2-Arylamino-6-ethynylpurines are cysteine-targeting irreversible inhibitors of Nek2 kinase

Article abstract of DOI:10.1039/d0md00074d

Renewed interest in covalent inhibitors of enzymes implicated in disease states has afforded several agents targeted at protein kinases of relevance to cancers. We now report the design, synthesis and biological evaluation of 6-ethynylpurines that act as covalent inhibitors of Nek2 by capturing a cysteine residue (Cys22) close to the catalytic domain of this protein kinase. Examination of the crystal structure of the non-covalent inhibitor 3-((6-cyclohexylmethoxy-7H-purin-2-yl)amino)benzamide in complex with Nek2 indicated that replacing the alkoxy with an ethynyl group places the terminus of the alkyne close to Cys22 and in a position compatible with the stereoelectronic requirements of a Michael addition. A series of 6-ethynylpurines was prepared and a structure activity relationship (SAR) established for inhibition of Nek2. 6-Ethynyl-N-phenyl-7H-purin-2-amine [IC50 0.15 μM (Nek2)] and 4-((6-ethynyl-7H-purin-2-yl)amino)benzenesulfonamide (IC50 0.14 μM) were selected for determination of the mode of inhibition of Nek2, which was shown to be time-dependent, not reversed by addition of ATP and negated by site directed mutagenesis of Cys22 to alanine. Replacement of the ethynyl group by ethyl or cyano abrogated activity. Variation of substituents on the N-phenyl moiety for 6-ethynylpurines gave further SAR data for Nek2 inhibition. The data showed little correlation of activity with the nature of the substituent, indicating that after sufficient initial competitive binding to Nek2 subsequent covalent modification of Cys22 occurs in all cases. A typical activity profile was that for 2-(3-((6-ethynyl-9H-purin-2-yl)amino)phenyl)acetamide [IC50 0.06 μM (Nek2); GI50 (SKBR3) 2.2 μM] which exhibited >5-10-fold selectivity for Nek2 over other kinases; it also showed > 50% growth inhibition at 10 μM concentration against selected breast and leukaemia cell lines. X-ray crystallographic analysis confirmed that binding of the compound to the Nek2 ATP-binding site resulted in covalent modification of Cys22. Further studies confirmed that 2-(3-((6-ethynyl-9H-purin-2-yl)amino)phenyl)acetamide has the attributes of a drug-like compound with good aqueous solubility, no inhibition of hERG at 25 μM and a good stability profile in human liver microsomes. It is concluded that 6-ethynylpurines are promising agents for cancer treatment by virtue of their selective inhibition of Nek2. This journal is

Full text of DOI:10.1039/d0md00074d

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RESEARCH ARTICLE
2-Arylamino-6-ethynylpurines are cysteine-
targeting irreversible inhibitors of Nek2 kinase†
Cite this: DOI: 10.1039/
d0md00074d
Christopher J. Matheson,‡a Christopher R. Coxon, ‡a Richard Bayliss, cd Kathy Boxall,‡b
Benoit Carbain,a Andrew M. Fry, c Ian R. Hardcastle,a Suzannah J. Harnor,
a
Corine Mas-Droux,d David R. Newell,e Mark W. Richards,c Mangaleswaran Sivaprakasam,a
a
David Turner,a Roger J. Griffin,§a Bernard T. Golding a and Céline Cano
*
Renewed interest in covalent inhibitors of enzymes implicated in disease states has afforded several agents
targeted at protein kinases of relevance to cancers. We now report the design, synthesis and biological evaluation
of 6-ethynylpurines that act as covalent inhibitors of Nek2 by capturing a cysteine residue (Cys22) close to the
catalytic domain of this protein kinase. Examination of the crystal structure of the non-covalent inhibitor 3-((6-
cyclohexylmethoxy-7H-purin-2-yl)amino)benzamide in complex with Nek2 indicated that replacing the alkoxy
with an ethynyl group places the terminus of the alkyne close to Cys22 and in a position compatible with the
stereoelectronic requirements of a Michael addition. A series of 6-ethynylpurines was prepared and a structure
activity relationship (SAR) established for inhibition of Nek2. 6-Ethynyl-N-phenyl-7H-purin-2-amine [IC50 0.15
μM (Nek2)] and 4-((6-ethynyl-7H-purin-2-yl)amino)benzenesulfonamide (IC50 0.14 μM) were selected for
determination of the mode of inhibition of Nek2, which was shown to be time-dependent, not reversed by
addition of ATP and negated by site directed mutagenesis of Cys22 to alanine. Replacement of the ethynyl group
by ethyl or cyano abrogated activity. Variation of substituents on the N-phenyl moiety for 6-ethynylpurines gave
further SAR data for Nek2 inhibition. The data showed little correlation of activity with the nature of the substituent,
indicating that after sufficient initial competitive binding to Nek2 subsequent covalent modification of Cys22 occurs
in all cases. A typical activity profile was that for 2-(3-((6-ethynyl-9H-purin-2-yl)amino)phenyl)acetamide [IC50
0.06 μM (Nek2); GI50 (SKBR3) 2.2 μM] which exhibited >5–10-fold selectivity for Nek2 over other kinases; it also
showed > 50% growth inhibition at 10 μM concentration against selected breast and leukaemia cell lines. X-ray
crystallographic analysis confirmed that binding of the compound to the Nek2 ATP-binding site resulted
in covalent modification of Cys22. Further studies confirmed that 2-(3-((6-ethynyl-9H-purin-2-
yl)amino)phenyl)acetamide has the attributes of a drug-like compound with good aqueous solubility, no
inhibition of hERG at 25 μM and a good stability profile in human liver microsomes. It is concluded that
6-ethynylpurines are promising agents for cancer treatment by virtue of their selective inhibition of Nek2.
Received 4th March 2020,
Accepted 2nd May 2020
DOI: 10.1039/d0md00074d
rsc.li/medchem
a Cancer Research UK Newcastle Drug Discovery Unit, Chemistry, School of Natural
and Environmental Sciences, Newcastle University, Newcastle upon Tyne, UK.
E-mail: celine.cano@ncl.ac.uk; Tel: +44 (0)191 208 7060
b Cancer Research UK Cancer Therapeutics Unit, The Institute of Cancer Research, Sutton, UK
c School of Molecular and Cellular Biology, The Astbury Centre for Structural
Molecular Biology, University of Leeds, UK
d Section of Structural Biology, The Institute of Cancer Research, Sutton, UK
e Cancer Research UK Newcastle Drug Discovery Unit, Translational and Clinical
Research Institute, Newcastle University Centre for Cancer, Faculty of Medical
Sciences, Newcastle University, Newcastle upon Tyne, UK
Introduction
The renaissance in the development of inhibitors that bind
covalently to a target enzyme either irreversibly or slowly
reversibly has yielded several licensed agents, especially in the
oncology area. As of 2019, six irreversible protein kinase
inhibitors have received FDA approval for use in oncology
(afatinib 1, osimertinib, neratinib
2
and ibrutinib,
† Electronic supplementary information (ESI) available: Kinase profiling, assay
details, synthetic procedures and characterisation data for all compounds;
including aniline precursors. All cell lines were supplied by the American Type
Culture Collection, Manassas, Virginia, United States. All animal experiments
performed were conducted under a UK Government Home Office License in
accordance with relevant national laws. All procedures were reviewed and
approved by the Newcastle University Animal Welfare Ethical Board and
performed according to institutional guidelines. See DOI: 10.1039/d0md00074d
‡ These authors contributed equally to the work described in this paper.
§ Deceased 24 September 2014.
dacomitinib and acalabrutinib, Fig. 1A). Afatanib (1), licensed
in 2013 for treatment of metastatic NSCLC through
irreversible inhibition of EGFR, and neratinib (2), approved in
2017 for treatment of HER2+ breast cancer, were designed as
modifications of gefitinib (3) by installation of electrophilic
acrylamide groups that target an active site cysteine. Protein
kinases are prominent targets for covalent inhibitors, which
owing to the high concentrations of ATP in cells (∼1–10 mM)
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may be more effective than competitive inhibitors because of
irreversible ATP-blockade.1 A range of warhead groups has
been employed in irreversible protein kinase inhibitors, for
example, acrylamides, alkynes, quinones and epoxides.2
Previously, our group discovered the vinyl sulfone (NU6300)
as the first irreversible inhibitor of CDK2 that was shown to
react covalently with an active site lysine residue (Lys89) in
the selectivity pocket.3
Nek2 over Plk1 (e.g., 5 IC50 = 0.36 μM), yet had poor cellular
activity,11 and a hybrid combining a core aminopyrazine
moiety with side-chains from the benzimidazole series 6 (IC50
= 0.022 μM)12 (Fig. 1B), which was potent and Nek2-selective.
As Nek2 contains a non-catalytic cysteine (Cys22) close to the
catalytic domain that is unique to the Nek family, this
residue can be targeted for irreversible binding. Only 10 other
kinases have
a cysteine in a
similar position.13 One
Nek2 is the closest human homologue of the protein
kinase encoded by NIMA (never-in-mitosis gene A) in the
fungus Aspergillus nidulans. The human protein is a serine–
threonine kinase with multiple functions including a role in
the mitotic spindle assembly. Overexpression of Nek2 may
result in deregulation of the mitotic machinery leading to
chromatid segregation errors, aneuploidy and chromosomal
instability, common genetic abnormalities observed in
tumour cells. Nek2 has been associated with tumorigenesis
through overexpression, which has been linked to Ewing's
osteosarcoma, diffuse large B-cell lymphoma, breast, ovarian
and colorectal tumours.4–9
Inhibition of Nek2 in a number of tumour cell lines
causes growth suppression and apoptosis. Reversible ATP-
competitive Nek2 kinase inhibitors have been reported based
on different structural classes, including: aminopyrazines
(e.g., 4 IC50 = 0.23 μM),10 but with poor Nek2-selectivity over
Plk1; benzimidazoles that exhibited >100-fold selectivity for
irreversible Nek2 inhibitor has been published (JH295, 7 IC50
= 0.77 μM),13 which targeted Cys22 via an N-arylpropiolamide
Michael acceptor and was reported to be selective against
other mitotic kinases, including CDK1, PLK1, Aurora B and
Mps1. Herein, we describe the design and synthesis of a new
class of selective tool compounds that covalently and
irreversibly inhibit the Nek2 protein kinase. Our studies
provide an insight into the role of Nek2 in cancer and
address the question as to whether Nek2 is truly an attractive
target for anti-cancer drug design.
Results and discussion
To improve understanding of the role of Nek2 in cancer, we
sought a selective inhibitor of Nek2 by targeting the active
site Cys22 residue. We have discovered covalent inhibitors
superior to compound 7, which was non-selective within a
subset of the kinome investigated here (see ESI†) and was
Fig. 1 A) Rationalisation of the modification of gefitinib (reversible) to afford afatinib and neratinib (irreversible). B) Reversible and irreversible
reported inhibitors of Nek2 kinase. For each case, covalent ‘warhead’ groups are shown in red and core scaffolds in blue.
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therefore inadequate for further studies. In previous work we
developed a series of 6-substituted-2-arylaminopurines that
reversibly inhibited Nek2 (IC50 = 0.27–24 μM) and were
selective (in some cases >10-fold vs. CDK2).14 This work
optimised interactions between the 2-arylamino group and
the specificity pocket of the enzyme and revealed that basic
or polar substituents e.g. tertiary amines, carboxamides,
ureas and sulfonamides conferred good potency (e.g., 8; IC50
= 19 μM (Nek2)). The crystal structure of 8 in complex with
Nek2 (Fig. 2A) revealed that the compound binds via a
classical hydrogen bonding triplet between the purine N9–H,
N3 and C2–NH, and the main chain of kinase hinge region
residues Cys89 and Glu87. Furthermore, the 6-alkoxy
substituent was adjudged to be non-critical for binding
affinity.14 Closer inspection of the structure of the
2-arylamino-6-alkoxypurine 8 bound to Nek2 revealed that the
P-loop cysteine (Cys22) is closely positioned to a putative
superimposed 6-ethynyl (cyan) substituent (4.4 Å) (Fig. 2A).
Moreover, the predicted angle of attack (74°) of cysteine SH
at the terminus of the alkyne suggests that only a small
realignment of the cysteine is required to achieve maximal
orbital overlap with the alkyne π*. Hence, replacement of the
6-position substituent with an ethynyl group was expected to
deliver an irreversible inhibitor (Fig. 2B).
control compounds, to explore their efficacy as irreversible
Nek2 inhibitors and potential applications.
a) Synthesis of 6-ethynylpurine probe molecules
Initially, two 6-ethynylpurines bearing 2-arylamino groups
were prepared (Scheme 1) as prototypes for irreversible
inhibition
of
Nek2.
