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Journal of Medicinal Chemistry
NHAc
>10
33
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3
4
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7
8
9
F
0.010
0.83
1.87
3.9
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46
47
48
49
50
Me
CF3
Cl
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F
0.050
0.074
6.1
Me
Et
NH2
NHAc
Cl
0.057
>10
0.14
0.14
>10
OMe
CF3
F
F
0.034
0.067
0.16
0.052
0.14
F
F
F
F
F
Me
Me
F
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Me
F
0.22
0.20
0.09
0.07
51
52
53
54
1
2
3
4
5
6
7
8
Me
Me
NH2
Me
Me
9
10
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NHAc
Table 3. Cyano-Heterocyclic Top Rings.
[Het]
PHDꢀ1 IC50 (ꢁM)
[Het]
PHDꢀ1
(ꢁM)
IC50
0.17
>10
>10
>10
0.28
55
56
57
58
61
0.084
0.27
1.28
62
63
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8
1.37
>10
1.8
59
65
9
10
>10
>10
60
66
67
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Table 4. Mouse Pharmacokinetic Profiles of Selected Compounds.
%P
PB
(mu
)
MD
Kp,u R1
Cl
Kinetic
Sol.
(ꢀM)
AUC0ꢀt
(ng.h/m
L)a
Cmax
cLo PS
PAMPA
(nm/s)
F%
40
B/P
(ꢀm)a
(mL/min/
kg)b
effl gP
A
u
uxc
17
176
0.80
108
71.3 0.95 1.11 0.8
2.59 54
44
246
39
1869
5.58
100 31.6
79.0 0.88 1.59 0.68 3.10 54
189
409
53
40038
14.8
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a Cassette PK, male C57BL6 mice, dosed 3 mg/kg p.o. in 0.5% methylcellulose. b Cassette
PK, male C57BL6 mice, dosed 0.5 mg/kg i.v. in DMA:Saline (50:50). c Absorption Systems
ExpressPlus BBB penetration potential determined using MDR1ꢀMDCK cell monolayers.
Pharmacokinetic profiles: The early lead compound 17 was found to have modest oral exposure
in mouse largely driven by high clearance (Table 4). This compound showed a moderate rate of
microsomal turnover in vitro (data not shown) which in part explains the high in vivo clearance.
Comparable brain exposure levels were seen (Kp,uu 1.11 and B/P 0.95) suggesting that this series
could be amenable to optimization for CNS indications if peripheral exposure levels could be
enhanced. Accordingly, incorporation of a methyl group in the meta position of the ‘top ring’
(39) improved oral exposure whilst maintaining good levels of CNS penetration. In addition to
reducing clearance through improved microsomal stability we observed that the methyl group
improves both the solubility and permeability presumably by reducing the planarity of the
molecule via the axial twist that this substituent imparts. The SAR data and crystal structure had
shown that the 7ꢀposition was amenable to modification, so we optimised substituents at this
position to tailor physicochemical and ADME properties. For example, the introduction of a 7ꢀ
amino group (53) was found to dramatically enhance peripheral exposure by further reducing
clearance. This might be attributed to the lowering of LogP or it could be masking a metabolic
softꢀspot. Although 53 exhibits excellent peripheral exposure, the compound does not partition
well into the brain (Kp,uu 0.39 and B/P 0.21). Similarly the 7ꢀNꢀacetamide (54) exhibits
reasonable peripheral exposure but poor brain levels (Kp,uu 0.02 and B/P 0.03). Both of these
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compounds have high PSA and they have reduced solubility and permeability relative to the 7ꢀH
analogue (39). It is well known that these factors impact a molecule’s ability to cross the bloodꢀ
brain barrier. In addition to adverse physicochemical properties for CNS penetration, these
compounds were shown to be substrates for MDR1 in an in vitro MDCKꢀMDR1 assay (efflux
ratios 12 and 99 respectively) suggesting that active efflux from the CNS could be a major
contributor to reduced CNS levels of free drug.
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Scheme 1. General Synthetic Routes to 1,2,4ꢀTriazoloꢀ[1,5ꢀa]pyridines
Scheme 2. Preparation of 5ꢀHaloꢀ1,2,4ꢀtriazoloꢀ[1,5ꢀa]pyridine Intermediates
(i) R2ꢀC(OMe)2NMe2, 70 – 110 °C, 1.25 – 12 h, EtOH or IPA or toluene; (ii) HONH2.HCl, rt –
50 °C, 1 – 2 h, IPA or MeOH; (iii) TFAA or Eaton’s reagent, 0.33 – 18 h, rt – 105 °C, THF.
