Synthesis and evaluation of heteroaryl substituted diazaspirocycles as scaffolds to probe the ATP-binding site of protein kinases

Article abstract of DOI:10.1016/j.bmc.2013.07.021

With the success of protein kinase inhibitors as drugs to target cancer, there is a continued need for new kinase inhibitor scaffolds. We have investigated the synthesis and kinase inhibition of new heteroaryl-substituted diazaspirocyclic compounds that mimic ATP. Versatile syntheses of substituted diazaspirocycles through ring-closing metathesis were demonstrated. Diazaspirocycles directly linked to heteroaromatic hinge binder groups provided ligand efficient inhibitors of multiple kinases, suitable as starting points for further optimization. The binding modes of representative diazaspirocyclic motifs were confirmed by protein crystallography. Selectivity profiles were influenced by the hinge binder group and the interactions of basic nitrogen atoms in the scaffold with acidic side-chains of residues in the ATP pocket. The introduction of more complex substitution to the diazaspirocycles increased potency and varied the selectivity profiles of these initial hits through engagement of the P-loop and changes to the spirocycle conformation, demonstrating the potential of these core scaffolds for future application to kinase inhibitor discovery.

Full text of DOI:10.1016/j.bmc.2013.07.021

Accepted Manuscript
Synthesis and Evaluation of Heteroaryl Substituted Diazaspirocycles as Scaf‐
folds to Probe the ATP-binding site of Protein Kinases
Charlotte E. Allen, Chiau L. Chow, John J. Caldwell, Isaac M. Westwood, Rob
L. van Montfort, Ian Collins
PII:
S0968-0896(13)00632-9
DOI:
http://dx.doi.org/10.1016/j.bmc.2013.07.021
BMC 10985
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Please cite this article as: Allen, C.E., Chow, C.L., Caldwell, J.J., Westwood, I.M., van Montfort, R.L., Collins, I.,
Synthesis and Evaluation of Heteroaryl Substituted Diazaspirocycles as Scaffolds to Probe the ATP-binding site of
Protein Kinases, Bioorganic & Medicinal Chemistry (2013), doi: http://dx.doi.org/10.1016/j.bmc.2013.07.021
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Synthesis and Evaluation of Heteroaryl Substituted
Diazaspirocycles as Scaffolds to Probe the ATP-binding site of
Protein Kinases
a
b
a
b
Charlotte E. Allen , Chiau L. Chow , John J. Caldwell , Isaac M. Westwood , Rob L. van
b
,a,
Montfort , Ian Collins *
a
b
Cancer Research UK Cancer Therapeutics Unit and Divisions of Cancer Therapeutics and
Structural Biology, The Institute of Cancer Research, 15 Cotswold Road, Sutton, SM2 5NG, U.K.
*
Corresponding author: Fax: +44 208 722 4216. E-mail: ian.collins@icr.ac.uk
Keywords
spirocycle, protein kinase, kinase inhibitor, selectivity
1

Graphical Abstract
Synthesis and Evaluation of Heteroaryl
Substituted Diazaspirocycles as Scaffolds to
Probe the ATP-binding site of Protein Kinases
Leave this area blank for abstract info.
Charlotte E. Allen, Chiau L. Chow, John J. Caldwell, Isaac M. Westwood, Rob L. van Montfort, Ian Collins
The Institute of Cancer Research, 15 Cotswold Road, Sutton, SM2 5NG, U.K.
P-loop
R
OH, CH OH
2
R = Ph, CH Ph
2
Hinge
peptide
N
HetAr
[]n
CO R, CONHR
2
[
]m
N
H
m, n = 0, 1
Ribose pocket
2