Subjecting
2-fluoro-6-chloro-(9-
tetrahydropyranyl)-purine (10)17 to Sonogashira cross-coupling
conditions afforded near quantitative yields of 11 when 3
mol% of PdIJPPh3)2Cl2 and 2 mol% of CuI were employed
with triisopropylsilylethyne (TIPS-ethyne, Scheme 1). The
tetrahydropyranyl group was removed by acidic hydrolysis of
11 to give 12 (96% yield) as a central scaffold for further
elaboration. Nucleophilic aromatic substitution (SNAr) of the
2-fluoro substituent by aniline nucleophiles under previously
established
conditions18
gave
2-arylamino-6-
triisopropylsilylethynylpurines (13, 14). The silyl group was
removed by treatment with fluoride (either TBAF or KF with
18-crown-6) affording 2-arylamino-6-ethynylpurines 23 and 24,
albeit in a modest isolated yield. It was noted that if the TIPS
moiety was removed from the ethynyl group at an earlier
stage, then nucleophilic aromatic substitution at the
2-position of the intermediate purine failed to proceed.
Previous reports, including our model study,15 have
indicated that 6-ethynyl- and 6-vinyl-purines react via
conjugate addition with nitrogen, oxygen and sulfur
nucleophiles facilitated in part by the electron deficient
purine.16 Such compounds were also found to exhibit
profound cytotoxicity in chronic myeloid leukaemia cell lines,
which was postulated to arise by attack of nucleophilic amino
acid residues on the electrophilic alkyne or alkene.16a
To build upon these foundations, a focussed series of
derivatives was prepared that combined side chains known to
confer potency in the competitive inhibitor class with a
‘warhead’ capable of covalently reacting with cysteine
residues. The synthesis of 2-arylamino-6-ethynyl- and 6-vinyl-
purines was therefore undertaken, along with appropriate
To establish preliminary SARs at the 2-position, a series of
compounds (25–32) was designed, bearing small alkyl, ether
and halide substituents on the 2-arylamino ring, intended to
probe the interaction of this structural element with the
kinase. To avoid intractable reaction mixtures, potentially
reactive heteroatoms (e.g. hydroxyl groups as in compound
18) required protection (e.g. for hydroxyl, TIPS was used and
removed concomitant with ethynyl group deprotection) prior
to participation in the SNAr step. In previous studies,
carboxamides (e.g., 8) and sulfonamides (e.g. 9) were
generally found to afford the greatest Nek2 inhibitory activity
and selectivity.14 The scope of this study was therefore
broadened through the synthesis of a number of more
complex analogues derived from the corresponding anilines
Fig. 2 A) X-ray structure (PDB code: 5 M51) of 2-arylamino-6-alkoxypurine 8 (grey) in complex with Nek2, superimposed with a putative
6-ethynylpurine (cyan) reveals that the alkyne is positioned for covalent interaction with cysteine 22. B) The proposed approach to deliver a
covalently-reactive Nek2 inhibitor.
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Scheme 1 General route to 6-ethynyl-2-arylaminopurines. Reagents and conditions: (a) triisopropylsilylacetylene, PdIJPPh3)Cl2, CuI, Et3N, THF, RT,
i
18 h; (b) TFA, PrOH, H2O, 100 °C, 2 h; (c) anilines, TFA, TFE, 80 °C, 18–27 h or MW 140 °C, 2 h; (d) TFA, reflux, 18 h (compounds 33 and 34); (e)
TBAF, THF, RT, 5 min or KF, 18-crown-6, THF, RT, 24 h.
(see ESI† for synthesis of the required anilines). Where
necessary, coupling of 4-methoxybenzyl (PMB)-protected
aminobenzamides to 2-fluoropurine (12) was followed by
removal of the PMB-group using TFA at reflux prior to alkyne-
deprotection (Scheme 1). This series of compounds allowed
for possible additional interactions between e.g. basic groups
of carboxamides (46–58) and the selectivity surface, which
was previously found to confer higher potency and selectivity
for Nek2 over other related kinases. This series included a
carboxamide derivative (58) with a gem-dimethyl group.
Additional carboxamides were generated through
coupling of 3- and 4-aminophenylacetic acid to
2-fluoropurine 12 under standard TFA/TFE-mediated
conditions. This resulted in the formation of trifluoroethyl
esters, which were hydrolysed under basic conditions to the
required 3- or 4-substituted acids 59 and 60 without
isolation (Scheme 1). The respective carboxylic acids were
transformed into carboxamides 66–68 using 1,1′-
carbonyldiimidazole (CDI) and the appropriate amine,
followed by acidic deprotection of PMB-groups as required,
and final removal of the silyl protecting groups (Scheme 2).
To explore the consequences of conformational restrictions
at the carboxamide side chain, a small series of purines
bearing lactam groups (73–76) was prepared by the
approach outlined in Scheme 1.
To probe the role of the carboxamide amino group of 66,
the ketone analogue 80 was synthesised. A modification of
the Dakin–West reaction (conversion of an amino-acid into a
ketoamide), can be used to convert a carboxylic acid with an
enolisable carbon at the α-position into a methyl ketone. This
method was employed to convert 3-nitrophenylacetic acid
into 3-nitrophenylacetone by treatment with acetic anhydride
in the presence of pyridine (modified Dakin–West
reaction19,20). Protection of the carbonyl group of
3-nitrophenylacetone by conversion into a dithiane with 1,3-
propanedithiol, was followed by reduction of the nitro group
to afford aniline 77. Coupling of 77 to the 2-fluoropurine 12
in the usual manner (Scheme 3) gave 78. Stepwise removal of
the dithiane (using [bisIJtrifluoroacetoxy)iodo]benzene) and
silyl (TBAF) protecting groups furnished the target inhibitor
(80) via intermediate 79.
The homosulfonamide analogues of 24 probing the
H-bond donor properties of the sulfonamide were prepared
by either i) direct SNAr reaction with the aniline according to
Scheme 1, affording the sulfone 82, or ii) from common
intermediate, 2,2,2-trifluoroethylsulfonate ester 83 according
to Scheme 4.21 For the latter, reaction with amines in the
presence
of
1,8-diazabicycloij5.4.0]undec-7-ene
under
microwave heating afforded sulfonamides in good to
excellent yield.21 Removal of all protecting groups was
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Scheme 2 Synthesis of carboxamide derivatives. Reagents and conditions: (a) i) 1 M NaOH, THF, RT, 18 h; ii) RNH2, CDI, DIPEA, DMF, RT, 18 h; (b)
TFA, reflux, 72 h; (c) TBAF, THF, RT, 5 min (followed in some cases by Amberlite 15 ion exchange resin, CaIJOH)2, THF, RT, 48 h).
completed under the optimised conditions described above
to give the target 6-ethynylpurines 87 and 88.
the enamine 89 (Scheme 5).14,16d Reduction24 of 89 using
sodium cyanoborohydride in the presence of 0.1 M HCl gave
tertiary amine 90. N-Oxidation of 90 using m-CPBA gave
N-oxide (91), which underwent
without heating, to give the desired 6-vinylpurine (92) in
The replacement of the 6-ethynyl group with a vinyl group
was performed to probe the conformation of the conjugate
acceptor within the binding pocket of Nek2 and to assess the
potential for non-specific reactivity and toxicity. Initial
attempts to synthesise the 6-vinylpurine analogues of 23
directly using a vinyltrifluoroborate salt and Suzuki–Miyaura
coupling failed. 6-Vinylpurines have been prepared from
thioethers by oxidation to the corresponding sulfoxide
followed by base-induced elimination.22 In our approach
based on an earlier study,23 to 6-vinylpurine (92) conjugate
addition of homopiperidine to 6-ethynylpurine (23) furnished
a
Cope β-elimination24
sufficient yield (30%).
b) Synthesis of control compounds
The 6-ethylpurine 96 was prepared as a control compound for
comparison with the conjugate acceptor 6-ethynylpurine (23).
6-Ethynyl-2-fluoro-9-tetrahydropyranylpurine (93) was directly
reduced with hydrogen in the presence of Lindlar's catalyst
Scheme 3 Synthesis of sulfonamides. Reagents and conditions: (a) 12, TFA, TFE, MW 140 °C, 2 h; (b) [bisIJtrifluoroacetoxy)iodo]benzene, MeOH,
H2O, RT, 10 min; (c) i) TBAF, THF, RT 5 min, ii) solid supported TBAF, THF, RT, 48 h.
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Scheme 4 Synthesis of sulfonamides. Reagents and conditions: (a) R2NH, DBU, THF, MW 160 °C, 15 min; (b) TFA, 70 °C, 3 h; (c) TFA, DCM, RT, 18
h; (d) i) TBAF, THF, RT, 5 min, ii) Amberlite 15 ion exchange resin, CaIJOH)2, THF, RT, 48 h.
with quinoline to the fully saturated 6-ethylpurine (94). The
THP protecting group was cleaved by acidic hydrolysis
followed by treatment with 4-aminobenzenesulfonamide
under standard SNAr conditions to produce 96 (Scheme 6).
c) Biological evaluation of probes
i. 6-Ethynylpurines are irreversible inhibitors of Nek2.
Initially, two simple 6-ethynylpurines, (23 and 24) were
investigated as prototype irreversible inhibitors of Nek2, and
6-Cyanopurine
(102)
was
prepared
by
heating
exhibited encouraging and equivalent potency (Nek2 IC50
∼
2-bromohypoxanthine (97) with aniline to afford N2-
phenylguanine (98), which was converted into the
6-chloropurine 99. The purine N-9/N-7 positions were
protected as a ∼3 : 1 mixture (N-9: N-7) of 4-methoxybenzyl-
substituted regioisomers. The major N-9-PMB isomer (100)
was isolated in 52% yield and treated with Et4NCN/DABCO to
furnish cyanopurine 101, from which the PMB group was
removed in hot TFA (Scheme 7).
140 nM). Subsequently, group of 2-arylamino-6-
a
ethynylpurines (25–32) was prepared and showed good to
moderate inhibition of Nek2 and that substitution at the
2-arylamino group was well tolerated. However, despite the
variation in the substituent, there was only
variation in inhibitory activity (Fig. 3A) and little useful SAR
understanding could be gained from this limited set of
a modest
Scheme 5 N-Oxidation-β-elimination approach to 6-vinylpurine 92. Reagents and conditions: (a) homopiperidine, THF, MW 100 °C, 10 min; (b)
NaBH3CN, TFA, THF, RT, 2 h, or NaBH3CN, 0.1 M HCl, RT, 5 h; (c) m-CPBA, DCM, RT, 2 h.
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covalent binders to Nek2. Further evidence was obtained
through the confirmation of time-dependent inhibition of
Nek2 kinase activity after treatment with 23 (Fig. 3C). To
confirm whether time-dependent inhibition was mediated
through Cys22, site-directed mutagenesis of this residue to
alanine (Nek2-C22A) was performed, resulting in only weak
competitive inhibition by 23 (IC50, Nek2-C22A = 3.5 μM), with
no increase in the degree of inhibition being observed with
time of incubation (Fig. 3D).
ii. Selectivity profiling. To improve the confidence that
Nek2 inhibition was responsible for observed cellular activity
and to assess potential off-target enzyme inhibition, a panel
of serine–threonine protein kinases was profiled against the
ethynylpurines 23 and 24 (Fig. 3E). Despite evidence of
modest Aurora A, CDK2, P70S6K and Chk2 inhibition,
generally >10-fold selectivity for Nek2 was maintained. In
agreement with earlier results, both 23 and 24 were poorly
active against Nek1 (23: IC50 = 20.8 μM; 24: 58% inhibition at
10 μM), which contains an alanine instead of the
corresponding cysteine residue in Nek2, garnering further
evidence of irreversible inhibition through cysteine-
adduction. Importantly, selectivity was maintained over all
seven other kinases tested (from a total of ten in the human
kinome) that contain active-site cysteine residues: Rsk1,2,3;
Msk1,2; Mekk1, Plk1 (ESI† Tables S1 and S2). Except for the
cysteine, the residues that line the ATP binding pocket are
divergent between these kinases and Nek2: the hinge
sequences are different, only Nek2 and Plk1 share a Phe
residue at the base of the pocket (Phe148 and Nek2). Of
interest, Nek2 also has a cysteine residue in the hinge motif
(Cys89) that could potentially be targeted with a covalent
warhead, but almost 100 human kinases have an equivalent
cysteine, and therefore this is not an attractive strategy to
develop a selective inhibitor.