Scheme 3. Synthetic Routes to 7ꢀAmino Analogues
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(i) 2M (aq.) LiOH, rt, 1 h, THF/MeOH (1:1); (ii) (PhO)2PON3, Et3N, 0.5 h, tꢀBuOH, 90 °C,
toluene; (iii) (4ꢀcyanoꢀ2ꢀmethylphenyl)boronic acid, PdCl2(dppf), Na2CO3, 100 °C, 2 h, 1,4ꢀ
dioxane/water (5:1); (iv) HCl, 50 °C, 2 h, 1,4ꢀdioxane.
Chemistry. The majority of compounds were accessed via corresponding 5ꢀhaloꢀ1,2,4ꢀtriazoloꢀ
[1,5ꢀa]pyridines using one of four routes (Scheme 1): direct SuzukiꢀMiyaura coupling (route A);
21 conversion to the triꢀnꢀbutyl or trimethyl stannane followed by Stille coupling (route B); 22
conversion to a boronic acid derivative followed by SuzukiꢀMiyaura coupling (route C) and
direct SNAr displacement (route D). In cases where the 5ꢀhaloꢀ1,2,4ꢀtriazoloꢀ[1,5ꢀa]pyridines
were not commercially available the scaffolds were typically synthesised from corresponding 2ꢀ
aminoꢀ6ꢀhalopyridines whereby the aminopyridine was condensed with a dimethylamide
dimethyl acetal and then treated with hydroxylamine hydrochloride. The hydroxyamidine was
cyclised under dehydrating conditions (e.g. TFAA or Eaton’s reagent) to give the corresponding
5ꢀhaloꢀ1,2,4ꢀtriazoloꢀ[1,5ꢀa]pyridine (Scheme 2).23 In the cases of 7ꢀamino substituted
compounds the amino group could be introduced in a couple of ways (Scheme 3). Starting from
the 5,7ꢀdichloro intermediate (68), accessed using the previously described route, the aryl ring
could be installed by regioselective SuzukiꢀMiyaura reaction at the 5ꢀposition. The 7ꢀchloro
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could then be displaced under BuchwaldꢀHartwig24 amination conditions to give 7ꢀalklyamino
products (e.g. 27) or by using modified Buchwald ‘amidation’ conditions25 to give 7ꢀaminoacyl
compounds (e.g. 28). Similarly, these modified Buchwald conditions could be used to prepare 7ꢀ
Nꢀtert-butoxycarbonylamino intermediates that could be deprotected to 7ꢀamino compounds
(e.g. 26). Alternatively, the 5ꢀchloroꢀ7ꢀcarboxylic ester intermediate (69), accessed using the
previously described route, could be converted to the 7ꢀNꢀtert-butoxycarbonylamino
intermediate (70) using a Curtius rearrangement. Subsequent SuzukiꢀMiyaura coupling and
deprotections for example, gave 53.
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Conclusion. We have identified a novel series of potent PHDꢀ1 inhibitors and we report the first
PHDꢀ1 isoform Xꢀray crystal structure with one of our lead compounds bound in to the active
site. The structural data shows that this chemotype operates through a unique monodentate
binding interaction with the active site iron in concert with other key interactions accessed via an
induced ‘Arg367ꢀout’ pocket. The observed SAR is consistent with many of the features
revealed by the Xꢀray crystal structure. Unlike many of the reported PHD inhibitors this scaffold
is highly amenable to optimization for good oral exposure, both peripherally and in the CNS
making these useful tool compounds to study in vivo pharmacological effects of PHD enzyme
inhibition and for further evaluation as potential drug candidates.
Experimental Section. The synthesis of key compounds is described below. Full experimental
procedures for the synthesis of all other compounds are described in the supplementary
information.