Abstract
With the success of protein kinase inhibitors as drugs to target cancer, there is a continued
need for new kinase inhibitor scaffolds. We have investigated the synthesis and kinase inhibition
of new heteroaryl-substituted diazaspirocyclic compounds that mimic ATP. Versatile syntheses
of substituted diazaspirocycles through ring-closing metathesis were demonstrated.
Diazaspirocycles directly linked to heteroaromatic hinge binder groups provided ligand efficient
inhibitors of multiple kinases, suitable as starting points for further optimization. The binding
modes of representative diazaspirocyclic motifs were confirmed by protein crystallography.
Selectivity profiles were influenced by the hinge binder group and the interactions of basic
nitrogen atoms in the scaffold with acidic side-chains of residues in the ATP pocket. The
introduction of more complex substitution to the diazaspirocycles increased potency and varied
the selectivity profiles of these initial hits through engagement of the P-loop and changes to the
spirocycle conformation, demonstrating the potential of these core scaffolds for future
application to kinase inhibitor discovery.
Abbreviations
AGC, protein kinases A, G and C; ATP, adenosine triphosphate; 9-BBN, 9-
borabicyclo[3.1.1.]nonane Boc, tert-butyloxycarbonyl; CAMK, calcium/calmodulin-dependent
kinases; Cbz, benzyloxycarbonyl; DFG, aspartate-phenylalanine-glycine sequence; DIBAL-H
diisobutylaluminium hydride; HA, heavy atom; LE, ligand efficiency; PKA, protein kinase A;
PKB, protein kinase B; RCM, ring-closing metathesis; S Ar, nucleophilic aromatic substitution;
N
TK, tyrosine kinase.
PDB Codes: 12-PKA, 3ZO1; 14-PKA, 3ZO2; 16-PKA, 3ZO3; 56-PKA, 3ZO4.
3

1.
Introduction
Protein kinases play a vital role in signal transduction pathways involved in cellular
1
-3
proliferation and survival, and are often implicated in deregulated signaling leading to cancer.
The development of protein kinase inhibitors to target the underlying mechanisms of cancer cell
4
,5
growth and survival has resulted in several licensed treatments. The design of specific targeted
inhibitors is important, but preclinical research and clinical experience also shows that inhibitors
with multi-target profiles can be effective through the ability to inhibit multiple signaling events
6
-8
simultaneously. There is a continued need for new protein kinase inhibitor scaffolds,
9
particularly those that can act as platforms for modification to vary selectivity profiles. Many
inhibitors have been designed to target the conserved ATP cofactor binding site, and both highly
specific and multi-targeted ligands can arise from this approach. Type I ATP-competitive
inhibitors recognize the active conformation of kinases, and typically possess a core
heteroaromatic ring system that forms hydrogen bonds to the hinge-peptide of the kinase in an
analogous fashion to ATP. Interactions in other regions of the kinase active site can be
4
, 10-12
introduced through extension of the core scaffold along appropriate vectors.
<
Figure 1>
We have previously described compounds such as 4-(4-chlorobenzyl)-1-(7H-pyrrolo[2,3-
d]pyrimidin-4-yl)piperidin-4-amine (1, Figure 1) which selectively inhibits PKB over other
1
3,14
kinases within the AGC family. Compound 1 closely resembles the pharmacophore defined
by ATP bound to the kinase (Figure 1A). The selectivity of 1 was dependent upon the
positioning of a basic primary amine in the ribose pocket, where it could interact with a glutamic
acidic residue in PKB and backbone carbonyl functionality, as well as exploiting a close contact
4

1
3
with a methionine side chain. 3-Aminopyrrolidine-based PKB inhibitors targeting these
1
5
structural features have also been reported.
We speculated that incorporation of the pendant primary amine into a second, spirocyclic ring
could provide new scaffolds for ATP-competitive kinase inhibition, and that the inhibition
1
2,16
profiles could be varied by modification of P-loop targeting substituents.
Diazaspirocycles
1
7
have been proposed as generally useful scaffolds for drug design, and these scaffolds
1
8
incorporate a potentially favourable solubilising element into the core of the inhibitors.
Compared to more typical, planar kinase inhibitor motifs, the rigid spirocyclic structures increase
the three-dimensional shape of molecules where they are present, which may be associated with
1
9,20
better physicochemical properties. Simple, unsubstituted aliphatic diazaspirocycles have been
incorporated into kinase inhibitors as peripheral substituents in the patent literature, but these are
2
1
most often seen as minor examples in much broader surveys of substituent amines. The use of
diazaspirocycles as core scaffolds to explore binding within the ATP-pocket, and the effect of
substitution of these scaffolds on the selectivity profile of kinase inhibitors, has not been
1
5,22
extensively detailed, or has involved the embedding of the spirocycles in non-aliphatic fused
1
5,23
structures, such as spiroindolines.
We therefore investigated the synthesis and kinase inhibition of heteroaryl-substituted
diazaspirocycles following the general design 2 (Figure 1B) capable of delivering a nitrogen
atom into the ribose pocket of the ATP binding site, while orienting a heteroaromatic group to
interact with the hinge peptide, and providing a platform for elaboration towards the P-loop.
Unsubstituted diazaspirocyclic scaffolds were chosen to vary the position of the basic nitrogen
relative to the point of attachment to the hinge-binding group. To each of the scaffolds, purin-6-
1
3
24
yl and isoquinolin-5-sulfonamide kinase heteroaromatic hinge binders previously exemplified
in AGC kinase inhibitors were attached, chosen to vary the degree and orientation of contact
5