Scheme 6 Synthesis of 6-ethylpurine 96. Reagents and conditions: (a)
Lindlar's catalyst, quinoline, H2, EtOAc, RT, 2 h; (b) TFA, 2-propanol,
water, 100 °C, 2 h; (c) 4-aminobenzenesulfonamide, TFA, TFE, 90 °C,
48 h.
compounds. It should be noted that reporting inhibitory
activity as IC50 may be inappropriate for irreversible
inhibitors due to the time-dependence of the covalent
reaction, although throughout this work activities are
reported as IC50 after 30 min incubation with Nek2 unless
otherwise stated.
Compounds 23 and 24 were selected as initial probes for
determination of the mode of 6-ethynylpurine-mediated
inhibition of Nek2. A kinase-inhibition reversibility assay
(Fig. 3B) was employed to assess whether Nek2 pre-incubated
with an inhibitor, e.g. 24 (30 min at 10 × IC50, calculated to
afford approximately 91% inhibition) could recover
enzymatic activity upon rapid dilution (×100) of the enzyme–
inhibitor complex with a solution containing ATP. Following
this dilution, a reaction progress curve was generated by
monitoring for levels of a phosphorylated substrate (Fig. 3B).
Compound 24 afforded only approximately 3% recovery of
iii. Preliminary cellular evaluation. The 2-arylaminopurines
23 and 24, were selected for further studies to investigate
initial cellular effects of inhibitors within this class.
Unfortunately, cellular growth-inhibition (U2OS, HeLa cell
activity, compared with 11% for
a control competitive
inhibitor, suggesting that 6-ethynylpurines are indeed
Scheme 7 Synthesis of cyanopurine 102. Reagents and conditions: (a) aniline, TFA, TFE, 80 °C, 18 h; (b) PhNMe2, POCl3, 115 °C, 1 h; (c)
4-methoxybenzyl chloride, K2CO3, DMF, 60 °C, 18 h; (d) Et4NCN, DABCO, MeCN, rt, 18 h; (e) TFA, 70 °C, 5 h.
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Fig. 3 Biochemical and cellular assessment of initial hit compounds 23 and 24 as irreversible inhibitors of Nek2: A) modification of the
2-arylamino motif. †Note: Nek2 IC50 values (uM) determined at 30 μM ATP concentration following a 30 minute incubation. B) Recovery of enzyme
activity of Nek2 following dilution of the enzyme–inhibitor complex with ATP for 24 and a known ATP-competitive inhibitor of Nek2. C) and D)
dose–response curves as a function of incubation time between 23 and Nek2-WT, and Nek2-C22A. E) Counter-screening of ethylnylpurines 23
and 24 against a panel of protein kinases (note: red = low inhibition, green = high inhibition). F) Cellular growth-inhibition by compounds 23 and
24 (a half maximal growth inhibitory concentration; b not determined). G) Comet assay results for cells treated with 23.
lines) was found to be disappointing for 24, which
compared poorly with the cellular efficacy (U2OS, HeLa
and MDA-MB-231 cell lines) observed for the parent
phenyl compound 23 (Fig. 3F). This was tentatively
attributed to the poor cell-permeability of the polar
sulfonamide group of 24. The growth inhibitory activity of
phenyl derivative 23 in the HEK293 cell line (GI50 = 0.1
uM) raises the possibility of non-specific toxicity through
action as a general electrophile, resulting in possible non-
specific DNA or protein binding. Given the proposed
mechanism of the 6-ethynylpurine inhibitors, it was
considered that they could act as DNA alkylators via a
similar mechanism. In order to determine the effect of
treatment with the inhibitor on cellular DNA damage, a
Comet assay was performed (Fig. 3G). MDA-MB-231 breast
cancer cells were treated with 23 for up to 96 h and
displayed similar olive tail moments to untreated cells,
confirming that DNA damage is not a major effect of this
inhibitor class.
Given the observed cellular activity and apparent
selectivity for Nek2 over other kinases, 6-ethynylpurine 23 was
considered a suitable starting point from which to conduct
modification to further improve inhibitor potency. According
to our previously reported findings,14 Nek2 competitive
inhibitors of the 6-alkoxypurines class bearing a basic group
at the 2-arylamino ring were found to confer good potency
and cellular activity. It was considered that this would
translate to the irreversible inhibitors class as the competitive
binding phase was crucial to achieving targeted and potent
inhibition. The parent 6-ethynylpurine irreversible inhibitor
(23) was modified at the 2-arylamino to produce a series
comprising carboxamides (46–58, 66–68, 73–76) and
sulfonamides (87 and 88), which were screened for inhibitory
activity against Nek2 (Table 1). In addition, 6-ethynylpurines
were evaluated for growth inhibitory activity in vitro against
the SKBR3 tumour cell line.
All purine derivatives tested exhibited inhibitory activity
against Nek2 with all but compound 74 possessing an IC50 of
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less than 0.13 μM. It is evident that the activity of the parent
3-substituted carboxamide 66 (IC50 = 0.062 0.01 μM) is not
affected by truncation (46, IC50 = 0.116 μM) or homologation
(47, IC50 = 0.079 μM) of the methylene spacer group.
compound 74 (Nek2; IC50 = 0.82 μM, SKBR3; GI50 = 1.3 0.2
μM) was the least active inhibitor in the Nek2 assay, but
proved one of the more potent growth inhibitors in SKBR3
cells. Off-target inhibition or non-specific toxicity are
common challenges to overcome within protein kinase
inhibitor design, and in particular for irreversible inhibitors.
As such, it was considered that these may play a role in the
conflicting cellular versus enzyme activity data, despite the
promising Comet assay data obtained for 23.
Reversing the carboxamide (57, IC50
complete removal of the heteroatom, through synthesis of
ketone 80 (IC50 0.076 μM) were inconsequential
modifications. Furthermore, moving the 2-arylamino ring
substituents from the 3- to the 4-position (e.g., 67; IC50
= 0.039 μM) and
=
=
0.093 μM) had little influence on the measured enzyme
inhibition. Sequential removal of the carboxamide hydrogen
Whilst the carboxamide derivative 66 was identified as
amongst the most potent inhibitors in this class and
inhibited growth of SKBR3 cell lines (Table 1), the
improvement in kinase inhibitory activity did not correlate
with the cellular growth inhibitory activity observed in U2OS
and HeLa (Table 2) cell lines. It was possible that this was
again a consequence of the more polar nature of the
2-arylamino side-chain limiting cellular permeability.
atoms
through
N-methylation,
affording
N-methylcarboxamide (48, IC50
=
0.076 μM) and N,N-
dimethylcarboxamide (68, IC50 = 0.088 μM) analogues of 66
had little effect on Nek2 inhibitory activity, which was true
for both 3- and 4-substituted carboxamides.
Conformational restriction of the carboxamide motif
appeared to be somewhat detrimental, with gem-dimethyl
Owing to concerns over lack of kinase selectivity of this
compound class, the inhibitory activity of 66 was profiled
against a panel of 48 kinases selected from commercially
available ProfilerPro® plates at 1 μM inhibitor concentration
(see ESI,† Table S1). Only two kinases other than Nek2 were
inhibited by greater than 50%, and IC50 values were
determined for these: Aurora A (IC50 = 0.08 μM) and BMX
(IC50 = 0.83 μM). It is notable that BMX kinase, a member of
the EGFR tyrosine kinase family, possesses a cysteine residue
within the ATP-binding domain that has been identified as a
potential target in the design of irreversible inhibitors.25 In
addition, both 23 and 66 were screened against 121 kinases
(National Centre for Protein Kinase Profiling, Dundee
University) at a single concentration of 1 μM (see ESI,† Table
S2). Greater than 50% inhibition of activity was observed for
nine of the kinases tested by 66, including Nek2, and IC50
values were determined (Table 3). Encouragingly, the IC50 of
66 against Nek2 was 18 nM in this assay, giving the
compound at least 5–10-fold selectivity for Nek2 over other
kinases, whilst AurA was only weakly inhibited by 66 in this
screen.
carboxamide 58 (IC50
= 0.21 μM) exhibiting a small,
approximate 3-fold reduction in inhibitory activity. Moreover,
it is likely that the conformational restriction imposed by a
bicyclic 2-arylamino system either disrupts favourable or
promotes unfavourable interactions within the ATP-binding
site of Nek2, as exemplified by compound 74 (IC50 = 0.82
μM). This suggests that the optimal 2-arylamino side-chain
characteristics are more linear with respect to the purine
scaffold, as indicated by comparison of the activity of 74 with
that of the regioisomeric analogue 73 (IC50 = 0.10 μM). In
contrast to the carboxamide examples, the N,N-
dimethylsulfonamide analogue (88, IC50 = 0.010 μM) lacking
an H-bond donor atom conferred a significantly improved
potency when compared with the primary sulfonamide (87,
IC50 = 0.130 μM). However, this may be simply a function of
physicochemical properties e.g., solubility.
Within this compound class, only rather limited SARs are
evident, with no discernible trend observed between the
activity of compounds against Nek2 and the degree and/or
nature of modification of the carboxamide group.
Presumably, all compounds excluding 74 exhibit sufficient
initial competitive binding affinity for Nek2 to allow the
purine to occupy the ATP-binding domain of the kinase and
enable subsequent covalent modification of Cys22 to occur.
The weaker activity observed for 74 does suggest that it is
possible to elucidate SARs for this compound class and that
potent activity is not common to all of the 6-ethynylpurines.
What these data do reveal, however, is that for irreversible
inhibitors in the 6-ethynylpurine class, there is a large degree
of tolerance for substitution on the 2-arylamino ring. This
indicates that significant structural changes should be
Interestingly, 23 displayed
a better overall selectivity
profile for Nek2 compared with 66. In addition to Nek2, only
two other kinases (MLK3 and TAK1) were inhibited by greater
than 50%. It is possible that the alternative target responsible
for the cellular activity of 23 is not included in the screen,
which constitutes only a fraction of the human kinome.
Conversely, the locus of cellular activity for 23 may not be a
kinase.
In keeping with the ultimate objective of developing a
targeted therapy, 66 was screened for cell growth inhibition
(10 μM, 72 h) against a panel of cell lines to identify specific
tumour types where depletion of Nek2 activity is growth
inhibitory (Fig. 4A–C). Previous findings in this area
indicated that cell lines derived from breast tumours and
leukaemias may be particularly sensitive to pharmacological
Nek2 inhibition. As was observed previously, the tumour cell
lines most sensitive to inhibitor treatment were those derived
from breast (MCF7, T47D, ZR75, SKBR3 and MDA-468) and
tolerated
in
order
to
enhance
pharmacokinetic/
physicochemical properties yet may promote concerns of
potential off-target toxicity. Coupled with this, all compounds
detailed in Table 1 exhibited growth inhibitory activity in the
SKBR3 cell line, yet there appeared to be a poor correlation
between the IC50 of compounds in the Nek2 kinase assay and
their activity in the SKBR3 cellular GI50 assay. In particular,
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Table 1 Examples of 2-arylamino-6-ethynylpurines synthesised and studied in this work
Compound
R
IC50 (μM) Nek2a
GI50 (μM) SKBR3
46
0.116
0.7 0.1
47
0.079
3.9 1.3
48
49
0.076
0.072
2.4 0.1
4.9 2.4
50
51
0.057
0.071
1.2 0.6
87.8
52
53
54
0.026
0.050
0.110
0.9 0.1
0.9 0.1
7.4 0.1
55
56
0.110
0.064
4.1 0.5
1.4 0.4
57
58
66
67
0.039
0.8 0.5
1.4 0.6
2.2 0.4
8.8 1.8
0.210
0.062 0.01
0.093
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Table 1 (continued)
Compound
R
IC50 (μM) Nek2a
GI50 (μM) SKBR3
68
0.088
0.4 0.2
73
74
0.100
0.820
10.3
1.3 0.2
75
0.010
—
76
80
0.042
0.076
0.8 0.1
—
82
87
0.057
0.130
1.3 0.0
4.1 2.2
88
0.010
—
a
All IC50 values are results obtained from n = 3 determinations. Nek2 IC50 values determined at 30 μM ATP concentration following a 30
minute incubation.
leukaemia (Rec-1 and RIVA), with all of these cell lines
exhibiting greater than 50% growth inhibition at a dose of 10
μM 66. GI50 values were determined for these cell lines, along
with HeLa and HCT116 (Fig. 4D and E), with the lymphoma
inhibition of C-Nap1 phosphorylation levels to approximately
20% of control at doses of 1 × GI50 (1.8 μM) (Fig. 5A),
indicating that cellular permeability was not a problem.
Despite showing negligible inhibition of cell growth, cells
treated with 66 have pC-Nap1 levels reduced to a comparable
degree at 0.07–0.14 × GI50 (5.0–10.0 μM). This strongly
suggests that growth inhibition observed in U2OS cells
following exposure to 23 arises via an alternative target.
