Model system for irreversible inhibition of Nek2: Thiol addition to ethynylpurines and related substituted heterocycles

Article abstract of DOI:10.1039/c3ob41806e

Recent studies have shown that irreversible inhibition of Nek2 kinase [(Never in mitosis gene a)-related kinase 2], overexpression of which is observed in several cancers, can be achieved using Michael acceptors containing an ethynyl group, which target the enzyme's cysteine 22 residue lying near the catalytic site. The model studies described herein demonstrate an analogous capture of the ethynyl moiety in a series of ethynyl-heterocycles (e.g. 6-ethynyl-N-phenyl-9H-purin-2-amine) by N-acetylcysteine methyl ester in the presence of 1,4-diazabicyclo[2.2.2]octane in either dimethyl sulfoxide or N,N-dimethylformamide. Kinetic studies showed a 50-fold range in reactivity with 7-ethynyl-N-phenyl-3H-[1,2,3]triazolo[4,5-d]pyrimidin-5-amine being the most reactive compound, whereas 4-ethynyl-N-phenyl-7H-pyrrolo[2,3-d]pyrimidin-2-amine was the least reactive. Studies of the isomeric compounds, 2-(3-((6-ethynyl-7- methyl-7H-purin-2-yl)amino)phenyl)acetamide and 2-(3-((6-ethynyl-9-methyl-9H- purin-2-yl)amino)phenyl)acetamide, revealed the N⁷-methyl isomer to be 5-fold more reactive than the 9-methyl isomer, which is ascribed to a buttressing effect in the N⁷-methyl compound. Comparison of the crystal structures of these isomers showed that the ethynyl group is significantly displaced away from the methyl group exclusively in the N ⁷-methyl isomer with an sp² bond angle of 124°, whereas the corresponding angle in the N⁹-methyl isomer was the expected 120°. The results of this study indicate heterocyclic scaffolds that are likely to be more promising for inhibition of Nek2 and other kinases containing a reactive cysteine. The Royal Society of Chemistry.

Full text of DOI:10.1039/c3ob41806e

Organic &
Biomolecular Chemistry
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PAPER
Model system for irreversible inhibition of Nek2:
thiol addition to ethynylpurines and related
substituted heterocycles†
Cite this: Org. Biomol. Chem., 2014,
12, 141
Honorine Lebraud, Christopher R. Coxon, Victoria S. Archard, Carlo M. Bawn,
Benoit Carbain, Christopher J. Matheson, David M. Turner, Celine Cano,
Roger J. Griffin, Ian R. Hardcastle, Ulrich Baisch,‡ Ross W. Harrington and
Bernard T. Golding*
Recent studies have shown that irreversible inhibition of Nek2 kinase [(Never in mitosis gene a)-related
kinase 2], overexpression of which is observed in several cancers, can be achieved using Michael acceptors
containing an ethynyl group, which target the enzyme’s cysteine 22 residue lying near the catalytic site.
The model studies described herein demonstrate an analogous capture of the ethynyl moiety in a series
of ethynyl-heterocycles (e.g. 6-ethynyl-N-phenyl-9H-purin-2-amine) by N-acetylcysteine methyl ester in
the presence of 1,4-diazabicyclo[2.2.2]octane in either dimethyl sulfoxide or N,N-dimethylformamide.
Kinetic studies showed a 50-fold range in reactivity with 7-ethynyl-N-phenyl-3H-[1,2,3]triazolo[4,5-d]-
pyrimidin-5-amine being the most reactive compound, whereas 4-ethynyl-N-phenyl-7H-pyrrolo[2,3-d]-
pyrimidin-2-amine was the least reactive. Studies of the isomeric compounds, 2-(3-((6-ethynyl-7-methyl-
7H-purin-2-yl)amino)phenyl)acetamide and 2-(3-((6-ethynyl-9-methyl-9H-purin-2-yl)amino)phenyl)acet-
amide, revealed the N7-methyl isomer to be 5-fold more reactive than the 9-methyl isomer, which is
ascribed to a buttressing effect in the N7-methyl compound. Comparison of the crystal structures of these
isomers showed that the ethynyl group is significantly displaced away from the methyl group exclusively
in the N7-methyl isomer with an sp2 bond angle of 124°, whereas the corresponding angle in the
N9-methyl isomer was the expected 120°. The results of this study indicate heterocyclic scaffolds that are
likely to be more promising for inhibition of Nek2 and other kinases containing a reactive cysteine.