between the ligand and the hinge-peptide, and the position of the spirocycles. Following
assessment of these compounds against a representative panel of protein kinases, versatile
synthetic routes were developed to introduce aromatic or polar functionality at two positions of a
[
5.5]-diazaspirocycle purine derivative with inhibitory activity, and thus to study the effect on
the inhibition profiles with these new substituted diazaspirocycles.
2.
Synthetic Chemistry
The 4-amino-4-benzylpiperidine scaffold 3 was made as a non-spirocyclic comparator,
2
5
following our previously reported synthesis (Scheme 1). Jenkins et al. have described robust
syntheses of the [5.5]-azaspirocycle 4, through ring-closing metathesis (RCM), and [4.5]-
azaspirocycle 5, through a bromine-mediated cyclisation, both starting from 1-benzylpiperidine-
1
7
4
-one. Commercially available [5.5]-azaspirocycles 6, 7 and [4.5]-azaspirocycle 8 provided
variations in the positions of the nitrogen atoms. Compounds 5 and 6 were subjected to a
protecting group exchange to generate scaffolds 9 and 10 with the basic nitrogen translocated.
<
<
Scheme 1>
Table 1>
Heteroaromatic groups were attached to the scaffolds 3-10 through S Ar reactions with 6-
N
1
3
24
chloropurine and sulfonylation with isoquinoline-5-sulfonyl chloride. N-Deprotection under
acidic conditions gave compounds 11-26 (Table 1). The unprotected 4-amino-4-benzylpiperidine
3
reacted regioselectively at the more nucleophilic piperidine nitrogen with 6-chloropurine and
isoquinoline-5-sulfonyl chloride to give 11 and 19, respectively, as confirmed by NMR and MS-
fragmentation analysis. With the exception of 21, diazaspirocycles were introduced with mono-
6

N-protection to avoid poor regioselectivity in the couplings. 3-Azaspiro[5.5]undecane, 1-oxa-
and 2-oxa-9-azaspiro[5.5]undecane purine derivatives 27-29 were prepared using similar
methods (Scheme 2).
<
Scheme 2>
The purine [5.5]-diazaspirocycle 12 was chosen for synthetic modification to introduce a
selection of hydrophobic and polar functional groups to the C-3 and C-4 positions of the 1,9-
1
7
diazaspiro[5.5]undecane. Compound 30 , an intermediate in the synthesis of 4, was
hydroborated with 9-BBN to give a chromatographically separable 1:1 mixture of alcohols 31
and 32 after oxidation (Scheme 3). Transfer hydrogenation was found to be optimum for the N-
benzyl deprotection, as 33 and 34 failed to react under standard hydrogenation conditions.
Introduction of the purine and isoquinoline sulfonamide substituents followed by N-Boc
deprotection gave compounds 33-36.
<
Scheme 3>
Racemic syntheses were developed to introduce aromatic substituents to the 1,9-
1
7
diazaspiro[5.5]undecane scaffold (Scheme 4). 4-Amino-4-allylpiperidine 37 was acylated with
acryloyl chloride to give a bis alkene, which underwent RCM with Grubbs I catalyst and
i
26
Ti(PrO) as an additive to form the unsaturated lactam 38. 1,4-Addition of cuprates introduced
4
2
7
phenyl and benzyl groups to the C-4 position of 38. The success of these reactions depended on
the copper (I) source and the concentration of the cuprate, with CuBr.SMe and 1M
2
concentration in THF identified as optimal for the formation of the 1,4-addition products 39 and
7