Cellular growth inhibition was not evident for 66, presumably
owing to improved compound selectivity.
v. Structural biology confirms covalent binding of
ethynylpurines. To examine the binding mode of
ethynylpurines to Nek2 and observe the covalent bond
directly, we solved X-ray co-crystal structures of Nek2 with
compounds 24 and 66, and also with the competitively-
binding control compounds 96 and 102 (see ESI,† Table S3).
C-Terminally his-tagged Nek2 1-273 protein was expressed,
purified and crystallized as previously described.26 Crystals of
Rec-1 (GI50 = 1.6 0.1 μM) and breast cancer SKBR3 (GI50
=
2.2 0.4 μM) cell lines being identified as particularly
sensitive to 66 treatment. Furthermore, 66 was also found to
be cytotoxic in SKBR3 cells; clonogenic assays with this cell
line gave LC50 values in the low micromolar range following
pre-incubation for as little as 3 hours prior to subculture
(Fig. 4F).
iv. C-Nap1 as a cellular biomarker for Nek2 inhibition. To
confirm that cellular Nek2 is inhibited upon treatment with
the ethynylpurines, e.g. 23, a cellular biomarker was required.
The phosphorylation state of C-Nap1 localised to the
centrosomes was used. As C-Nap1 is a substrate only for
Nek2, this can be used as a direct measure of cellular Nek2
activity. Encouragingly, cells treated with 23 displayed
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Table
2 Enzyme versus cellular inhibitory activity of unsubstituted (23), homocarboxamide (66) and homosulfonamide (87) 2-arylamino-6-
ethynylpurines
IC50 (μM)
GI50 (μM)
Compound
R
Nek2a
U2OS
HeLa
23
66
Ph
0.14 0.1
0.062 0.01
2.1 1.0
71.0
1.2 0.5
47.1 2.1
87
0.13
N.D.
N.D.
a
All IC50 values are results obtained from n = 3 determinations. Nek2 IC50 values determined at 30 μM ATP concentration following a 30
minute incubation; N.D. not determined.
Table 3 IC50 values for kinases inhibited by 66 by greater than 50% in the National Centre for protein kinase profiling screen. Colour code: green
(relatively potent inhibitor), red (relatively weak inhibitor)
inhibitor-bound Nek2 were generated by soaking ADP-bound
Nek2 crystals in mother-liquor supplemented with each of
the inhibitor compounds for several hours before cryo-
protection and cryo-cooling. Diffraction data were collected
at Diamond Light Source and ESRF and X-ray crystal
structures were solved by molecular replacement and refined
as previously described.26 The structure of Nek2 in complex
with the 6-ethynylpurine 24 was solved to 2 Å resolution
(Fig. 6A) and revealed that the triplet of hydrogen bonds
between the purine and the kinase hinge region (Cys87 and
Glu87) is maintained in an almost identical binding mode to
the previously reported reversible binding class of
6-cyclohexylmethoxypurines. Having established that the
ethynyl motif present in 23 and 24 may be responsible for
covalent adduction, the cysteine residue (Cys22) present in
the catalytic domain of Nek2 was found to be well positioned
for the proposed conjugate addition to the bound inhibitor.
Furthermore, crystal structure analysis (Fig. 6B) clearly
showed a covalent bond between the sulfur of Cys22 and
inhibitor 24, supporting the mechanism of irreversible
modification proposed in Fig. 6D. X-ray crystallographic
analysis confirmed that 66 also bound to the Nek2 ATP-
binding site in the expected manner, with covalent
modification of Cys22 evident (Fig. 6C). Potential interactions
between the carboxamide side-chain of inhibitor 66 and the
carboxylic acid side-chain of Asp93 were also identified
(Fig. 6C), suggesting that it should be possible to attain
greater binding-affinity through appropriate substitution at
the 2-arylamino-group. It may be that the hinge interaction is
also crucial for activation of 6-ethynylpurines towards
nucleophiles, by partial protonation of N-3 of the purine
which accepts a hydrogen bond upon binding. The terminal
alkyne should consequently be more susceptible to
a
Michael-type addition by an active site nucleophile (e.g., Cys-
SH, Fig. 6D). It is likely that the conjugate addition proceeds
via an allene intermediate that tautomerises to afford a
thioenol-ether adduct.
vi. Evaluation of control compounds. To dissect the role of
the covalent binding ethynyl group from the putative initial
reversible binding 2-arylaminopurine, a series of ethynyl
group-replacements were introduced at the 6-position of 23
and 24. In each case, control compound 92, 96 and 102 that
were predicted to make the same interactions with the kinase
active-site, but lack the covalent modifying ethynyl functional
group, exhibited significantly lower inhibitory activity in
biochemical assays making them important tool compounds
(Table 4).
Perhaps the most intriguing result was related to the
6-vinylpurine 92, which is reported to undergo conjugate
addition reactions with thiols, albeit at a slightly slower rate
than 6-ethynylpurines.15 92 was found to be a very weak Nek2
inhibitor (17% inhibition at 10 μM) compared with ethynyl
analogue 23 (IC50 = 0.14 μM). This indicates that the
geometry of the conjugate-acceptor substituent at the purine
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Fig. 4 A) Growth inhibition of solid tumour cell lines treated with 10 μM 66 for 72 hours. B) Growth inhibition of solid tumour cell lines treated
with 10 μM 66 for 72 hours. C) Growth inhibition of leukaemia cell lines treated with 10 μM 66 for 72 hours. D and E) Growth inhibition of cell lines
identified as being sensitive to 66 following 72 h exposure to inhibitor (n = 3). F) Cytotoxicity of 66 in SKBR3 cells following incubation for a range
of times.
6-position is critical, underlining the importance of correctly
aligning the conjugate acceptor group with Cys22. However,
it is possible that due to lower thiol-reactivity, 92 requires a
longer incubation time within the Nek2 active site to be
equipotent with 23. This outcome is of critical importance
and alleviates the fear that unwanted side-reactions would
occur indiscriminately.
The saturated 6-ethylpurine (96) is a non-conjugate
acceptor, lacking an alkene/alkyne purine 6-substituent
required for Michael-type addition reactions. Compound 96
was found to be a weak inhibitor of Nek2 (IC50 = 46.8 μM),
but was shown to bind in an identical orientation in the
Nek2 active-site as the 6-ethynyl derivative 24 (Fig. 7). The
crystal structure of Nek2-96 complex superimposed with
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Fig. 5 A) Inhibition of C-Nap1 phosphorylation in U2OS cells by 66 compared with 23. B) Dose-dependent reduction of pC-Nap1 levels for
selected carboxamides within the 6-ethynylpurine series.
Nek2-24 (Fig. 7) revealed that the covalent link between 24
and Cys-22 of Nek2 is accompanied by a relative movement
of the N-terminal lobe towards the C-terminal lobe. This
movement of the N-terminal lobe is not evident when the
competitive inhibitor (96) is in complex with Nek2.
The nitrile group of 102 retains both the linear geometry of
the ethynyl group is not expected to undergo conjugate
addition by nucleophiles such as thiols in the same manner
possessed the attributes of a drug-like compound. The purine
was found to have good aqueous solubility, no inhibition of
hERG (human ether-a-go-go-related gene product) was
observed at 25 μM, and 66 exhibited a good stability profile
in both human and mouse liver microsomes, despite the
electrophilic 6-ethynyl group, probably reflecting the low
lipophilicity of the compound (cLogP
= 1.4). Studies
employing the Caco-2 cell line allowed an estimation of
compound permeability and efflux to be determined. The
direction [A → B] equates to compound passing from the
gastrointestinal tract into the blood, with [B → A] relating to
drug efflux in the reverse direction. The inhibitor showed low
permeability and high efflux, suggesting that the oral
bioavailability of 66 may be poor. It is possible, however, that
the inherent permeability of the compound is good, but that
it is also a substrate for drug efflux transporters.
as 6-ethynylpurines. 102 was measured to be
a weak
competitive inhibitor of Nek2 (IC50 = 12 μM) and was
approximately 200-fold less potent against Nek2 than the
equivalent 6-ethynylpurine (23). This result suggested that the
competitive binding component only contributes weakly to
the activity observed in the 6-ethynylpurine series, assuming
that 23, 24 and 102 make similar binding interactions with
Nek2. Superposition of the crystal structure of 102 in complex
with Nek2 confirmed that a near identical binding-orientation
arose compared with the benchmark 6-ethynylpurine 24
(Fig. 8A). The covalent adduction of 24 can clearly be seen by
the thioenol ether formed between 24 and Cys22. This is
absent in the crystal structure of the cyano compound,
indicating that no covalent interaction arose although the
same mode of binding of the shared scaffold into the
nucleotide-binding pocket of Nek2 was observed and the
triplet of hydrogen bonds between the purine and the kinase
hinge motif was preserved (Fig. 8B and C). Significantly,
in vitro growth inhibition assays showed that 102 (GI50 > 100
μM) has no effect on cell viability in three human tumour
cell-lines, WT U2OS, MDA-MB-231 and HeLa cells, as well as
in the ‘normal’ human embryonic kidney (Hek293) cell-line.
vii. In vitro pharmacokinetics of 6-ethynylpurines. Further
studies to characterise 66 were undertaken, including
preliminary in vitro ADME testing (Cyprotex Discovery,
Macclesfield, UK) (Table 5). These studies suggested that 66
In vivo modelling. The promising in vitro results prompted
the evaluation of 66 in in vivo models for both
pharmacokinetics (PK) and antitumour efficacy. In
a
preliminary PK evaluation, 66 was administered at a single
dose to female CD1 mice. The compound was rapidly cleared
from the blood following intravenous administration, with a
low area under the curve (AUC), high clearance and a short
half-life (T1/2). No inhibitor was detected in blood samples for
animals treated orally. Compound 66 has poor permeability
and is readily effluxed in the Caco-2 model which translates
into poor oral bioavailability due to poor absorption. To
understand the rapid clearance of 66, stability studies were
performed for the compound in buffered solutions at pH 2,
7.4 and 10, and in a solution containing 5 mM glutathione
(GSH), which approximates to intracellular concentrations.
Samples of 66 in the three solutions were incubated at 37 °C
and the concentration of 66 determined by HPLC at time
points over 18 h. It is likely that 66 is poorly soluble in
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Fig. 6 Interaction of irreversible inhibitors 24 and 66 with Nek2: A) crystal structure of 24 (cyan) bound to Nek2 indicating interactions with the
hinge-region of the kinase (pink) and B) crystal structure of Nek2 (green) in complex with 24 (cyan) including electron density map (2Fo-Fc)
contoured at 1.0σ (mesh). C) Crystal structure Nek2 (green) in complex with 66 including electron density map (2Fo-Fc) contoured at 1.0σ (mesh).
A potential interaction with the side chain of Asp93 is indicated. D) Proposed mechanism of covalent adduction of cysteine-22 by ethynylpurines.
solutions at higher pH, and therefore the concentration only
becomes consistent once the sample is incubated at 37 °C for
10 minutes. It was evident that under the assay conditions, 66
is consumed in a time dependent manner in the presence of
glutathione. This suggests that reaction with glutathione may
contribute to the rapid clearance observed for 66.
Notwithstanding the reactivity of compound 66 with
glutathione, in the mechanistic cellular C-Nap1 assay activity
was nonetheless observed with a number of compounds, and
therefore instability in the presence of glutathione at
physiological concentrations does not preclude cellular activity.
to the high degree of structural homology within the catalytic
domain. However, targeting specific nucleophilic residues
close to the catalytic site provides a means to discriminate.
In this context, it is therefore encouraging that over 200
protein kinases contain a targetable cysteine residue close to
the catalytic site.2,27–29 Covalent binding of an enzyme
inhibitor has the potential advantages of prolonged duration
of action and enhanced pharmacological efficacy, which
allows lower dosing and reduced off-target effects. However,
this advantage may not be relevant for Nek2 protein, which
has a short cellular half-life of 45–75 minutes.30 There is
concern that covalent binding agents will exhibit non-specific
activity and off-target toxicity (e.g. promotion of immune
responses31) owing to their perceived ability either to react
almost randomly with biological nucleophiles, e.g. proteins
and nucleic acids, or to be quenched by glutathione.
Conclusions
Achieving selectivity between specific members of the same
or closely-related protein kinase families is challenging owing
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Table 4 Replacement of the ethynyl covalent-acceptor motif
Compound
R
X
IC50 (Nek2)a
23
H
0.145 μM
24
0.140 μM
92
96
H
H
17% at 10 μM
46.8 μM
Et
102
12 μM
All IC50 values are results obtained from n = 3 determinations.a % inhibition or IC50 determined after 30 min.
Fig. 7 Overlay of the ATP-binding domain of Nek2 in complex with 24 (green) and 96 (grey).