Received 4th September 2013,
Accepted 29th October 2013
DOI: 10.1039/c3ob41806e
www.rsc.org/obc
kinase inhibition is a common feature of the majority
of therapeutic kinase inhibitors.2,3 An alternative approach
Introduction
Modulation of protein kinase activity by small-molecule inhibi- entails the use of irreversible kinase inhibitors (enzyme
tors is a validated therapeutic approach for the targeted treat- inactivators).4–6 These function by virtue of a covalent reaction
ment of cancer and other diseases.1 The majority of kinase between a nucleophilic amino-acid within the ATP-binding site
inhibitors developed to date are heterocyclic compounds that and an electrophilic group of the inhibitor. However, until
occupy the ATP-binding domain of the kinase through non- recently, potential toxicity problems associated with in-
covalent interactions with amino-acid residues, thereby serving discriminate reactivity, has limited this approach. Recognition
as competitive inhibitors. Although selectivity for a particular of the potential therapeutic advantages of covalent inhibitors,
protein kinase is often partially achievable by exploitation of which include a prolonged inhibition of the target enzyme and
subtle differences within the ATP-binding site, off-target hence reduced dosing, has resulted in a resurgence of interest.7
Importantly, high selectivity for
a particular kinase is
achievable by exploitation of the initial non-covalent inhibitor
binding to orientate the electrophilic ‘warhead’, followed by
covalent reaction with a serine or cysteine residue that may be
present only in a small number of kinases.8
As part of a programme to develop inhibitors of Nek2, a
kinase implicated in leukaemia, breast, cervical, ovarian, and
prostatic cancer,9 we identified 2-arylamino-6-ethynylpurines
as time-dependent inactivators. Subsequent studies have
Newcastle Cancer Centre, Northern Institute for Cancer Research,
School of Chemistry, Bedson Building, Newcastle University, Newcastle upon Tyne,
NE1 7RU, UK. E-mail: bernard.golding@ncl.ac.uk; Fax: (+44)(0)19 1222 6929;
Tel: (+44)(0)19 1222 6647
†Electronic supplementary information (ESI) available. CCDC 949816 (9) and
949815 (10). For ESI and crystallographic data in CIF or other electronic format
see DOI: 10.1039/c3ob41806e
‡Current address: Department of Chemistry, University of Malta, Msida, MSD 2080,
Malta.
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demonstrated that these compounds bind irreversibly to Nek2,
by conjugate addition of the Cys-22 residue located close to
the ATP-binding site, to the terminus of the electrophilic
Results
NMR measurements
alkyne to afford a (Z)-thioalkenyl-purine.10 Irreversible inhi- Reactions were carried out in hexadeuterodimethyl sulfoxide
1
bition of Nek2 kinase has also recently been described using (DMSO-d6) and monitored by H qNMR. Initial attempts were
an N-arylpropiolamide as Michael acceptor for attack by this made using D2O as the reaction solvent so as to mimic the
cysteine residue.11 These results are compliant with the know- physiological environment more closely, but this was unsuitable
ledge that ethynyl groups12 with a carbonyl, nitro or cyano sub- owing to the poor solubility of the reactants. Thus, DMSO-d6
stituent or attached to a suitable heterocycle (e.g. pyridine13,14 was chosen as solvent for its solubilising properties and the
or pyrimidine15) readily undergo 1,4-addition reactions, fact that the residual solvent peak at δ 2.50 ppm (1H NMR) did
especially with soft nucleophiles such as thiols. Purines bearing not interfere with analyses. A concentration of 6 mmol dm−3