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General Procedures. All solvents and chemicals were used as purchased without further
purification. The progress of all reactions was monitored on Waters ZQꢀ2000 system UPLC
systems. Ultra Performance Liquid Chromatography (UPLC) with UV (photodiode array)
detection over a wide range of wavelengths, normally 220ꢀ450 nm, using a Waters (trade mark)
Acquity UPLC system equipped with Acquity UPLC BEH, HSS or HSS T3 C18 columns
(2.1mm id x 50mm long) operated at 50 or 60 °C. Mobile phases typically consisted of
acetonitrile mixed with water containing either 0.1% formic acid, 0.1% TFA or 0.025%
ammonia. Mass spectra were recorded with a Waters SQD single quadrupole mass spectrometer
using atmospheric pressure ionization. Proton (1H) NMR spectra were recorded on a Bruker
Avance 400 or a Bruker Avance 300 using deuterated solvents specified in the experiments.
Chemical shifts are given in parts per million (ppm) (δ relative to residual solvent peak).
Preparative HPLC was performed using Agilent Technologies 1100 Series system or a Waters
autopurification LC/MS system or a Shimadzu semi prep HPLC system, typically using Waters
19 mm id x 250 mm long C18 columns such as XBridge™ or SunFire™ 5 ꢁm materials at room
temperature. Mobile phases typically consisted of acetonitrile mixed with water containing
either 0.1% formic acid or 0.1% ammonia, unless otherwise stated. Super Critical Fluid
Chromatography (SFC) chiral separations were performed on a Waters prep 30/MS system,
using a flow rate of 30 mL/min, temperature of 40 °C and a pressure of 100 bar. Mobile phases
typically consisted of supercritical CO2 and a polar solvent such as methanol, ethanol or
isopropanol. Column type and eluent are detailed for individual examples. All final compounds
were determined to be >95% purity by LC/MS.
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4-{[1,2,4]Triazolo[1,5-a]pyridin-5-yl}benzonitrile (17). A stirred suspension of 5ꢀbromoꢀ
[1,2,4]triazolo[1,5ꢀa]pyridine [CAS 143329ꢀ58ꢀ2] (2.00 g, 10.10 mmol), (4ꢀcyanophenyl)boronic
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acid [CAS 126747ꢀ14ꢀ6] (1.484 g, 10.10 mmol), tetrakis(triphenylphosphine)palladium(0)
(0.817 g, 0.707 mmol) and cesium carbonate (3.95 g, 12.12 mmol) in 1,4ꢀdioxane (28 mL) and
water (5.6 mL) was placed under an inert atmosphere with 3 x N2/vacuum cycles. The reaction
was heated to 100 °C for 3 hrs. The reaction was diluted with EtOAc whilst hot and was filtered
through Celite, washing with further amounts of EtOAc. The filtrate was concentrated in vacuo.
The crude product was recrystallized from hot EtOH to afford 4ꢀ{[1,2,4]triazolo[1,5ꢀa]pyridinꢀ5ꢀ
yl}benzonitrile (17) as a white solid (909 mg, 4.13 mmol, 45%). 1H NMR (400 MHz, DMSOꢀd6)
δ ppm 7.49 ꢀ 7.53 (m, 1 H), 7.78 ꢀ 7.84 (m, 1 H), 7.94 ꢀ 7.98 (m, 1 H), 8.05 ꢀ 8.10 (m, 2 H), 8.20
ꢀ 8.25 (m, 2 H), 8.58 (s, 1 H), MS ES+: 221
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3-Methyl-4-{[1,2,4]triazolo[1,5-a]pyridin-5-yl}benzonitrile (39). A suspension of 5ꢀbromoꢀ
[1,2,4]triazolo[1,5ꢀa]pyridine [CAS 143329ꢀ58ꢀ2] (1.75 g, 8.81 mmol), (4ꢀcyanoꢀ2ꢀ