However, this belief ignores the strict stereoelectronic Experimental section
requirements for efficient reaction of a protein nucleophile
Chemicals and solvents
with an electrophilic reagent,15 and thus so far adverse
properties of covalent inhibitors have been found to be
minimal. Nevertheless, the development of covalent
inhibitors must recognise the potential for off-target effects
and be biomarker-focussed throughout. In this paper we have
shown that selective covalent binding to Nek2 can be
achieved with ethynylpurines, which exhibit numerous
positive features as potential anticancer agents and therefore
provide ample justification for the pursuit of this protein
target with small molecule inhibitors.
All chemical reagents were purchased from the Aldrich
Chemical Company, Apollo Scientific or Alfa Aesar Chemicals
and were of the highest available purity. Chemicals were used
as supplied with no further treatment. If chemicals used were
stated as dry/anhydrous, they were stored in SureSeal™
septum-sealed bottles and removed under an inert nitrogen
environment, with the reaction being carried out under the
relevant inert atmosphere. Palladium catalysts were stored
and measured out under an inert atmosphere.
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Fig. 8 Interaction with competitively binding control compound 102 with Nek2: A) alignment of crystal structures of Nek2 (green) covalently
bound to 24 (cyan) and Nek2 (red) non-covalently bound to 102 (magenta). B) Space-filling representation of 102 (magenta) bound into the
nucleotide-binding pocket. C) Crystal structure of 102 (magenta) bound to Nek2, including electron density map (2Fo-Fc) contoured at 1.0σ
(mesh). Hydrophobic residues lining the pocket, the hinge residues Glu87 and Cys89, and unadducted Cys22 are indicated.
Table 5 In vitro ADME properties for 66 (Cyprotex Discovery, Macclesfield, UK)
Microsomal stability
Microsomal stability (human)
Microsomal stability (mouse)
CLint (μL min−1 mg−1
)
S.E. CLint
0.681
T1/2 (min)
n
CLint (μL min−1 mg−1
)
S.E. CLint
1.82
T1/2 (min)
n
6.21
223
5
10.2
136
5
Solubility
Estimated precipitation range (μM)
Lower bound
100
Upper bound
Calculated mid-range
>100
>100
Caco-2 permeability
Papp (× 10−6 cm−1
SD
n
Mean Papp
)
Mean recovery
(%)
(× 10−6 cm−1
)
1
2
Direction
A → B
B → A
0.0534
4.88
0.0421
5.29
0.0477
5.08
0.00799
0.293
2
2
57.6
64.0
Chromatography
Biotage automated chromatography using pre-packed silica
cartridges. A Biotage SP4 automated flash purification system
was used with UV monitoring at 298 nm and compound
collection at 254 nm. Biotage KP-NH cartridges were
employed for the separation of secondary, tertiary, and
heterocyclic amines; using a primary amine (propyl amine)
bonded silica. When stated, compounds were purified via
semi-preparative HPLC, using an ACE 5 Phenyl 150 × 21.2
mm column using an Agilent 1200 Modular Preparative
HPLC system.
Reaction monitoring and compound identification was aided
using thin layer chromatography (TLC) and retardation factor
(Rf) values. TLC was conducted with Merck aluminium
backed Si F254, NH2 F254s and RP-18 F254s plates. Fluorescent
compounds were visualised under short wave (254 nm) UV
irradiation. Compound purification was achieved using
medium pressure ‘Flash’ column chromatography, with the
use of Davisil silica 40–60 μm as the stationary phase, or
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Analytical techniques
Et2O (300 mL), before being removed from the column and
dried in a vacuum oven at 40 °C.
All melting points were determined using a Stuart Scientific
SMP3 or a Stuart Scientific SMP40 melting point apparatus
1
and are uncorrected. H and 13C nuclear magnetic resonance
General procedure A: TFA/TFE coupling of anilines with
2-fluoropurines using conventional heating
(NMR) spectra were obtained as solutions in deuterated
solvents DMSO-d6, MeOD or CDCl3 using a Bruker Avance III
500 spectrometer recording at 500 MHz. Chemical shifts (δ)
are reported in parts per million (ppm) and the spin-
multiplicity abbreviated as: s (singlet), d (doublet), t (triplet),
q (quartet), quin (quintet), sept (septet), m (multiplet), or br
(broad), with coupling constants (J) given in Hertz (Hz).
Liquid chromatography – mass spectrometry (LC–MS) was
carried out on a Micromass Platform LC running in both
positive and negative electrospray mode with a PDA 240–400
nm detector using a Waters Symmetry Shield RP18 3 μm, 4.6
× 20 mm column with a flow rate of 3.0 mL min−1.
Alternatively, a Waters Acquity UPLC system was used, with a
Waters SQD ESCi source using an Acquity UPLC BEH C18 1.7
μm, 2.1 × 50 mm column with a flow rate of 0.6 mL min−1.
The mobile phase used was 0.1 % v/v formic acid (aq.)/MeCN.
Fourier transform infrared (FTIR) spectra were obtained
using a Bio-Rad FTS 3000MX diamond ATR as a neat sample.
Ultraviolet (UV) absorption data were collected using a
Hitachi U-2800A spectrophotometer in ethanol. High-
resolution mass spectra were performed by the ESPRC UK
National Mass Spectrometry Facility, Swansea University,
Singleton Park, Swansea, SA2 8PP. The purity of final
compounds was assessed by reversed-phase HPLC; all tested
compounds were >95% purity. HPLC instrument, Agilent
1200 equipped with a photodiode array detector (190–400
nm). Sample temperature, ambient; injection volume, 5 μL;
flow rate, 1 mL min−1. 5% to 100% MeCN gradient over 9
min and an isocratic hold at 100% MeCN for 2.5 min, before
returning to initial conditions. Mobile phase A = 0.1%
ammonia in water or 0.1% formic acid in water, mobile
phase B = MeCN. Column: Waters XSELECT CSH C18, 3.5
μm, 4.6 mm × 150 mm or Waters XTerra RP18, 5 μm, 4.6 mm
× 150 mm. Column maintained at ambient temperature.
TFA (2.5–5.0 equiv.) was added to a solution of the purine
substrate (1.0 equiv.) and the required aniline (2.0 equiv.) in
TFE (10 mL mmol−1). The reaction mixture was heated at
reflux for 24 h unless otherwise stated, after which the
solution was cooled and evaporated to dryness. The resulting
residue was dissolved in EtOAc (10 mL mmol−1) and washed
with a saturated aqueous solution of NaHCO3 (5 mL mmol−1)
and brine (5 mL mmol−1). The combined aqueous layers were
extracted with EtOAc (10 mL mmol−1), and the combined
organic extracts were dried (MgSO4) and concentrated in
vacuo to give the crude product for chromatographic
purification.
General procedure B: removal of TIPS-protecting groups
using TBAF
TBAF (1.2 equiv.) was added to a solution of the TIPS-
protected substrate (1.0 equiv.) in THF (10–20 mL mmol−1).
The reaction mixture was stirred at RT for 5 min before being
concentrated in vacuo and the crude residue purified by
chromatography to afford the target compound.
6-Chloro-2-fluoro-9-(tetrahydro-2H-pyran-2-yl)-9H-purine
(10)33
Part 1. To a stirred solution of HBF4 (48% aqueous, 120
mL) at 0 °C, was added 2-amino-6-chloropurine (6.0 g, 35.0
mmol). Over 20 min, a solution of NaNO2 (4.9 g, 70.0 mmol)
in water (200 mL) was added dropwise, ensuring the
temperature remained close to 0 °C. The pale yellow solution
was raised to RT and stirred for 18 h. The resulting solution
was neutralised to pH 7 in an ice bath at 0 °C by addition of
Na2CO3 (6.00 g) in water (200 mL). The crude material was
purified by chromatography on silica (10% MeOH/DCM) to
afford 6-chloro-2-fluoropurine as a white crystalline solid
(4.52 g, 75%); mp 171–173 °C (lit.,34 mp 174 °C); λmax (EtOH/
nm) 393; IR (cm−1) 2964, 2785, 1735, 1581; 1H NMR (500
MHz, DMSO-d6) 8.60 (1H, s, H-8), 13.9 (1H, s, NH-9); LRMS
(ES+) m/z 172.6 [M + H]+.
Part 2. 3,4-Dihydropyran (60 μL, 0.58 mmol) was added
dropwise over 10 min to a vigorously stirred solution of
6-chloro-2-fluoropurine (100 mg, 0.58 mmol) and (rac)-
camphorsulfonic acid (5 mg, 0.02 mmol) in EtOAc (50 mL) at
65 °C. The temperature was maintained at 65 °C for 18 h.
The resulting bright yellow solution was neutralised to pH 7
by careful addition of aqueous NH3 solution, until a cloudy
suspension persisted. The crude mixture was washed with
brine (2 × 30 mL) and the aqueous phase was re-extracted
with EtOAc (2 × 30 mL). The combined organic extracts were
dried (Na2SO4) and purified by chromatography on silica
(30% EtOAc/petrol). The desired compound was isolated as a
pale yellow oil which solidified on refrigeration (110 mg,
75%); mp 69–70 °C (lit.33 mp not available); λmax (EtOH/nm)
Microwave assisted synthesis
When stated, reactions were carried out under microwave
irradiation, in sealed vessels, using a Biotage Initiator Sixty
with robotic sample bed. Samples were irradiated at 2.45
GHz, able to reach temperatures of 60–250 °C with a rate of
heating at 2–5 °C s−1, and pressures of up to 20 bar.
TBAF scavenger resin32
Amberlite 15 Ion exchange resin (SO3H, 100 mL) was loaded
into a column and washed with water (400 mL). The column
was eluted with sat. calcium hydroxide solution whilst the
pH of the eluent was monitored. Once the initially pH neutral
eluent became strongly basic, the column was eluted with
water until the pH of the eluent returned to neutral. The
resin was washed with DCM (300 mL), THF (300 mL) and
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269; IR (cm−1) 3125, 2954, 2872, 2028, 1577; 1H NMR (300
MHz, DMSO-d6) 1.60 (2H, m, CH2), 1.74 (1H, m, CH), 2.01
(2H, m, CH2), 2.32 (1H, s, CH), 3.71 (1H, t, J = 12.0 Hz, CH),
4.01 (1H, d, J = 12.0 Hz, CH), 5.64 (1H, d, J = 12.0 Hz, CH),
8.25 (1H, s, H-8); LRMS (ES+) m/z 257.7 [M + H]+.
2-Fluoro-9-(tetrahydro-2H-pyran-2-yl)-6-((triisopropylsilyl)-
ethynyl)-9H-purine (11). An oxygen-free solution of 6-chloro-2-
fluoro-9-(tetrahydro-2H-pyran-2-yl)-9H-purine (10) (50 mg, 1.95
mmol), bisIJtriphenylphosphine)palladiumIJII) chloride (41 mg,
3.00 mol%) and copper iodide (7 mg, 2.00 mol%) in THF (10
mL) was degassed by bubbling nitrogen through the solution
synthesised following general procedure A using: 2-fluoro-6-
((triisopropylsilyl)ethynyl)-9H-purine (12) (0.156 g, 0.49 mmol)
and 4-aminobenzenesulfonamide (0.17 g, 0.98 mmol). The
compound was purified using reversed phase column
chromatography (C18 silica; 25% to 95% MeCN/water + 0.1%
HCOOH), followed by trituration of the resulting oil using
DCM, to obtain the product as a yellow solid (70 mg, 30%);
m.p.163–165 °C; λmax (EtOH/nm) 361.0, 291.0, 286.5, 215.5;
1
IR (cm−1) 3327, 2944, 2867, 1569, 1531, 1368, 1149; H NMR
(500 MHz, DMSO-d6) 1.15 (21H, m, SiIJCHIJCH3)2)3), 7.17 (2H,
s, SO2NH2), 7.70 (2H, d, J = 9.0 Hz, H-2′ and H-6′), 7.95 (2H,
d, J = 9.0 Hz, H-3′ and H-5′), 8.33 (1H, s, H-8); HRMS calcd.
for C22H31N6O2SSi [M + H]+ 471.19874, found 471.19420.
6-Ethynyl-N-phenyl-9H-purin-2-amine (23). The TIPS-
protected purine 13 (0.330 g, 0.84 mmol) and TBAF (1 M in
THF, 930 μL, 0.93 mmol) were reacted in THF (10 mL)
according to general procedure B, with purification by
chromatography on silica (EtOAc) to give the desired product
as a yellow solid (0.201 g, 100%); Rf 0.27 (EtOAc); mp 140–160
°C (decomposed); λmax (EtOH/nm) 243; IR (cm−1) 3414, 3111,
3072, 2920, 2110, 1704; 1H NMR (500 MHz, DMSO-d6) 4.86
(1H, s, CCH), 6.91–6.96 (1H, m, H-4′), 7.29 (2H, dd, J = 7.6
and 8.0 Hz, H-3′ and H-5′), 7.80 (2H, dd, J = 2.0 and 8.0 Hz,
H-2′ and H-6′), 8.30 (1H, s, H-8), 9.70 (1H, s, NH), 13.17 (1H,
br, NH-9); HRMS calcd. for C13H10N5 (ES+) m/z 236.0934 [M +
H]+, found 236.0931.
in
a
sealed Biotage microwave vial for
5
min.