a 6-ethynyl or 6-vinyl moiety also undergo conjugate addition for the substrate heterocycle was found to give a homogeneous
reactions with nitrogen, oxygen and sulfur nucleophiles.16,17
solution and was chosen for all experiments. The resonance
To assess the reactivity of selected heterocycles substituted from the ethynyl proton of the ethynyl-heterocycles (or a suit-
with an ethynyl or vinyl group (1–12), and hence their potential able alternative resonance for 12) was used as the reference
as inhibitors of Nek2 kinase, we have performed a model signal with N,N-dimethylformamide (DMF) as internal refer-
study in which these compounds were reacted with N-acetyl- ence. N-Acetylcysteine methyl ester 13 was in 10-fold excess
cysteine methyl ester (13) (Scheme 1). By modifying the core over the ethynyl- or vinyl-substrate with 1,4-diazabicyclo[2.2.2]-
1
heterocycle of the molecules and substituents, surprising differ- octane (DABCO) in catalytic amount. In the H NMR spectra,
1
ences in reactivity were observed using the quantitative H NMR small resonances corresponding to the disulfide 14 from oxi-
(qNMR) method.18–22 The model system developed will have dation of 13 were observed, but did not interfere with the
broad application for the comparative evaluation of candidate kinetic analyses. The difficulty of avoiding this oxidation due
covalent enzyme inhibitors that react with cysteine residues.
to DMSO and/or O2 has been discussed.23
Kinetic studies
To achieve sufficiently fast reactions, it was found necessary to
add a catalytic quantity (optimally 0.3 mol equiv.) of DABCO to
generate the thiolate of N-acetylcysteine methyl ester 13.
DABCO was chosen as base due to its simple NMR profile and
good solubility in DMSO-d6. Considering the pKa values of ter-
tiary amines and thiols in DMSO,24 the pKa of DABCO (9.06)25 is
probably less than that of 13 and so only a small equilibrium
concentration of the corresponding thiolate 13− is formed.
The concentration of DABCO was assumed to be constant
throughout the reaction. Thiol 13 was employed in 10-fold
excess and so its concentration was essentially constant. It is
also assumed that a rapid pre-equilibrium converts 13 into
13−. The equation of the reaction rate was therefore pseudo-
first order (eqn (1) and (2), sm = heterocyclic starting
material).26
d½smꢁ
dt
K½13ꢁ0½DABCOꢁe
ꢀ
¼ k″½smꢁ
ð1Þ
½DABCOþꢁe þ K½DABCOꢁe
d½smꢁ
ꢀ
¼ kapp½smꢁ
ð2Þ
dt
The apparent rate constant (kapp) was determined experi-
mentally from the slope of line obtained by plotting ln([sm]/
[sm] at t = 0) against time (eqn (3)). Typical plots are shown as
Fig. 1. From the apparent rate constant, the value of the half-
26
life of the reaction was assessed by dividing ln 2 by kapp
.
½smꢁ
ln
¼ ꢀkapp
t
ð3Þ
½smꢁt¼0
The kinetic data obtained is summarised in Table 1. It is
Scheme 1 Reaction of 6-ethynylpurine 6 with N-acetylcysteine methyl
ester 13. Reagents and conditions: (a) 13, DABCO in DMF, room temp./18 h. assumed that all compounds follow the same rate law.
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Table 1 Kinetic results
Compounds
Structures
kapp/10−4 s−1
t1/2/min
1
0.30 0.02
5.49 0.28a,b
17.07 0.94a,c
390
2
3
21.0
6.7
Fig. 1 Ln([6]/[6] at t = 0) versus time showing a pseudo first order
reaction.
4
5
4.57 0.46
23.8
26.6
In the absence of the thiol 13 there was no reaction
between DABCO and 6. There was also no reaction after
addition of methanol to the mixture of 6 and DABCO. Reaction
of 5 with 13 without DABCO gave kapp ∼ 2 × 106 s−1
(t1/2/h ∼ 100).