methylphenyl)boronic acid [CAS 313546ꢀ18ꢀ8] (1.56 g, 9.69 mmol), PdCl2(dppf) (0.65 g, 0.881
mmol) and sodium carbonate (1.87 g, 17.62 mmol) in 1,4ꢀdioxane (24 mL) and water (4.9 mL)
was flushed with N2 and heated to 100 °C for 1h. The reaction was cooled and partitioned
between EtOAc and water. The organic phase was collected, washed with brine, dried (phase
separator) and concentrated in vacuo. The resulting residue was purified by flash
chromatography (0ꢀ70% EtOAc in petroleum ether on basic silica). The appropriate fractions
were collected and concentrated in vacuo. The resulting residue was recrystallized from hot
EtOH to afford 3ꢀmethylꢀ4ꢀ{[1,2,4]triazolo[1,5ꢀa]pyridinꢀ5ꢀyl}benzonitrile (39) as a white solid
(991 mg, 4.23 mmol, 48%). 1H NMR (400 MHz, DCMꢀd2) δ ppm 2.19 (s, 3 H), 7.05 (d, J = 7.07
Hz, 1 H), 7.54 (d, J = 7.83 Hz, 1 H), 7.64 ꢀ 7.77 (m, 3 H), 7.85 ꢀ 7.92 (m, 1 H), 8.33 (s, 1 H), MS
ES+: 235
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4-[7-Amino-[1,2,4]triazolo[1,5-a]pyridin-5-yl]-3-methylbenzonitrile (53). A solution of
methyl 2ꢀaminoꢀ6ꢀchloropyridineꢀ4ꢀcarboxylate [CAS 1005508ꢀ80ꢀ4] (10.0 g, 53.6 mmol) and
DMFꢀDMA (7.18 mL, 53.6 mmol) was heated to 70 °C for 2h. More DMFꢀDMA (7.18 mL, 53.6
mmol) was added and the reaction was heated for a further 4h. The reaction was cooled to 50 °C
and hydroxylamine hydrochloride (3.72 g, 53.6 mmol) was added. The reaction was stirred at 50
°C for 2h. The reaction was concentrated in vacuo and the resulting residue was triturated with
EtOH. The precipitate was filtered and dried under vacuum to afford methyl 2ꢀchloroꢀ6ꢀ(N'ꢀ
hydroxyformimidamido)isonicotinate (12.0 g, 98%). 1H NMR (400 MHz, DMSOꢀd6) δ ppm 3.32
(s, 3 H), 7.24 (d, J = 1.14 Hz, 1 H), 7.59 (d, J = 1.14 Hz, 1 H), 7.72 (d, J = 9.73 Hz, 1 H), 10.02
(d, J = 9.73 Hz, 1 H), 10.37 ꢀ 10.43 (m, 1 H), MS ES+: 230
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A solution of methyl 2ꢀchloroꢀ6ꢀ(N'ꢀhydroxyformimidamido)isonicotinate (12.0 g, 52.3 mmol)
in THF (105 mL) was treated with TFAA (14.8 mL, 105 mmol). The reaction was heated to 40
°C for 18h. The reaction was quenched and basified with saturated (aq) NaHCO3 and partitioned
between EtOAc and water. The organic was collected, dried (phase separator) and concentrated
in vacuo. The resulting residue was purified by flash chromatography (0ꢀ100% EtOAc in
petroleum ether on SiO2) to afford methyl 5ꢀchloroꢀ[1,2,4]triazolo[1,5ꢀa]pyridineꢀ7ꢀcarboxylate
(69) (6.44g, 58%). 1H NMR (400 MHz, MeOHꢀd4) δ ppm 4.03 (s, 3 H), 7.88 (d, J = 1.52 Hz, 1
H), 8.43 (d, J = 1.52 Hz, 1 H), 8.66 (s, 1 H), MS ES+: 212.
A solution of methyl 5ꢀchloroꢀ[1,2,4]triazolo[1,5ꢀa]pyridineꢀ7ꢀcarboxylate (69) (6.44 g, 30.4
mmol) in methanol (51 mL) and THF (51 mL) was treated with LiOH (2M aq) (30.4 mL, 60.9
mmol). The reaction was stirred at room temperature for 1h. The reaction was acidified with 2N
HCl (30 mL) and the resulting precipitate was filtered, washed with MeOH and dried to afford 5ꢀ
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chloroꢀ[1,2,4]triazolo[1,5ꢀa]pyridineꢀ7ꢀcarboxylic acid (5.15 g, 86%). 1H NMR (400 MHz,
DMSOꢀd6) δ ppm 7.79 (d, J = 1.52 Hz, 1 H), 8.35 (d, J = 1.39 Hz, 1 H), 8.79 (s, 1 H), 13.98 (br.