Triisopropylsilylacetylene (0.50 mL, 2.20 mmol) and
triethylamine (0.70 mL, 4.90 mmol) were added to the
mixture which was again degassed for 15 min. The solution
quickly became dark red and stirring was continued at room
temperature for 18 h. The black-brown suspension was
filtered through Celite®, eluting with MeOH (3 × 20 mL). The
product was purified by chromatography on silica (10%
EtOAc/petrol) and isolated as a viscous yellow oil (78 mg,
99%); λmax (EtOH/nm) 303.5; IR (cm−1) 3433, 2945, 2865,
2705, 1702; 1H NMR (500 MHz, DMSO-d6) 1.15 (21H, m,
SiIJCHIJCH3)2)3 and SiIJCHIJCH3)2)3), 1.60 (2H, m, CH2), 1.75
(1H, m, CH), 1.99 (2H, m, CH2), 2.19 (1H, s, CH), 3.74 (1H, t,
J = 12.0 Hz, CH), 4.16 (1H, d, J = 12.0 Hz, CH), 5.69 (1H, d, J
= 12.0 Hz, CH), 8.30 (1H, s, H-8); LRMS (ES+) m/z 403.4
[M + H]+.
2-Fluoro-6-(2-(triisopropylsilyl)ethynyl)-9H-purine (12). TFA
(3 mL) was added to a solution of THP-protected purine 11
(0.644 g, 1.60 mmol) in IPA (15 mL). Water (3 mL) was added
and the solution was heated to reflux for 2 h. The mixture
was cooled and neutralised (conc. NH3) before being
extracted with EtOAc (3 × 50 mL) and the combined organic
extracts dried (MgSO4) and concentrated. The resulting
residue was purified by chromatography on silica (30%
EtOAc/petrol) to give the desired product as a pale yellow oil
(0.461 g, 91%); Rf 0.25 (7 : 3 petrol/EtOAc); λmax (EtOH/nm)
302; IR (cm−1) 2945, 2866, 2361, 2000, 1584; 1H NMR (500
MHz, DMSO-d6) 1.12–1.21 (21H, m, SiIJCHIJCH3)2)3), 8.68 (1H,
s, H-8), 13.89 (1H, br, NH-9); HRMS calcd. for C16H24FN4Si
(ES+) m/z 319.1749 [M + H]+, found 319.1752.
N-Phenyl-6-((triisopropylsilyl)ethynyl)-9H-purin-2-amine
(13). According to general procedure A, the title compound
was prepared using: 2-fluoro-6-((triisopropylsilyl)ethynyl)-9H-
purine (12) (0.70 g, 2.2 mmol) and aniline (0.40 mL, 4.4
mmol). The compound was isolated after chromatography
(silica: 5% MeOH/DCM) followed by reversed phase column
chromatography (C18 silica; 25% to 95% MeCN/water + 0.1%
HCOOH), as a yellow oil (0.44 g, 47%); λmax (EtOH/nm) 276;
IR (cm−1) 3389, 2361, 2021; 1H NMR (500 MHz, CDCl3) 1.13
(21H, m, SiIJCHIJCH3)2)3), 7.02 (1H, t, J = 7.5 Hz, H-4′), 7.33
(2H, dd, J = 7.4, 7.5 Hz, H-3′ and H-5′), 7.80 (2H, d, J = 7.4 Hz,
H-2′ and H-6′), 10.42 (1H, s, NH); LRMS (ES+) m/z 392.0 [M +
H]+.
4-(6-Ethynyl-9H-purin-2-ylamino)benzenesulfonamide (24).
Following general procedure B, the title compound was
prepared using: TBAF solution (1.0 M in THF, 170 μL, 0.17
mmol) and 4-(6-((triisopropylsilyl)ethynyl)-9H-purin-2-ylamino)-
benzenesulfonamide (14) (50 mg, 0.11 mmol) in THF (3 mL)
to complete the deprotection after 5 min. The title compound
was purified by chromatography on silica (10% MeOH/DCM)
and isolated as a beige solid (18 mg, 50%); mp 156–158 °C;
λmax (EtOH/nm) 356.0, 292.5, 215.5; IR (cm−1) 3347, 3255,
1
2920, 2848, 2118, 1568, 1529, 1477, 1128; H NMR (500 MHz,
DMSO-d6) 4.90 (1H, s, CC–H), 7.16 (2H, s, SO2NH2), 7.17
(2H, d, J = 9.0 Hz, H-2′ and H-6′), 7.93 (2H, d, J = 9.0 Hz, H-3′
and H-5′), 8.36 (1H, s, H-8); HRMS calcd. for C13H11N6O2S [M
+ H]+ 315.0664, found 315.0687.
2-(3-(6-(2-(Triisopropylsilyl)ethynyl)-9H-purin-2-ylamino)-
phenyl)acetic acid (59). 2-Fluoropurine intermediate 12 (1.06
g, 3.32 mmol) and 3-aminophenyl acetic acid (1.00 g, 6.64
mmol) were reacted with TFA (1.28 mL, 16.6 mmol) in TFE
(25 mL) according to general procedure A. Upon completion
of the reaction, the reaction solvent was removed in vacuo
and the resultant residue was dissolved in THF (20 mL) and
1 M NaOH solution (15 mL). The mixture was stirred at RT
for 18 h before the THF was removed in vacuo. The aqueous
solution was then taken to pH 3 with 4 M HCl solution and
extracted with EtOAc (3 × 75 mL). The combined organic
extracts were dried (Na2SO4) and concentrated, and the
resulting orange residue was purified by chromatography on
silica (9 : 1 DCM/MeOH) followed by chromatography on
reverse phase silica (9 : 1 MeOH/H2O + 0.1% HCOOH). The
desired product was obtained as an orange oil/gum (0.952 g,
4-(6-((Triisopropylsilyl)ethynyl)-9H-purin-2-ylamino)-
benzenesulfonamide (14). The title compound was
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64%); Rf 0.32 (9 : 1 DCM/MeOH); λmax (EtOH/nm) 275; IR
(cm−1) 2972, 2360, 2340, 1977, 1702; 1H NMR (500 MHz,
DMSO-d6) 1.13–1.22 (21H, m, SiIJCHIJCH3)2)3), 3.51 (2H, s,
CH2CO2H), 6.82–6.85 (1H, m, H-6′), 7.22 (1H, dd, J = 7.7 and
7.8 Hz, H-5′), 7.65–7.68 (1H, m, H-2′), 7.72–7.76 (1H, m, H-4′),
8.25 (1H, s, H-8), 9.71 (1H, s, NH), 12.33 (1H, br, CO2H),
13.10 (1H, br, NH-9); HRMS calcd. for C24H32N5O2Si (ES+) m/z
450.2320 [M + H]+, found 450.2320.
2921, 2107, 1658; 1H NMR (500 MHz, DMSO-d6) 4.76 (1H, s,
CCH), 6.77–6.81 (1H, m, H-6′), 6.83 (1H, s, CONHH′), 7.15
(1H, dd, J = 8.0 and 8.1 Hz, H-5′), 7.38 (1H, s, CONHH′), 7.47–
7.50 (1H, m, H-2′), 7.68–7.73 (1H, m, H-4′), 8.19 (1H, s, H-8),
9.52 (1H, s, NH), 13.06 (1H, s, NH-9); HRMS calcd. for
C15H13N6O (ES+) m/z 293.1149 [M + H]+, found 293.1145.
2,2,2-TrifluoroethylIJ3-((6-((triisopropylsilyl)ethynyl)-9H-
purin-2-yl)amino)phenyl)methanesulfonate
(83).
N-(4-Methoxybenzyl)-2-(3-(6-(2-(triisopropylsilyl)ethynyl)-
9H-purin-2-ylamino)phenyl)acetamide (61). CDI (0.140 g, 0.86
mmol) and DIPEA (150 μL, 0.86 mmol) were added to a
solution of the carboxylic acid 59 (0.193 g, 0.43 mmol) in dry
DMF (5 mL). The mixture was stirred at RT for 1.5 h, at which
point 4-methoxybenzylamine (223 μL, 1.72 mmol) was added.
Following a further 18 h stirring at RT, the solvent was
removed in vacuo and the resulting residue was purified by
chromatography on KP-NH silica (19 : 1 DCM/MeOH) gave the
desired compound as a yellow oil/gum (0.238 g, 99%); Rf 0.48
(19 : 1 DCM/MeOH, KP-NH); λmax (EtOH/nm) 277; IR (cm−1)
2360, 2153, 2120, 1980; 1H NMR (500 MHz, DMSO-d6) 1.12–
1.25 (21H, m, SiIJCHIJCH3)2)3), 3.42 (2H, s, COCH2), 3.71 (3H, s,
OCH3), 4.20 (2H, d, J = 5.8 Hz, NHCH2), 6.85 (2H, d, J = 8.8
Hz, H-3″ and H-5″), 6.86–6.91 (1H, m, H-6′), 7.17 (2H, d, J =
8.8 Hz, H-2″ and H-6″), 7.20 (1H, dd, J = 7.9 and 8.0 Hz, H-5′),
7.59–7.62 (1H, m, H-2′), 7.74–7.78 (1H, m, H-4′), 8.24 (1H, s,
H-8), 8.44 (1H, t, J = 5.8 Hz, NHCH2), 9.65 (1H, s, NH), 12.06
(1H, br, NH-9); HRMS calcd. for C32H41N6O2Si (ES+) m/z
569.3055 [M + H]+, found 569.3057.
2-(3-(6-(2-(Triisopropylsilyl)ethynyl)-9H-purin-2-ylamino)-
phenyl)acetamide (63). PMB-amide 61 (0.232 g, 0.41 mmol)
was dissolved in TFA (6 mL) and the resulting solution was
heated at reflux for 72 h. The reaction mixture was
evaporated to dryness and the resulting residue was dissolved
in EtOAc (8 mL) and washed with a saturated aqueous
solution of NaHCO3 (2 × 4 m) and brine (4 mL). The
combined aqueous layers were extracted with EtOAc (8 mL)
and the combined organic extracts were dried (MgSO4),
concentrated in vacuo and the residue purified by
chromatography on KP-NH silica (19 : 1 DCM/MeOH) gave the
desired compound as a yellow oil/gum (0.170 g, 90%); Rf 0.24
(19 : 1 DCM/MeOH, KP-NH); λmax (EtOH/nm) 276; IR (cm−1)
3282, 2965, 2943, 2362, 1669; 1H NMR (500 MHz, DMSO-d6)
1.13–1.21 (21H, m, SiIJCHIJCH3)2)3), 3.34 (2H, s, COCH2), 6.83–
6.86 (1H, m, H-6′), 6.88 (1H, s, CONHH′), 7.19 (1H, dd, J = 8.5
and 8.7 Hz, H-5′), 7.42 (1H, s, CONHH′), 7.57–7.60 (1H, m, H-
2′), 7.72–7.76 (1H, m, H-4′), 8.23 (1H, s, H-8), 9.65 (1H, s,
NH), 13.08 (1H, s, NH-9); HRMS calcd. for C24H33N6OSi (ES+)
m/z 449.2480 [M + H]+, found 449.2480.
2-Fluoropurine intermediate 12 (0.416 g, 1.31 mmol) and
aniline 160 (0.704 g, 2.62 mmol) were reacted with TFA (504
μL, 6.54 mmol) in TFE (10 mL) according to general
procedure A. The resulting orange oil was purified by
chromatography on reverse phase silica (19 : 1 MeOH/H2O +
0.1% HCOOH) followed by chromatography on silica (7 : 3
petrol/EtOAc) to give the desired compound as a yellow/
orange oil/gum (0.506 g, 68%); Rf 0.34 (9 : 1 MeOH/H2O,
+0.1% HCOOH, C18); λmax (EtOH/nm) 368, 277; IR (cm−1)
2950, 2365, 2161, 2011, 1967, 1601; 1H NMR (500 MHz,
DMSO-d6) 1.13–2.23 (21H, m, SiIJCHIJCH3)2)3), 4.85 (2H, s, Ar–
CH2), 4.95 (2H, q, J = 8.7 Hz, F3CCH2O), 7.00–7.04 (1H, m, H-
6′), 7.34 (1H, dd, J = 7.9 and 8.1 Hz, H-5′), 7.74–7.77 (1H, m,
H-2′), 7.96–8.00 (1H, m, H-4′), 8.28 (1H, s, H-8), 9.84 (1H, s,
NH), 13.12 (1H, br, NH-9); HRMS calcd. for C25H33F3N5O3SSi
(ES+) m/z 568.2020 [M + H]+, found 568.2015.