4
0. Compounds 39 and 40 were reduced using an excess of LiAlH , followed by N-benzyl
4
deprotection to give the diamine intermediates in good yields. S Ar and sulfonylation reactions
N
of the diamines were regioselective for reaction at the less hindered piperidine nitrogen, but were
inefficient. While purines 41 and 42 were isolated after chromatography, the corresponding
isoquinoline compounds were not obtained.
<
Scheme 4>
An alternative RCM strategy was investigated to install substituents to position C-3 of the 1,9-
2
6,28
diazaspiro[5.5]undecane.
Amine 37 was acylated with freshly prepared 2-benzylacryloyl
2
9
chloride (Scheme 4). The RCM using Grubbs I catalyst was poorly reproducible, but Hoveyda-
Grubbs II catalyst gave the tri-substituted lactam 43 reliably in good yield (66%). Filtration
through amine-functionalised ion-exchange resin was found to be an effective method for the
3
0
removal of ruthenium residues following the RCM. Hydrogenation of 43 reduced the alkene
while keeping the benzyl protecting groups intact. The lactam was reduced using excess LiAlH4,
followed by N-debenzylation to give 44 in good yield. A regioselective S Ar reaction with 6-
N
chloropurine gave compound 45.
When applying the above route to introduce phenyl substitution at C-3, the RCM reaction
i
using Hoveyda-Grubbs II catalyst with Ti(PrO) generated 46 in low yields with unpredictable
4
reaction times of between 24-72 hours. This was surprising, as phenyl-substituted dienes are
3
1
reported to be metathesised efficiently. The RCM was attempted using the less sterically
hindered Grubbs catalyst dichloro[1,3-bis(2-methylphenyl)-2-imidazolidinylidene](2-
isopropoxyphenylmethylene)ruthenium (II), which has been reported to give increased activity in
3
2
the formation of trisubstituted olefins, and 46 was isolated in good yield (76%). Hydrogenation
8

of 46 removed the double bond, but in contrast to lactam 43, the N-benzyl protecting group of
the piperidine ring was also cleaved. Reduction of the lactam was difficult, with LiAlH4,
DIBAL-H, BH .THF or 9-BBN giving either unreacted starting material or mixtures of starting
3
material and the partially reduced hemiaminal. We speculated that the 3-phenyl substituent
adjacent to the lactam carbonyl was sterically impeding nucleophilic attack. Therefore, the
3
3
smaller and more reactive reducing agent AlH was prepared in-situ from LiAlH and AlCl ,
3
4
3
and gratifyingly gave full reduction of the amide. Removal of the remaining N-benzyl group was
achieved by transfer hydrogenolysis to give the diamine 47. The regioselective S Ar reaction of
N
4
7 with 6-chloropurine yielded 48.
To prepare the C-3 ester derivative of the 1,9-diazaspiro[5.5]undecane, intermediate 37 was
alkylated using methyl 2-(bromomethyl)acrylate to give bis-alkene 49 in good yield (Scheme 4).
The RCM of 49 was first attempted with Grubbs I catalyst in the presence of pTsOH, according
2
8
to a method outlined by Prusov and Maier but no conversion of the starting material was
i
observed with this catalyst, nor with Hoveyda-Grubbs II catalyst and Ti(PrO) . However,
4
dichloro[1,3-bis(2-methylphenyl)-2-imidazolidinylidene](2-isopropoxyphenylmethylene)
ruthenium (II) gave full conversion of the bis-alkene to the cyclised product 50 in good yield.
The spirocycle 50 was hydrogenated to reduce the alkene and remove the N-benzyl protecting
groups. A subsequent S Ar reaction with 6-chloropurine gave 51.
N
The ester 51 was converted to the carboxylic acid 52 and hydroxymethyl derivative 53
(
Scheme 4). The methyl, dimethyl and morpholine amides 54-56 were prepared by hydrolysis of
the ester 51 to the lithium salt of the acid followed directly by amine couplings. Exhaustive
hydrogenation removed the N-benzyl protecting groups and reduced the alkenes, prior to S Ar
N
reactions to give 57-59.
9