4.36 0.42c,d
Reaction between 6-ethynylpurine 6 and N-acetylcysteine
methyl ester 13
6
2.44 0.05
4.50 0.91c
1.99 0.05
14.88 0.29c
3.00 0.12
1.39 0.13
47.3
26.5
58.0
7.8
The qNMR study of 6 revealed the formation of one major iso-
meric product. To confirm which isomer, E (15) or Z (16), was
formed, the reaction between 6-ethynylpurine 6 and N-acetyl-
cysteine methyl ester 13 was carried out on a larger scale,
using DMF as solvent, to recapitulate the conditions of the
kinetic experiments. The E isomer 15 was isolated pure,
whereas the Z isomer 16 was obtained as an inseparable
mixture with the product 17 arising from further attack by 13
on the initial adduct(s) (ratio of 15 : 16 ∼ 9 : 1).
7
8
9
10
11
38.6
83.8
By comparison of the spectra from the isolated compounds
and the 1H qNMR experiment, the major isomer obtained in
the kinetic experiments was also the E isomer 15 (Fig. 2) at
every time point. The alkene protons from 15 were not easily
1
distinguished in the H qNMR spectra, as the resonance at δ
12
1.04 0.05c,e
111.6
6.88 ppm (vCHS) overlapped with the signal from one aro-
matic proton and the resonance of the other alkene proton
was obscured by the NH signal of the acetamido moiety. A
small resonance at δ 8.55 ppm corresponded to the NH of the
acetamido moiety of the Z isomer 16. The ratio between the
E and Z isomers was estimated as 9 : 1 (E : Z) by comparison of
their acetamido NH signals. It is reported that thiolate attack
on an ethynyl-ketone affords mainly the Z-isomer.27 Consider-
ing the reaction between 6 and 13, it can be postulated that
a The DABCO concentration was decreased to 0.03 equiv. b Two
experiments. c Four scans were used for the acquisition of the spectra
and data were recorded every 3 min. d The spectra were recorded every
5.8 min. e The spectra were recorded every 4.1 min.
formation of an allene intermediate is followed by proton opposite side of the thioether group, then Z-isomer 16 will
transfer from protonated DABCO to give the thioethenyl be formed. However, this reaction pathway ‘pushes’ the
product (Scheme 2). If the proton approaches from the thioether group toward the purine, which may render
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Fig. 2 Comparison of 1H NMR spectra from the reaction between 6 and 13 (A: 1H NMR of mixture of 16 and 17; B: 1H NMR of 15; C: 1H qNMR of 6).
Scheme 2 Mechanism for formation of the Z/E isomers.
Scheme 3 Postulated mechanism for Z/E isomerisation.
preferable the alternative mode of protonation leading to the
thermodynamically stable E-isomer 15. A further possibility is
that the Z-isomer 16 is the kinetic product, but undergoes
rapid isomerisation to E-isomer 15 (Scheme 3).
Discussion
Kinetic studies of the reactions between ethynyl-heterocycles
and N-acetylcysteine methyl ester
In the absence of the thiol 13 there was no reaction As mentioned above, 2-arylamino-6-ethynylpurines were shown
between DABCO and 6. Addition of methanol to the mixture of to bind covalently to the Nek2 kinase via Cys-22 through a
6 and DABCO also gave no reaction. Attempted reaction of Michael-type addition. A model study of this biologically
5 with 13 in the absence of DABCO gave a very slow reaction important reaction, using 13 to mimic the thiol of Cys-22, was
(t1/2 ∼ 100 hours) at 24 °C.