N-(4-Methoxybenzyl)-1-(3-((6-((triisopropylsilyl)ethynyl)-9H-
purin-2-yl)amino)phenyl)methanesulfonamide
(84).
Trifluoroethyl sulfonate ester 83 (0.215 g, 0.38 mmol),
4-methoxybenzylamine (64 μl, 0.49 mmol) and DBU (115 μL,
0.76 mmol) were combined in dry THF (3 mL) in a sealed
vial. The reaction mixture was heated under microwave
irradiation at 160 °C for 15 min, before being evaporated to
dryness. The resulting residue was dissolved in DCM (4 mL)
and washed with a saturated aqueous solution of NaHCO3 (4
mL), after which the biphasic mixture was passed through an
Isolute® phase separator and the organic phase concentrated
in vacuo. The crude residue was purified by chromatography
on silica (19 : 1 DCM/MeOH) gave the desired compound as a
yellow oil (0.224 g, 97%); Rf 0.44 (19 : 1 DCM/MeOH); λmax
(EtOH/nm) 276; IR (cm−1) 2361, 2341, 2162, 1992, 1969, 1609;
1H NMR (500 MHz, DMSO-d6) 1.13–1.22 (21H, m,
SiIJCHIJCH3)2)3), 3.72 (3H, s, OCH3), 4.05 (2H, d, J = 6.0 Hz,
NHCH2), 4.23 (2H, s, Ar–CH2), 6.87 (2H, d, J = 8.7 Hz, H-3″
and H-5″), 6.91–6.95 (1H, m, H-6′), 7.23 (2H, d, J = 8.7 Hz, H-
2″ and H-6″), 7.28 (1H, dd, J = 8.0 and 8.2 Hz, H-5′), 7.55 (1H,
t, J = 6.0 Hz, NHCH2), 7.72–7.75 (1H, m, H-2′), 7.87–7.91 (1H,
m, H-4′), 8.26 (1H, s, H-8), 9.76 (1H, s, NH), 13.06 (1H, s, NH-
9); HRMS calcd. for C31H41N6O3SSi (ES+) m/z 605.2725 [M +
H]+, found 605.2723.
2-(3-(6-Ethynyl-9H-purin-2-ylamino)phenyl)acetamide (66).
The TIPS-protected purine 63 (0.165 g, 0.37 mmol) was
reacted with TBAF (1 M in THF, 0.55 mL, 0.55 mmol) in THF
(4 mL) according to general procedure B. The residue was
purified by semi-preparative HPLC (17 : 3 H2O/MeCN), to give
the desired compound as a yellow solid (0.084 g, 0.28 mmol,
76%); Rf 0.32 (17 : 3 DCM/MeOH, KP-NH); mp 250–270 °C
(decomposed); λmax (EtOH/nm) 274; IR (cm−1) 3359, 3170,
(3-((6-((Triisopropylsilyl)ethynyl)-9H-purin-2-yl)amino)-
phenyl)methanesulfonamide (85). PMB-sulfonamide 84
(0.221 g, 0.37 mmol) was dissolved in TFA (6 mL) and the
resulting solution was heated at reflux for 3 h. The reaction
mixture was evaporated to dryness and the resulting residue
was dissolved in EtOAc (7 mL) and washed with a saturated
aqueous solution of NaHCO3 (2 × 4 mL) and brine (4 mL).
The combined aqueous layers were extracted with EtOAc (7
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mL) and the combined organic extracts were dried (MgSO4),
concentrated in vacuo and the residue purified by
chromatography on KP-NH silica (19 : 1 DCM/MeOH) to give
the desired compound as a yellow oil/gum (0.108 g, 60%); Rf
0.21 (19 : 1 DCM/MeOH, KP-NH); λmax (EtOH/nm) 276; IR
(cm−1) 3367, 2943, 2865, 2160, 2021, 1606; 1H NMR (500
MHz, DMSO-d6) 1.13–1.23 (21H, m, SiIJCHIJCH3)2)3), 4.21 (2H,
s, Ar–CH2), 6.84 (2H, s, SO2NH2), 6.95–6.98 (1H, br, H-6′), 7.13
(1H, dd, J = 7.9 and 8.0 Hz, H-5′), 7.68–7.71 (1H, m, H-2′),
7.90 (1H, ddd, J = 1.0, 1.9 and 8.0 Hz, H-4′), 8.25 (1H, s, H-8),
9.75 (1H, s, NH), 13.07 (1H, s, NH-9); HRMS calcd. for
C23H33N6O2SSi (ES+) m/z 485.2149 [M + H]+, found 485.2147.
( 3 - ( ( 6 - E t h y n y l - 9 H - p u r i n - 2 - y l ) a m i n o ) p h e n y l ) -
methanesulfonamide (87). TBAF (1 M in THF, 0.15 mL, 0.15
mmol) was added to a solution of TIPS-protected purine 85
(60 mg, 0.12 mmol) in THF (5 mL). The reaction mixture was
stirred at RT for 5 min before being diluted with THF (12
mL) and the TBAF scavenger resin (0.60 g, 10× w/w) added.
The resulting suspension was agitated at RT for 48 h, before
being filtered and the filtrate concentrated in vacuo. The
resulting residue was purified by chromatography on silica
(9 : 1 DCM/MeOH) to afford the target compound as a pale
yellow solid (23 mg, 58%); Rf 0.29 (9 : 1 DCM/MeOH); mp
190–210 °C (decomposed); λmax (EtOH/nm) 273.5, 359.0; IR
(cm−1) 3361, 3248, 2976, 2809, 2707, 2114, 1701, 1610, 1583,
1491; 1H NMR (500 MHz, DMSO-d6) 4.22 (2H, s, SO2CH2),
4.85 (1H, s, CCH), 6.86 (2H, s, SO2NH2), 6.94–6.98 (1H, m,
H-6′), 7.30 (1H, dd, J = 7.7 and 8.0 Hz, H-5′), 7.64–7.68 (1H,
m, H-2′), 7.88–7.92 (1H, m, H-4′), 8.29 (1H, s, H-8), 9.72 (1H,
s, NH), 13.13 (1H, br, NH-9); HRMS calcd. for C14H13N6O2S
(ES+) m/z 329.0815 [M + H]+, found 329.0821.
(E)-6-(2-(Azepan-1-yl)vinyl)-N-phenyl-9H-purin-2-amine
(89)14. A solution of 6-ethynyl-N-phenyl-9H-purin-2-amine (23)
(50 mg, 0.21 mmol) and homopiperidine (480 μL, 4.23 mmol)
in anhydrous THF (2 mL) was subjected to microwave
heating at 100 °C for 10 minutes in a sealed nitrogen flushed
microwave vial (2–5 mL capacity). The cooled solution was
partitioned between EtOAc (20 mL) and saturated NaHCO3
solution (20 mL). The organic extract was concentrated in
vacuo to a yellow/orange syrup which was subjected to
purification by chromatography on KP-NH silica (9.5 : 0.5
DCM/MeOH), yielding the title compound as a yellow solid
(69 mg, 98%); mp 135–137 °C (lit.,14 mp 135–137 °C); λmax
(EtOH/nm) 360, 238, 254; IR (cm−1) 3030, 2921, 2850, 1629,
1559; 1H NMR (500 MHz, CDCl3) 1.49 (4H, m,
homopiperidine CH2), 1.64–1.71 (4H, m, homopiperidine
CH2), 3.28–3.38 (4H, m, homopiperidine CH2), 5.49–5.52 (1H,
d, J = 15.0 Hz, CHCH), 6.95–6.99 (1H, t, J = 10.5 Hz, H-4′),
7.19 (1H, s, H-8), 7.22–7.26 (2H, dd, J = 9.5, 10.5 Hz, H-3′ and
H-5′), 7.32 (1H, br s, NH), 7.44–7.46 (2H, d, J = 9.5 Hz, H-2′
and H-6′), 8.23–8.26 (1H, d, J = 15.0 Hz, CHCH), 12.89 (1H,
br s, NH-9); HRMS calcd. for C19H22N6 [M + H]+ 335.1975,
found 335.1979.
was added sodium cyanoborohydride solution (1 M, in THF,
1.95 mL, 1.95 mmol) followed by TFA (3 μL, 0.04 mmol) or
HCl (aq) (1 M, 40 μL, 0.04 mmol). Stirring was continued for
2 h (TFA) or 5 h (HCl), after which the solution was
partitioned between EtOAc (20 mL) and saturated NaHCO3
solution (10 mL). The organic extract was dried (Na2SO4)
before purification by chromatography on KP-NH silica (9.5 :
0.5 DCM/MeOH) to yield a pale yellow solid (41 mg, 30%);
mp 47–49 °C; λmax (EtOH/nm) 326, 277, 238; IR (cm−1) 2928,
1
2858, 1582; H NMR (500 MHz, DMSO-d6) 1.32–1.33 (4H, m,
homopiperidine CH2), 1.39–1.40 (4H, m, homopiperidine
CH2), 2.53–2.56 (4H, t, J = 5.5 Hz, homopiperidine NCH2),
2.86–2.91 (2H, t, J = 8.0 Hz, CH2), 2.92–2.98 (2H, t, J = 8.0 Hz,
CH2), 6.69–6.72 (1H, t, J = 8.5 Hz, H-4′), 7.05–7.08 (2H, dd, J =
7.5 and 8.5 Hz, H-3′ and H-5′), 7.65–7.66 (2H, d, J = 7.5 Hz,
H-2′ and H-6′), 7.97 (1H, s, H-8), 9.21 (1H, br s, NH); HRMS
calcd. for C19H25N6 [M + H]+ 337.2135, found 337.2138.
N-Phenyl-6-vinyl-9H-purin-2-amine (92). m-CPBA (titrated
as 62%, 30 mg, 0.108 mmol) was added in one portion to a
stirred solution of 6-(2-(azepan-1-yl)ethyl)-N-phenyl-9H-purin-2-
amine (90) (30 mg, 0.09 mmol) in anhydrous DCM (2 mL).
The colourless solution instantly became bright yellow. After
2 h at room temperature the reaction was diluted with DCM
(5 mL) and washed with saturated NaHCO3 solution (4 mL).
The crude product was purified by chromatography on silica
(9.5 : 0.5 DCM/MeOH) to yield a yellow solid (6.4 mg, 30%);
mp 121–123 °C; UV λmax (EtOH) 270, 207; IR (cm−1) 1578,
1532, 1496, 1439, 1392, 1349; 1H NMR (500 MHz, DMSO-d6)
5.84–5.86 (1H, dd, J = 1.5 and 12.5 Hz, cis CHCHH), 6.86–
6.89 (1H, dd, J = 1.5 and 17.5 Hz, trans CHCHH), 7.01–7.04
(1H, t, J = 8.5 Hz, H-4′), 7.08–7.14 (1H, dd, J = 12.5 and 17.5
Hz, CHCH2), 7.24 (1H, s, H-8), 7.27–7.30 (2H, dd, J = 8.5
and 7.5 Hz, H-3′ and H-5′), 7.52–7.54 (2H, d, J = 7.5 Hz, H-2′
and H-6′); HRMS calcd. for C13H12N5 [M + H]+ 238.0509,
found 238.0511.
6-Ethynyl-2-fluoro-9-(tetrahydro-2H-pyran-2-yl)-9H-purine
(93)17. The TIPS-protected purine 11 (68 mg, 0.169 mmol)
and TBAF (1 M in THF, 203 μL, 0.203 mmol) were reacted in
THF (3 mL) according to general procedure B, with
purification by chromatography on silica (15–100% EtOAc/
petrol) to give the desired product as an off-white solid (41
mg, 100%); Rf 0.32 (50% EtOAc/petrol); 1H NMR (500 MHz,
CDCl3) 1.58–1.63 (1H, m, CH), 1.63–1.78 (2H, m, CH2), 1.90–
1.99 (1H, m, CH), 1.99–2.06 (1H, m, CH), 2.07–2.12 (1H, m,
CH), 3.68–3.74 (1H, m, CH), 3.71 (1H, s, CCH), 4.09–4.14
(1H, m, CH), 5.65 (1H, dd, J = 2.6 and 10.8 Hz, NCH), 8.26
(1H, s, H-8); LRMS (ES+) m/z 247.0 [M + H]+.