s., 1 H), MS ES+: 198
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A suspension of 5ꢀchloroꢀ[1,2,4]triazolo[1,5ꢀa]pyridineꢀ7ꢀcarboxylic acid (1.00 g, 5.06 mmol) in
toluene (15 mL) was treated sequentially with TEA (1.058 mL, 7.59 mmol), tertꢀbutanol (0.484
mL, 5.06 mmol) and diphenyl phosphorazidate (1.091 mL, 5.06 mmol). The reaction was heated
to 90 °C under N2 for 30 min. The reaction was concentrated in vacuo and the resulting residue
was purified by flash chromatography (0ꢀ100% EtOAc in petroleum ether on SiO2) to afford tertꢀ
butyl (5ꢀchloroꢀ[1,2,4]triazolo[1,5ꢀa]pyridinꢀ7ꢀyl)carbamate (70) (1.02 g, 75%). 1H NMR (400
MHz, DCMꢀd2) δ ppm 1.53 (s, 9 H), 6.94 ꢀ 7.06 (m, 1 H), 7.39 (d, J = 2.02 Hz, 1 H), 7.68 (d, J =
2.02 Hz, 1 H), 8.25 (s, 1 H), MS ES+: 213
A suspension of tertꢀbutyl (5ꢀchloroꢀ[1,2,4]triazolo[1,5ꢀa]pyridinꢀ7ꢀyl)carbamate (70) (0.200 g,
0.744 mmol), (4ꢀcyanoꢀ2ꢀmethylphenyl)boronic acid [CAS 313546ꢀ18ꢀ8] (120 mg, 0.744 mmol),
PdCl2(dppf) (54.5 mg, 0.074 mmol) and sodium carbonate (158 mg, 1.489 mmol) in 1,4ꢀdioxane
(2 mL) and water (0.400 mL) was flushed with N2 and heated to 100 °C for 2h. The reaction was
cooled to room temperature and partitioned between EtOAc and water. The organic was
collected, washed with brine, dried (phase separator) and concentrated in vacuo. The resulting
residue was purified by flash chromatography (0ꢀ100% EtOAc in petroleum ether on basic silica)
to afford tertꢀbutyl (5ꢀ(4ꢀcyanoꢀ2ꢀmethylphenyl)ꢀ[1,2,4]triazolo[1,5ꢀa]pyridinꢀ7ꢀyl)carbamate
(194 mg, 75%). 1H NMR (400 MHz, DMSOꢀd6) δ ppm 1.52 (s, 9 H), 2.10 (s, 3 H), 7.15 ꢀ 7.23
(m, 1 H), 7.61 ꢀ 7.74 (m, 1 H), 7.82 ꢀ 7.90 (m, 1 H), 7.92 ꢀ 8.02 (m, 2 H), 8.31 (s, 1 H), 10.10 (s,
1 H), MS ES+: 294 (MꢀtBu).
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A solution of tertꢀbutyl (5ꢀ(4ꢀcyanoꢀ2ꢀmethylphenyl)ꢀ[1,2,4]triazolo[1,5ꢀa]pyridinꢀ7ꢀ
yl)carbamate (0.194 g, 0.555 mmol) and HCl (4M in 1,4ꢀdioxane) (0.694 mL, 2.78 mmol) in
1,4ꢀdioxane (5 mL) was heated to 50 °C for 2h. The reaction was concentrated in vacuo and the
resulting residue was purified by SCXꢀ2 cationic exchange cartridge, loading and washing with
MeOH and eluting with 2M NH3 in MeOH. The resulting residue was purified by reverse phase
preparative HPLC eluted with acetonitrile / water (with 0.1% ammonia) to afford 4ꢀ(7ꢀaminoꢀ
[1,2,4]triazolo[1,5ꢀa]pyridinꢀ5ꢀyl)ꢀ3ꢀmethylbenzonitrile (53) as a white solid (77 mg, 0.303
mmol, 54.5 % yield). 1H NMR (400 MHz, DCMꢀd2) δ ppm 2.18 (s, 3 H), 4.39 (br. s., 2 H), 6.40
(d, J = 2.15 Hz, 1 H), 6.83 (d, J = 2.27 Hz, 1 H), 7.47 (d, J = 7.83 Hz, 1 H), 7.59 ꢀ 7.69 (m, 2
H), 8.01 (s, 1 H) MS ES+: 250
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N-[5-(4-Cyano-2-methylphenyl)-[1,2,4]triazolo[1,5-a]pyridin-7-yl]acetamide (54). DMFꢀ