investigated using time-resolved qNMR with the optimised
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reaction conditions described above. 1H qNMR was used to lower than the rate constant value obtained for the parent
measure the concentration of ethynyl-heterocycles by monitor- 6-ethynylpurine 5. It is possible that the electron-donating char-
ing the change in intensity of the ethynyl proton of 1–11 and acter of the N-methyl group from the conjugated side chain of
Ha of 12, relative to the DMF internal standard, with respect to compound 11 increases the overall electron density of the
time (see ESI†). The nature of the heterocycle to which the purine core, reducing the electrophilicity of the 6-ethynyl
ethynyl moiety was attached was shown to be the critical influ- group and hence reactivity towards the thiolate. Replacement
ence on the rates of the thiol addition reactions (see Table 1) of the electron-donating phenylamino moiety by a 2-benzyl
with other factors discussed below being of lesser significance. group showed no significant gain in reactivity for 4 compared
with its isostere 5. Compounds 6, 7 and 8 bearing a substitu-
Effect of the core heterocycle
ent on the aniline ring were also studied by 1H qNMR to
The impact of the core heterocycle on the kinetics of the explore the potential impact of aniline ring substituents on
Michael type-addition of the thiolate onto an ethynyl moiety is the reactivity of the 6-ethynyl moiety. Adding an amide chain
evident from a comparison of the rate constants for pyrrolopyri- to the phenylamino group as in 10 showed a small decrease in
midine 1, pyrazolopyrimidine 2 and triazolopyrimidine 3. The reactivity compared to 5, possibly because of a small electron-
latter reacted significantly faster than purine 5. To monitor donor effect from the CH2 group,31 which would reduce the
accurately these reactions, the concentration of DABCO was acceptor property of the ethynyl moiety. The comparable reac-
decreased to 0.03 equiv. However, as the reactivity of triazolo- tivities of 5 and 7, the latter having an electron-withdrawing
pyrimidine was still high, the number of scans for this reaction cyano group,31 suggest that the major effect on reactivity is the
was lowered from 8 to 4 to allow for monitoring every 3 minutes nature of the heterocyclic core. This conclusion is supported
and to provide enough data for sufficiently accurate results. For by the data for 8 in which the electron-donating morpholino
2 and 3, with 0.03 equiv. DABCO, the E : Z ratio was 9 : 1. Pyrrolo- group32 makes the ethynyl group only 2-fold less reactive.
pyrimidine 1 proved to be particularly unreactive, and after
5 h the reaction had proceeded to only approximately 50% con-
sumption of starting material. This low reactivity can be attri-
buted to the higher electron density of the heterocycle relative to
4, rendering the ethynyl moiety less electrophilic towards attack
by the thiolate. The more electron-deficient the heterocycles
were, the more reactive the compounds were. This observation
10, which was comparable to 6 with respect to reactivity. A but-
correlated with calculations of the effect of aza-substitution on
Effect of methylation at the 7- and 9-position of 6
Compounds 9 and 10, like the analogues 4 and 11, lack one of
the NH functions of the parent purine 5, which are implicated
in binding to Nek2.10 Surprisingly, the 7-methyl compound 9
appeared to be 5-fold more reactive than its 9-methyl analogue
tressing effect33 and steric strain relief34 can be postulated as
the electron densities at the ring atoms of the heterocyclic
cores of 1, 2 and 3 compared with the π-deficient purine 5.28
the reason for activation of the ethynyl moiety in 9. The methyl
group at the N-7 position buttresses the ethynyl group result-
ing in a direct effect on bond angles.33 This imposes steric
Comparison of ethynyl and vinyl at 6-position
strain, which is relieved by the attack of the thiolate. This pos-
tulate was confirmed by X-ray crystallography. When the
methyl group was positioned at the 7-position, the angle
between the ethynyl group and the purine core was 124.15(15)°
and 122.89(13)° (there are two independent molecules in the
asymmetric unit of the crystal structure), whereas when the
To assess the relative importance of ethynyl versus vinyl at the
6-position of a purine, 6-ethynyl-N-phenyl-9H-purin-2-amine 5
and N-phenyl-6-vinyl-9H-purin-2-amine 12 were studied by
qNMR. The addition of the thiolate to vinyl analogue 12 was
found to be slower (4×) than its 6-ethynyl analogue 5. The
difference in reactivity between 5 and 12 is in accordance with
studies carried out on methyl propiolate, which is more reac-
methyl was located at the 9-position the corresponding angle
is almost exactly ideal sp2 geometry with 119.8(3)° (Fig. 3).