6-Ethyl-2-fluoro-9-(tetrahydro-2H-pyran-2-yl)-9H-purine
(94). Lindlar's catalyst (10 mg, 20% w/w) was suspended in a
stirred solution of 93 (50 mg, 0.20 mmol) and quinoline (20
μL, 0.16 mmol) in EtOAc (5 mL) under a balloon of H2. After
2 h at room temperature the reduction was complete and the
suspension was filtered through a plug of Celite, eluting with
methanol (30 mL). Volatiles were removed in vacuo and the
crude residue purified by chromatography on silica (50%
6-(2-(Azepan-1-yl)ethyl)-N-phenyl-9H-purin-2-amine
(90).
To a stirred solution of (E)-6-(2-(azepan-1-yl)vinyl)-N-phenyl-
9H-purin-2-amine (89) (130 mg, 0.39 mmol) in THF (3 mL)
EtOAc/petrol). The pure compound was isolated as
a
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colourless oil (50 mg, 99%); λmax (EtOH/nm) 264; IR (cm−1)
2946, 2860, 2364, 2338, 1604; 1H NMR (500 MHz, DMSO-d6)
1.35 (3H, t, J = 7.5 Hz, CH3), 1.59 (2H, m, tetrahydropyran
CH2), 1.97 (1H, m, tetrahydropyran CH), 1.99 (2H, d, J = 10.5
Hz, tetrahydropyran CH2), 2.50 (1H, m, tetrahydropyran CH),
3.01 (2H, q, J = 7.5 Hz, CH2), 3.73 (1H, m, tetrahydropyran
CH), 4.04 (1H, d, J = 10.5 Hz, tetrahydropyran CH), 5.69 (1H,
d, J = 10.5 Hz, tetrahydropyran CH), 8.74 (1H, s, H-8); LRMS
(ES+) m/z 251.0 [M + H]+.
6-Ethyl-2-fluoro-9H-purine (95). TFA (3 mL) was added to a
solution of 6-ethyl-2-fluoro-9-(tetrahydro-2H-pyran-2-yl)-9H-
purine (94) (0.18 g, 0.72 mmol) in IPA (15 mL). Water (3 mL)
was added and the solution was heated to reflux for 2 h. The
mixture was cooled and neutralised (conc. NH3) before being
extracted with EtOAc (3 × 50 mL) and the combined organic
extracts dried (MgSO4) and concentrated. The resulting
residue was purified by chromatography on silica (25%
EtOAc/petrol). The compound was isolated as a white solid
(0.117 g, 98%); mp 146–148 °C; λmax (EtOH/nm) 269; IR
(cm−1) 1676, 1616, 1573; 1H NMR (400 MHz, DMSO-d6) 1.30
(3H, t, J = 7.5 Hz, CH3), 3.00 (2H, q, J = 7.5 Hz, CH2), 8.17 (1
H, s, H-8); LRMS (ES+) m/z = 167.7 [M + H]+.
added dropwise on to crushed ice in an ice bath with rapid
stirring [CAUTION – VERY EXOTHERMIC]. Once addition
was complete and the ice had melted, the homogeneous
solution was neutralised to pH 7 by slow addition of NaOH
solution (1.0 M), maintaining rapid stirring in an ice bath.
The aqueous mixture was extracted with EtOAc (2 × 20 mL).
The combined organic extracts were dried (Na2SO4) and
purified by chromatography on silica (50% EtOAc/petrol). The
title compound was isolated as a white solid (0.67 g, 46%);
mp 172–174 °C (lit.32 155–160 °C); λmax (EtOH/nm) 329, 272;
IR (cm−1) 3399, 3289, 1627, 1601, 1571, 1540; 1H NMR (500
MHz, DMSO-d6) 6.89–6.92 (1H, t, J = 7.5 Hz, H-4′), 7.23–7.26
(2H, dd, J = 7.4 and 7.5 Hz, H-3′ and H-5′), 7.71–7.73 (2H, d, J
= 7.4 Hz, H-2′ and H-6′), 8.20 (1H, s, H-8), 9.81 (1H, br s, NH),
13.20 (1H, br s, NH-9); LRMS (ES+) m/z 246.06 [M + H]+.
6-Chloro-9-(4-methoxybenzyl)-N-phenyl-9H-purin-2-amine
(100). 4-Methoxybenzylchloride (0.33 mL, 2.43 mmol) was
added dropwise to a stirred solution of 6-chloro-N-phenyl-9H-
purin-2-amine (99) (0.15 g, 0.6 mmol) and K2CO3 (0.25 g, 1.83
mmol) in anhydrous DMF (15 mL). The resulting solution
was gently warmed to 60 °C under nitrogen for 18 h. Upon
addition of water (50 mL) and brine (6 mL) to the mixture, a
white precipitate was observed. The mixture was extracted
with DCM (2 × 30 mL) and the combined organics dried
(Na2SO4). The N-9 regioisomer was separated by column
chromatography on silica (50% EtOAc/petrol) as a white
crystalline solid (114 mg, 52%); mp 166–168 °C; λmax (EtOH/
4-(6-Ethyl-9H-purin-2-ylamino)benzenesulfonamide
(96).
The title compound was synthesised according to general
procedure A using: 6-ethyl-2-fluoro-9H-purine (95) (81 mg,
0.49 mmol) and 4-aminobenzenesulfonamide (0.17 g, 0.98
mmol). The compound was isolated after purification by
chromatography on silica (50% EtOAc/petrol) as a white solid
(33 mg, 21%); mp 291–293 °C; λmax (EtOH/nm) 318, 287, 212;
IR (cm−1) 3377, 3060, 2852, 1388, 1158; 1H NMR (500 MHz,
DMSO-d6) 1.35 (3H, t, J = 6.0 Hz, CH3), 3.00 (2H, q, J = 6.0
Hz, CH2), 7.14 (2H, br s, SO2NH2), 7.69 (2H, d, J = 7.5 Hz, H-
2′ and H-6′), 7.99 (2H, d, J = 7.5 Hz, H-3′ and H-5′), 8.17 (1H,
s, H-8), 9.89 (1H, br s, NH); HRMS calcd. for C13H15N6O2S [M
+ H]+ 319.0971, found 319.0979.
1
nm) 274, 225; IR (cm−1) 3286, 2834, 1599, 1511; H NMR (500
MHz, DMSO-d6) 3.74 (3H, s, OCH3), 5.35 (2H, s, CH2), 6.93–
6.95 (2H, d, J = 9.0 Hz, H-3″ and H-5″), 6.98–7.01 (1H, t, J =
8.5 Hz, H-4′), 7.30–7.34 (2H, dd, J = 8.0 and 8.5 Hz, H-3′ and
H-5′), 7.35–7.38 (2H, d, J = 9.0 Hz, H-2″ and H-6″), 7.76–7.79
(2H, d, J = 8.0 Hz, H-2′ and H-6′), 8.04 (1H, s, H-8), 9.80 (1H,
br s, NH); LRMS (ES+) m/z 365.1 [M + 1]+.
6-Cyano-9-(4-methoxybenzyl)-2-(phenylamino)-9H-purine-6
(101). 6-Chloro-9-(4-methoxybenzyl)-N-phenyl-9H-purin-2-amine
(100) (0.10 g, 0.27 mmol) was suspended in anhydrous MeCN
(10 mL) and stirred at room temperature. Addition of
tetraethylammonium cyanide (86 mg, 0.55 mmol) followed by
DABCO (61 mg, 0.55 mmol) afforded a yellow homogenous
solution which was stirred for a further 18 h under nitrogen.
Excess cyanide was hydrolysed by addition of aqueous
ammonium hydroxide solution (32% v/v, 30 mL) with stirring
for an additional 1 h. The crude mixture was partitioned
between DCM (20 mL) and brine (20 mL). The organic layer
was isolated and dried (Na2SO4) before purification by
column chromatography on silica (50% EtOAc/petrol). The
required compound was obtained as a bright yellow solid (62
N2-Phenylguanine 2,2,2-trifluoroacetate salt (98). To a
suspension of 2-bromohypoxanthine (1.00 g, 4.7 mmol) and
aniline (0.9 mL, 9.40 mmol) in TFE (40 mL) was added TFA
(1.80 mL, 23.5 mmol). The mixture was heated to reflux
under nitrogen for 24 h. The mixture was filtered hot, washed
with EtOH (3 × 50 mL g−1), and air-dried for 30 min. The
filtrate was evaporated in vacuo and the solid was
recrystallised from EtOH to obtain the title compound as a
white solid (1.17 g, 73%); mp 229–231 °C; λmax (EtOH/nm)
272; IR (cm−1) 3332, 3128, 2943, 2756, 2555, 2387, 1678, 1572;
1H NMR (300 MHz, DMSO-d6) 7.07 (1H, t, J = 7.5 Hz, H-4′),
7.36 (2H, dd, J = 7.5, 8.0 Hz, H-3′ and H-5′), 7.62 (2 H, d, J =
8.0 Hz, H-2′ and H-6′), 7.94 (1H, s, H-8), 8.46 (1H, br s, NH),
9.00 (1H, br s, NH); HRMS calcd. for C11H10N5O [M + H]+
228.0881, found 228.0880.
mg, 62%); mp 242–244 °C; λmax (EtOH) 356, 276; IR (cm−1
)
4000, 3470, 3293, 3180, 2258, 1611, 1584, 1511, 1471, 1245;
1H NMR (500 MHz, DMSO-d6) 3.73 (3H, s, OCH3), 5.36 (2H, s,
CH2), 6.93–6.95 (2H, dd, J = 8.0, 8.5 Hz, H-3′ and H-5′), 6.98–
7.01 (1H, t, J = 8.5 Hz, H-4′), 7.34–7.39 (4H, m, H-2″, H-3″, H-
5″ and H-6″), 7.73–7.75 (2H, d, J = 8.0 Hz, H-2′ and H-6′), 8.66
(1H, s, H-8), 10.11 (1H, br s, NH); LRMS (ES+) m/z 357.2 [M +
H]+.
6-Chloro-N-phenyl-9H-purin-2-amine (99)35
.
N2-
Phenylguanine trifluoroacetate salt (98) (2.00 g, 5.87 mmol)
and N,N-diethylaniline (1.9 mL, 11.73 mmol) was suspended
in neat POCl3 (30 mL) at room temperature. The reaction
mixture was heated at 115 °C for 60 min under a nitrogen
atmosphere. The resulting yellow solution was carefully
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6-Cyano-2-(phenylamino)-9H-purine (102). 6-Cyano-9-(4-
methoxybenzyl)-2-(phenylamino)-9H-purine (101) (50 mg, 0.14
mmol) was dissolved in TFA (2 mL). The deep orange
solution was heated at 70 °C for 5 h. The reaction mixture
was concentrated in vacuo and the resulting orange oil was
redissolved in EtOAc (2 mL). Residual TFA was neutralised by
washing the organic phase with aqueous NaHCO3 (2 × 2 mL).
The organic extract was dried (Na2SO4) and purified using
reversed phase column chromatography on silica (70%
MeOH/H2O + 0.1% HCOOH) to obtain a bright yellow solid
(13 mg, 40%); mp 247–249 °C (decomposed); λmax (EtOH/nm)
387, 272; IR (cm−1) 3389, 2255, 1601, 1537, 1496, 1396, 1348;
preparation; C. M-D., data collection and analysis of crystal
structures; D. R. N., project conception, experimental design
and interpretation and data analysis; M. W. R., crystal
structure refinement, validation and data deposition; M. S.,
synthesis and characterization of compounds; D. T., synthesis
and characterization of compounds; R. J. G., project
conception, experimental design and interpretation and data
analysis; B. T. G., experimental design and interpretation,
data analysis and manuscript preparation; C. C.,
experimental design and interpretation, data analysis and
manuscript preparation.
1H NMR (500 MHz, DMSO-d6) 7.01 (1H, t, J = 8.5 Hz, H-4′), Conflicts of interest
7.34 (2H, dd, J = 8.0 and 8.5 Hz, H-3′ and H-5′), 7.76 (2H, d, J
There are no conflicts of interest to declare.
= 8.0 Hz, H-2′ and H-6′), 8.52 (1H, s, H-8), 9.99 (1H, br s, NH),
13.60 (1H, br s, NH-9); HRMS calcd. for C12H8N6 [M+]+
236.0738, found 236.0734.
Acknowledgements
This research was supported by a grant from Cancer Research
UK (Grant Reference C2115/A21421). RB acknowledges
funding from Cancer Research UK (Grant References C24461/
A10285, C24461/A23302). The use of the EPSRC Mass
Spectrometry Service at the University of Wales (Swansea) is
also gratefully acknowledged. Similarly, we thank beamline
staff at The Diamond Light Source who provided excellent
facilities for data collection.
Accession codes
X-ray co-crystal structures of Nek2 with compounds 24
(6SGD) and 66 (6SGH), and also with the competitively-
binding control compounds 96 (6SGI) and 102 (6SGK) have
been deposited with the PDB. Authors will release the atomic
coordinates and experimental data upon article publication.
Ethical statement
References
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