3
.
Results and Discussion
Compounds were tested for protein kinase inhibition in a panel of 24 enzymes containing
representative members of the major kinome family subclasses, using a commercially available
3
4,35
microfluidic assay format. Given the small size of most of the compounds 11-27 (MW range
2
58-381, median MW = 308), the inhibitors were tested at a concentration of 30 μM, so that
-
significant activity (>50% inhibition) would be indicative of ligand efficient binding (>0.3 kcal
1
-1 36
37
HA ). The ATP concentration used was approximately the K
for each enzyme tested. To
m,[ATP]
3
8
determine the variability and reproducibility of the assay, the pan-AGC kinase inhibitor H-89
was included as an internal control in each assay plate. Over multiple assay runs during
compound screening, this control gave high reproducibility with low standard errors in the mean
%
inhibition (Figure 2). This gave confidence in the robustness of the assay for single-point
screening. The determination of % inhibition at a single fixed concentration, rather than full IC50
or K values for each compound against each kinase, imposes limitations on the interpretation of
d
the data. The non-linear relationship between % inhibition and concentration does not permit
differentiation between compounds with similar high % inhibitions (e.g. between 95-100%),
even though there may be significant differences in the IC , and thus selectivity cannot be
5
0
accurately quantified. Nevertheless, the method provides an efficient means to identify
potentially useful activity and distinguish major changes in selectivity patterns that we and others
3
9-42
have previously used to assess kinase inhibition by new screening sets.
<
Figure 2>
The simple heteroaryl-substituted diazaspirocycles 12-18 and 20-26 (Table 1) showed activity
profiles that were apparently dominated by the hinge-binder component (Figure 2). The purines
1
0

1
2-18 were most promiscuous, inhibiting enzymes in the AGC, TK, CAMK, and CMGC
families, with the highest activities against PKA and other AGC kinases. This was expected
1
3
since these compounds were closest in structure to the PKA/PKB inhibitors such as 1, and
compounds 12-18 generally showed similar activity patterns to the 4-amino-4-benzylpiperidin-1-
yl purine 11. The isoquinolines 20-26 showed a reduced spread of activity, inhibiting only
certain AGC and some CAMK kinases. The 4-amino-4-benzylpiperidinyl isoquinoline 19 also
showed a decrease in the spread of activity relative to the comparator purine 11. The strong
inhibition of PKA may reflect the similarity of the general design 2 to known isoquinoline-5-
2
4
sulfonamide PKA inhibitors. 5-(2,8-Diazaspiro[5.5]undecan-2-ylsulfonyl)isoquinoline (25)
showed inhibition of CHK1 as well as PKA in the screen, and this was confirmed by IC50
4
3
determination (25; CHK1 IC = 12.8 μM; PKA IC = 3.6 μM).
5
0
50
We had speculated that the protonated amine in the diazaspirocycles might provide a binding
interaction to the kinase ribose pocket even with suboptimal hinge binding elements. However,
our screening data indicates that the presence of an appropriate and correctly oriented hinge
binder is critical if the binding contribution of the amine is to be realised, and that the presence
of the protonated amine does not automatically confer promiscuous inhibition. This is consistent
with a Free-Wilson analysis of the binding contributions of the functional groups in an ATP-
competitive 2-(4-(1H-pyrazol-4-yl)phenyl)ethanamine PKA/PKB inhibitor that adopted a similar
4
4
binding pose to 1.
The diazaspirocyclic substituent did modulate the activity of compounds within the hinge-
binder sets. For the purine analogs, piperidin-4-yl derived scaffolds with a nitrogen atom
proximal to the spirocentre were less promiscuous than those with the more distal nitrogen
(
compare 12, 13 to 14, 15). The purines bearing piperidin-3-yl substituted scaffolds (16, 17) were
less active still. For the isoquinoline analogues the reverse was observed, whereby the 4-
1
1

piperidinyl scaffolds with proximal nitrogens (20, 21) were active at more enzymes in the screen
than the compounds with the nitrogen displaced one atom further around the second ring (22,
2
3). Derivatives of the [4.5]-azaspirocycle 5 (e.g. 18, 26), which had the most extreme difference
in positioning of the basic nitrogen compared to the comparator 4-amino-4-benzylpiperidines 11
and 19, showed very limited activity.
As the purines 12 and 14 showed high activity in the initial screen, IC determinations were
5
0
carried out against the 24 kinases using the microfluidic assay format and were found to be
consistent with values estimated from the single-point percentage inhibition data using the Hill
equation, validating the use of the single concentration screen to assess the library
(
Supplementary Material, Table S1). Compound 14 was more potent across the panel than 12.
Both compounds exhibited highest activity against PKA (IC = 80 nM 14, 1 μM 12), with high
5
0
ligand efficiencies (LE = 0.48 14, 0.41 12). The IC values for 14 in the small panel spanned
5
0
more than 3 orders of magnitude (0.08 – 183 μM), confirming meaningful selectivity between
the most and least inhibited kinases tested. Although the inhibitory activity of these relatively
low MW compounds is weak, it is efficient and comparable to hits of similar size that have been
1
3,14,44
successfully progressed to potent inhibitors.
Thus the screening set 12-27 based on two
points of the simple pharmacophore 2 generated novel hits with potentially useful differences in
their activity profiles.
<
Figure 3>
High resolution crystal structures were obtained of PKA in complex with the purines 12, 14
2
4,45
and 16. The use of PKA as a surrogate of other AGC family kinases is well established. The
binding mode for 12 (PDB 3Z01; Figure 3A) showed the purine hydrogen bonding to the hinge
1
2