tive than the corresponding α,β-unsaturated ester, methyl acry-
late, because of increased electrophilicity at the β-carbon.29
Effect of the group at the 2-position
Purine kinase inhibitors often exhibit three hydrogen bonds
between the ATP-binding domain of the kinase and 2-NH, N-3
and N9-H (acting as donor, acceptor and donor, respectively) of
the heterocycles.30 Relevant to this feature, varying the 2-aryl-
amino group attached to the purine was essential to structure–
activity relationships studied in a Nek2 project aiming to
improve potency and selectivity of inhibitors.10 To determine
a potential influence of the 2-NH for the reactivity of the
6-ethynyl group, 2-benzyl-6-ethynyl-9H-purine 4, where the NH
was replaced by an isosteric CH2 group, and 6-ethynyl-N-
methyl-N-phenyl-9H-purin-2-amine 11, where the nitrogen was
methylated, were studied. The rate constant for 11 was 4-fold Fig. 3 X-Ray crystal structures of 9 (left) and 10 (right).
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One may speculate that other effects such as intermolecular number of experiments. Longitudinal relaxation (T1) measure-
interactions are responsible for this change of geometry. ments were carried out employing a standard 180°–90° inver-
However, only weak attractions between the ethynyl proton sion recovery pulse sequence. The delay between transients
and either the keto oxygen or the nitrogen atom on the 7-posi- was held constant at 10 s whilst the inversion delay was
tion of the neighbouring molecule are observed. In both cases increased from 1 × 10−6 s (effectively zero) until full 90° relax-
these interactions are of comparable strength lying between ation of the slowest relaxing 1H nucleus was observed resulting
2.26(2) Å and 2.41(4) Å and therefore, cannot be responsible in zero net signal for the aforementioned proton. The observed
for the above-mentioned effect.
T1 was multiplied by five to allow for 99% signal recovery
resulting in an overall relaxation delay time of 36 seconds.
1H qNMR experiments were carried out using a composite
90°–180°–180° pulse, at a constant temperature of 24.00
0.01 °C.37 Samples were retained within the spectrometer for
Conclusions
In a project directed towards the development of Nek2 inhibi- the duration of the experiment and maintained at a steady
tors, 6-ethynyl- and 6-vinyl-heterocyclic compounds were 20 Hz rotation throughout. The experiment was controlled using
shown to exhibit irreversible binding via a Michael addition-
a multi-acquisition automation program, which included
type reaction with Cys-22.10 To assess the reactivity of the Fourier-transform and basic phase-correcting commands. Data
6-ethynyl- and vinyl-heterocycles towards the attack of Cys-22, were processed using standard Bruker phase-correcting algo-
model studies were conducted using an excess of N-acetylcys- rithm. Line broadening was maintained at 0.30 Hz throughout
teine methyl ester 13 with a catalytic amount of DABCO, to and all baseline correction was applied from 10.0 to 0.0 ppm.
mimic the Cys-22 residue. Attack of the thiolate on ethynyl- Signals were integrated between defined chemical shift values
purine 6 showed formation of both E and Z isomers 15 and 16, at appropriate intervals throughout the course of the experi-
respectively, in a ratio 9 : 1. This stereochemical outcome ment. Microsoft Excel 2007 was used to manipulate raw data
differs from that observed10 for the irreversible inhibition of and to show data graphically. The measured concentration was
Nek2 with compound 6. In the case of the enzyme, the Z plotted on a graph as ln(C/C0) versus time with the slope being
stereochemistry may be determined by the positioning of the the apparent rate constant (kapp) of the studied reaction. Each
Cys-22 and a proton source relative to the ethynyl group of 6. reaction was repeated three times unless stated otherwise.
For both the enzymatic and model systems the direction of
General procedure for kinetic studies
attack of the thiolate on the ethynyl group is presumably gov-
erned by the need to adopt a Bürgi–Dunitz approach35 (cf. The ethynyl substituted heterocycle (690 µL from a stock solu-
Scheme 2). Varying the heterocyclic core revealed significant tion in DMSO-d6 containing 4.2 µmol of purine) was added to
changes in reactivity from the most reactive compound of the an excess of N-acetylcysteine methyl ester (7.48 mg, 42 µmol).