DMA (5.13 mL, 38.3 mmol) was added to a solution of 4,6ꢀdichloropyridinꢀ2ꢀamine [CAS
116632ꢀ24ꢀ7] (5.00 g, 30.7 mmol) in EtOH (150 mL). The reaction was heated to 85 °C for 75
mins. The reaction mixture was allowed to cool to room temperature then solvent was removed
in vacuo to give N'ꢀ(4,6ꢀdichloropyridinꢀ2ꢀyl)ꢀN,Nꢀdimethylmethanimidamide as a brown oil
which was taken on to the next step without purification. Hydroxylamine hydrochloride (2.99 g,
43.0 mmol) was added to a solution of N'ꢀ(4,6ꢀdichloropyridinꢀ2ꢀyl)ꢀN,Nꢀ
dimethylmethanimidamide (6.70 g, 30.7 mmol) in MeOH (133 mL) under nitrogen. The reaction
was stirred at room temperature for 1 h and then concentrated in vacuo. The residue was
triturated with water and the solid collected by filtration, washing with water. The solid was
dried under vacuum overnight to afford Nꢀ(4,6ꢀdichloropyridinꢀ2ꢀyl)ꢀN'ꢀ
hydroxymethanimidamide (6.12 g, 97 %). 1H NMR (400 MHz, DMSOꢀd6) δ ppm 7.13 (s, 2 H)
7.68 (d, J = 10 Hz, 1 H) 9.89 (d, J = 10 Hz, 1 H) 10.44 (s, 1 H).
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(Nꢀ(4,6ꢀDichloropyridinꢀ2ꢀyl)ꢀN'ꢀhydroxymethanimidamide (6.12 g, 29.7 mmol) and Eaton’s
reagent (30 mL) were combined and heated to 105 °C for 20 min. The reaction mixture was
allowed to cool to room temperature, diluted with ice water and basified with solid K2CO3 to pH
8. The resulting solution was extracted twice with EtOAc, and the organic layers were combined
and dried over MgSO4, filtered, and concentrated in vacuo. The residue was recrystallized from
MTBE to afford 5,7ꢀdichloroꢀ[1,2,4]triazolo[1,5ꢀa]pyridine (68) (4.63 g, 83%). 1H NMR (300
MHz, DMSOꢀd6) δ ppm 7.76 (d, J = 2 Hz, 1 H) 8.16 (d, J = 2 Hz, 1 H) 8.66 (s, 1 H), MS ES+:
188.
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A suspension of 5,7ꢀdichloroꢀ[1,2,4]triazolo[1,5ꢀa]pyridine (68) (2.00 g, 10.6 mmol), (4ꢀcyanoꢀ
2ꢀmethylphenyl)boronic acid (1.79 g, 11.13 mmol), tetrakis(triphenylphosphine)palladium(0)
(0.61 g, 0.53 mmol) and Na2CO3 (0.888 g, 8.38 mmol) in DME (80 mL) and water (16 mL) was
deꢀgassed and refilled with N2. The reaction was heated to reflux for 2h. The reaction was poured
into water and extracted twice with EtOAc. The organics were combined, dried (phase separator)
and concentrated in vacuo. The resulting residue was purified by flash chromatography (0ꢀ100%
EtOAc in petroleum ether on basic silica) to afford 4ꢀ(7ꢀchloroꢀ[1,2,4]triazolo[1,5ꢀa]pyridinꢀ5ꢀ
yl)ꢀ3ꢀmethylbenzonitrile (1.6 g, 57%). 1H NMR (400 MHz, DMSOꢀd6) δ ppm 2.12 (s, 3 H), 7.46
(d, J = 1.83 Hz, 1 H), 7.70 (d, J = 7.94 Hz, 1 H), 7.87 (d, J = 7.63 Hz, 1 H), 7.96 (s, 1 H), 8.22
(d, J = 2.14 Hz, 1 H), 8.53 (s, 1 H), MS ES+: 269.