peptide as expected, and the nitrogen of the azaspirocycle interacting with the side chain
carboxylate of Glu127, a residue located in the ribose pocket and postulated to be involved in
4
6
communicating ATP-binding to substrate-peptide binding residues. Compounds 14 and 16
(
PDB 3Z02, 3ZO3; Figure 3B, C) bound similarly to the hinge region but displayed an
alternative interaction in the ribose pocket, with the azaspirocycle nitrogen interacting with the
side chain carboxylate of Asp184 from the DFG motif, and close to Gln171 and the backbone
carbonyl of Glu170. As the DFG aspartate residue is highly conserved across the kinome due to
its role in the enzyme catalytic function, the different interactions of the nitrogen atoms are
consistent with the increased promiscuity of 14 in the panel compared to 12.
The inhibitory activities of the purines 12 and 13 across the small panel correlated to an extent
with the identity of the residue equivalent to Glu127 in PKA (Figure 2). This was determined by
sequence alignment and overlay of publically available crystal structures of the kinase domains.
For most of the 24 kinases the relevant residue corresponded to the gatekeeper + 7 position in the
kinase domain sequence, which occupies a position at the start of the first α -helix in the C-
terminal subdomain. However for the four members of the CMGC family tested and the tyrosine
kinase CK1d, there are differences in the conformation of the peptide loop between the
gatekeeper residue and the start of the α-helix, such that the gatekeeper + 6 residues occupy this
space. Activity was generally higher for kinases with glutamate at this position (12 and 13, >50%
inhibition for 6/8 kinases with Glu at this position), with less likelihood of inhibition when the
glutamate was replaced by aspartate or non-acidic residues (12, <50% inhibition for 12/16; 13
<
50% inhibition for 11/16 kinases with non-glutamate residues at this position). Targeting the
presence of the glutamate residue at the gatekeeper + 7 position is thus potentially useful to
direct the activity profiles of the purine-diazaspirocyclic inhibitors towards a sub-set of kinases.
These results confirm that appropriately positioned basic amines within the core pharmacophore
1
3

can contribute positively to potency and selectivity. No similar correlation of activity was seen
for the isoquinoline-diazaspirocyclic compounds, which is consistent with the different vector
expected for the spirocycle relative to the hinge when attached to this topologically distinct
2
4
hinge-binder.
For all three purine compounds 12, 14 and 16 refinement of the ligand electron density
indicated the [5.5]-spirocyclic scaffolds to adopt chair-chair conformations on binding to PKA.
Predicted conformations of purines 13, 15, 17 and 18 bound to PKA were generated through
structural modification of the most closely related ligand structure bound to PKA, followed by
energy minimization of the ligand structure within the PKA protein, using the MMFF94X force
4
7
field in the Molecular Operating Environment (MOE) program. This modelling suggested that
the size of the distal heterocyclic ring (piperidine vs. pyrrolidine), would not significantly affect
the binding modes, so that [4.5]-azaspirocycles 13 and 15 closely mimicked the binding of 12
and 14, respectively. The [4.5]-azaspirocycle 18 was predicted to be unable to simultaneously
interact with an acidic residue in the main ribose pocket region and maintain key hinge-binding
interactions, consistent with the poor activity in the screen. The amine of 16 was shown to bind
to the DFG aspartate and this compound showed a more promiscuous profile than 17 which was
predicted to contact the non-conserved glutamate residue.
To confirm the importance of the basic piperidine nitrogen, the N-Cbz-protected precursors of
1
4 and 22 were profiled and showed reduced activity compared to the corresponding unprotected
piperidines (data not shown). This is potentially due to unfavorable steric clashes of the bulky
substituent in the relatively tight ATP binding site of many kinases, as well as the removal of
interactions to the protonated amines. Interestingly, in this screening panel Aurora A kinase was
best able to tolerate the N-substitution. In the purine series, the 3-azaspiro[5.5]undecane 27, and
1
-oxa- and 2-oxa-9-azaspiro[5.5]undecanes 28 and 29 were also prepared (Scheme 2 and Figure
1
4