series (triazolopyrimidine 3) to the least reactive compound The solution temperature was maintained at approximately
(pyrrolopyrimidine 1). Methylation at the N-7 position of the 24 °C (water-bath) before addition of a DMSO-d6 solution
6-ethynylpurine 6 afforded the third most reactive compound (9) (10 µL) containing DABCO (0.14 mg, 1.26 µmol) and DMF
of the series, which was explained by a buttressing interaction (0.33 µL, 4.2 µmol) to afford a final 6-substituted heterocycle
between the ethynyl moiety and methyl group, rendering it concentration of 6 mM in a total volume of 700 µL. The NMR tube
more reactive than 10. The relative reactivities of 6-ethynyl- containing the reagents was quickly inverted several times to
and 6-vinyl-heterocycles towards a thiolate, as determined in aid mixing and dissolution. The thoroughly mixed solution
this paper, will aid the design of potent irreversible inhibitors was inserted into the NMR machine cavity and the acquisition
of Nek2 and other kinases containing an active site cysteine of quantitative 1H NMR data was immediately initiated. The
thiol.
time between the addition of the DABCO/DMF-solution and
the completion of the first 1H qNMR experiment was moni-
tored and subsequent time intervals between experiments
were calculated based on the defined parameters.
Experimental
Typical examples
Quantitative 1H NMR (1H qNMR) measurements were obtained
using a Bruker Avance III 500 MHz (11.74 τ) spectrometer oper- 2-(3-((6-Ethynyl-9H-purin-2-yl)amino)phenyl)acetamide 6 (690 µL
ating at 500.303 MHz, equipped with a 5 mm broadband from stock solution DMSO-d6, 1.23 mg, 4.2 µmol) was
observe (BBO) probe. Experimental set-up and acquisitions treated according to general procedure. Disappearance of the
were controlled using Topspin 2.136 (Bruker, Rheinstetten, singlet at 4.75–4.86 ppm was monitored as a function of time
Germany). Precision 5 mm diameter NMR tubes obtained using the 1H qNMR method outlined. Each spectrum was
from Bruker were used for all acquisitions. Experiments were recorded every 10 min using 8 scans per experiment.
carried out in DMSO-d6 using solutions of a known concen-
2-Phenylamino-6-vinylpurine 14 (690 µL from stock solution
1
tration. H NMR spectra were acquired as the summation of 4 in DMSO-d6, 1.0 mg, 4.2 µmol) was treated according to
transients and 1 equilibrating transient (signal unrecorded). general procedure. Disappearance of the characteristic vinyl
Spectral data were collected at regular intervals for a defined proton (Ha) signal at 5.90–5.96 ppm was monitored as a
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function of time. Each spectrum was recorded every 15 min
using 8 scans per experiment.
7 J. Singh, R. C. Petter, T. A. Baillie and A. Whitty, Nat. Rev.
Drug Discovery., 2011, 10, 307–317.
2-(3-((6-Ethynyl-7-methyl-7H-purin-2-yl)amino)phenyl) acet-
amide 9 (690 µL from stock solution DMSO-d6, 1.29 mg,
4.2 µmol) was treated according to the general procedure. Dis-
appearance of the singlet at 4.95–5.05 ppm was monitored as a
function of time using the 1H qNMR method outlined. Each
spectrum was recorded every 3 min using 4 scans per
experiment.
8 Q. Liu, Y. Sabnis, Z. Zhao, T. Zhang, S. J. Buhrlage,
L. H. Jones and N. S. Gray, Chem. Biol., 2013, 20,
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For further examples see the ESI.†
X-ray crystal structure determination
X-ray crystallographic data were collected on an Oxford Diffrac-
tion Gemini A Ultra diffractometer at 150 K using Cu Kα radi- 12 R. M. LoPachin, T. Gavin, A. DeCaprio and D. S. Barber,
ation (λ 1.54184 Å). Analytical numeric absorption Chem. Res. Toxicol., 2012, 25, 239–251.
=
corrections using a multifaceted crystal model based on 13 S. Nishikawa, M. Sato, H. Kojima, C.-e. Suzuki, N. Yamada,
expressions derived by R. C. Clark & J. S. Reid38 were applied,
based on symmetry-equivalent and repeated reflections. Struc-
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