A solution of 4ꢀ(7ꢀchloroꢀ[1,2,4]triazolo[1,5ꢀa]pyridinꢀ5ꢀyl)ꢀ3ꢀmethylbenzonitrile (0.250 g,
0.930 mmol), acetamide (0.110 g, 1.861 mmol), cesium carbonate (0.606 g, 1.861 mmol),
Pd2(dba)3 (0.043 g, 0.047 mmol) and dicyclohexyl(2',4',6'ꢀtriisopropylꢀ[1,1'ꢀbiphenyl]ꢀ2ꢀ
yl)phosphine (0.044 g, 0.093 mmol) in 1,4ꢀdioxane (4 mL) was degassed with nitrogen for 5
mins. The reaction mixture was heated in a sealed vial at 110 °C for 1.5 hours. The reaction was
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removed from heating and allowed to cool, diluted in EtOAc (30 mL), washed with water (10
mL) and brine, dried (MgSO4) and concentrated in vacuo. The crude product was absorbed onto
Celite and purified by column chromatography on basic silica, eluted with 0ꢀ100% [9:1
EtOAc/MeOH]/petroleum ether. The resulting residue was further purified by reverse phase
preparative HPLC eluted with acetonitrile / water (with 0.1% ammonia) to afford Nꢀ(5ꢀ(4ꢀcyanoꢀ
2ꢀmethylphenyl)ꢀ[1,2,4]triazolo[1,5ꢀa]pyridinꢀ7ꢀyl)acetamide (54) as a white solid (46.8 mg,
0.161 mmol, 17% yield). 1H NMR (400 MHz, DMSOꢀd6) δ ppm 2.11 (s, 3 H) 2.15 (s, 3 H) 7.20
(d, J = 2 Hz, 1 H) 7.67 (d, J = 8 Hz, 1 H) 7.86 (d, J = 8 Hz, 1 H) 7.94 (s, 1 H) 8.23 (d, J = 2 Hz,
1 H) 8.35 (s, 1 H) 10.56 (s, 1 H), MS ES+: 292.
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ASSOCIATED CONTENT
Supporting Information
Contains PHDꢀ1 activity assay protocols; PAMPA and MDR1ꢀMDCKꢀpgp efflux protocols;
synthetic procedures and analytical data for all novel compounds (in Microsoft Word file
format). Compound SMILES strings and associated activity and ADME data (in csv file format).
This material is available free of charge via the Internet at http://pubs.acs.org
Accession Codes
PDB code for PHDꢀ1 bound with 17 is 5V1B and for PHDꢀ2 bound with 17 is 5V18. Authors
will release the atomic coordinates and experimental data upon article publication.
AUTHOR INFORMATION
Corresponding Author
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* Phone, +44 (0)1223 336 427; Eꢀmail: drkerryjenkins@yahoo.co.uk
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Present Address
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* Wren Therapeutics Ltd., c/o University of Cambridge, Department of Chemistry, Lensfield
Road, Cambridge, United Kingdom, CB2 1EW.
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Author Contributions
All authors have given approval to the final version of the manuscript.
ACKNOWLEDGMENT
We would like to acknowledge the contributions from Michael Bestwick for stimulating
discussion on pharmacokinetics, and Johannes Grosse for his support and guidance throughout
this program. Also we would like to thank Mark Portsmouth (analytical), Suzi Cowan (NMR),
Paul Strutt (HPLC/UPLC) and Divya Lad (PAMPA) for their support and hard work.
XꢀRay crystallographic images and 2D interaction diagram generated using Molecular Operating
Environment (MOE), 2013.08; Chemical Computing Group Inc., 1010 Sherbooke St. West,
Suite #910, Montreal, QC, Canada, H3A 2R7, 2016.
ABBREVIATIONS
2ꢀOG, 2ꢀoxoglutarate; B/P, Brain / Plasma ratio; CAS, Chemical Abstracts Service registry
number; dba, dibenzylideneacetone; DMFꢀDMA, dimethylformamideꢀdimethyl acetal; dppf,
1,1'ꢀbis(diphenylphosphino)ferrocene; Eaton’s reagent, 7.7 wt% phosphorus pentoxide solution
in methanesulfonic acid; ES, electrospray ionization; EtOAc, ethyl acetate; EtOH, ethanol; Het,
heterocycle; HIF, HypoxiaꢀInducible Factor; HRE, Hypoxia Response Elements; IPA, Isopropyl
alcohol; Kp,uu, unbound brainꢀtoꢀplasma concentration ratios; MDCK, MadinꢀDarby canine
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kidney; MDR1, Multidrug Resistance Protein 1; PHD, Prolylhydroxylase Domain; pKa,
logarithmic acid dissociation constant; SCXꢀ2, silylated propyl phosphonic acid Strong Cationic
Exchange Resin; SNAR, nucleophilic aromatic substitution; Sol., Solubility; TEA, triethylamine;
VHL, von HippelꢀLindau.
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