2
). Removal of the basic amine (27) reduced activities in the panel with the exception of Aurora
A kinase, emphasizing the importance of the basic functionality in the binding of this set of
compounds. Replacement of the nitrogen proximal to the spirocentre with oxygen to give 28
recovered some of the AGC and CAMK kinase inhibitory activity, with potent inhibition of FYN
and Aurora A, thus heteroaryl-substituted oxazaspirocycles may be of future interest as scaffolds
for targeting the ribose-pocket of a different spectrum of kinases.
Based on its inhibitory profile and binding mode in PKA, purine analogue 12 was chosen to
assess the effects of adding P-loop interacting substituents at position C-3 and C-4 of the 1,9-
diazaspiro[5.5]undecane scaffold. It was expected that the activity profile of 12 could be
sensitive to different functionality in these positions and therefore hydroxyl, phenyl, benzyl,
carboxylate and carboxamide groups were targeted. The purines 33, 35, 41, 42, 45, 48, 51-53,
5
7-59 and two related isoquinoline analogs (34, 36) were tested for kinase inhibition at 30 μM
for consistency with previous data (Figure 2), since the increase in size was limited (MW range
288-385).
The introduction of hydroxyl groups in 33 and 35 maintained the activities of 12, except there
was a reduction in TK inhibition (Figure 2). The hydroxymethyl substituted analogue 53 also
maintained the original AGC kinase activity, with a reduction in activity across the other kinases
in the panel. Therefore, the addition of hydroxyl substituents to the purine [5.5]-azaspirocycle
1
2, led to a slight increase in specificity towards the AGC kinases in the small panel tested. The
presence of a glutamate at the gatekeeper +7 position continued to be associated with generally
higher activity. The C-3 and C-4 hydroxyl elaborated scaffolds were also attached to the
isoquinoline-5-sulfonamide hinge-binder, to give 34 and 36. Compared to the parent isoquinoline
derivative 20, compounds 34 and 36 showed increased inhibition of AGC kinases, in particular
for RSK1, PKA and MSK1. In contrast to the purine analogues, isoquinolines 20, 49 and 51
1
5

showed no inhibition of PKB isoforms (AKT1 and AKT2), despite the high sequence similarity
4
5
of these proteins with PKA.
Phenyl and benzyl substituents were introduced at the C-4 position of the 1,9-
diazaspiro[5.5]undecane, to give 41 and 42, Compounds 41 and 42 showed increased activity
towards the AGC sub-family compared to 12. This was consistent with the activity profile of the
4-benzyl-4-aminopiperidin-1-yl purine inhibitor 11. There was an appreciable increase in TK
activity for the phenyl substituted analogue 41, but this was less marked for the benzyl
substituted compound 42 (Figure 2). For compounds 11, 41 and 42 the addition of the P-loop
targeting aromatic group reduced the influence on the activity pattern of the presence of
glutamate at the gatekeeper + 7 residue, so that non-polar residues in this position became more
frequently associated with higher activities.
Elaboration at the C-3 position of 12 to give 45 and 48 resulted in a reduction in overall
activity, but maintained some selectivity towards the AGC kinases. The C-3 position of 12 was
also functionalised to give the methyl ester 51 and the amides 57-58. The ester and amide
analogues showed reduced inhibitory activity compared to the parent purine [5.5]-azaspirocycle
1
2 (Figure 2) but enhanced selectivity for the AGC kinases. The acid 52 also had less activity
against the kinases in the panel, but altered the profile of the undecorated [5.5]-azaspirocycle 12
4
3
towards AurA and RSK1 (AurA IC = 6.1 μM).
5
0
The single-concentration inhibitory activities of 41, 42 and 45 were determined against a 96-
member kinase panel distributed across eight of the kinome sub-families, with the majority of
kinases representing the four main sub-families (AGC, CAMK, CMGC and TK) (Supplementary
Material, Table S2). This broader analysis confirmed that the C-4 elaborated phenyl 41 and
benzyl 42 purine [5.5]-azaspirocycles possessed a degree of selectivity towards the AGC
kinases. However, there was still significant inhibition of the kinases in the TK and CAMK sub-
1
6

families by 41 and in the CAMK sub-family by 42. A reduction in overall activity was confirmed
for the C-3 benzyl substituted compound 45, particularly in the AGC kinase sub-family
compared to 41 and 42. Although 12, 41, 42 and 45 have a broad range of inhibitory activities,
these screening data show that variation of the positioning of a lipophilic group interacting with
the P-loop can vary the pattern of inhibition.
<
Figure 4>
The substituted inhibitors 41, 42, 45, 48 were modelled in the ATP-binding site of PKA
starting from the bound conformation of 12 in PKA chimera. This suggested that the aryl rings of
4
1 and 42 could project along a vector towards the P-loop, however the extra carbon of the
benzyl group of 42 would enable a closer interaction (Figure 4A), consistent with the increased
inhibition of AGC and other kinases within the panels by 42. Modelling of compounds 45 and 48
also showed the substituents approaching the P-loop, however they would likely require
additional substitution to efficiently bind without compromising hinge peptide and ribose pocket
binding interactions (Figure 4B). A crystal structure of 41 bound to PKA was determined (PDB
3
ZO4; Figure 3D) and showed interesting differences between the binding of 12 and 41, and
with the prediction of 41 binding. A key variation was in the conformation of the spirocyclic
core scaffold. Whereas the electron density for 12 bound to PKA was consistent with a chair-
chair conformation of the spirocycle, the first piperidine ring of 41 adopted a twist-boat
conformation. This led to the projection of the equatorial phenyl substituent along a more direct
vector to the P-loop than was predicted from the model based on the chair-chair conformation of
1
2 (Figure 4A). The phenyl substituent of 41 in PKA occupied a similar space to the terminal 4-
chloro substituent of 1 in PKA-PKB chimeric protein (Figure 1) and sat within a hydrophobic
1
7

environment created by the P-loop backbone and the side chain of Val57. The spirocyclic amine
1 formed hydrogen bonds directly to the side chain of Glu127 and the backbone carbonyl of
4
Glu170, with a water-mediated contact to the side chain of Gln171. The distortion of the
azaspirocycle conformation from a chair-chair (12) to twist-boat-chair (41) would suggest that
any additional energy of binding obtained by 41 from contacting the P-loop could be offset by
4
3
induced strain in the ligand. This was supported by the measured IC for inhibition of PKA by
5
0
4
1 (IC = 149 nM) which, although substantially increased over 12 (IC = 1000 nM), was less
5
0
50
improved than that of the C-4 benzylated analogue 42 (IC = 31 nM) where binding to the P-
5
0
loop was expected to be compatible with maintaining a lower energy chair-chair conformation of
the spirocycle.
In the above analysis, the changes in activity profile could be related in part to specific
interactions of the ligands in the ATP site. However, the degree to which the distinct activity
profiles of fragments or low-to-moderate molecular weight hit compounds such as those
described here can be maintained and exploited during elaboration to larger molecular weight
3
9,40
species is not yet fully defined.
While the results of this study are encouraging for the
identification of starting points with different spectra of kinase inhibitory activities, to realize the
8
,9
controlled design of ATP-competitive kinase inhibitors with specific polypharmacology would
require more information on the inherent stability, or otherwise, of kinase selectivity profiles
during optimization of other important molecular properties.
4
.
Conclusions
Heteroaryl-substituted diazaspirocyclic compounds were prepared as potential ATP-
competitive kinase inhibitors based on the pharmacophore defined by the 4-aminopiperidin-1-yl
purine PKB inhibitor 1 and ATP, and in particular to exploit the interactions of basic nitrogen
1
8

atoms in the ribose pocket. Several compounds showing ligand efficient inhibition of multiple
kinases were identified, validating the design. The nature of the adenine-mimic largely
determined the inhibitory activities shown by simpler spirocyclic compounds, with structural
similarity to 1 conferring a similar activity profile, particularly inhibition of AGC kinases.
However, varying the hinge-binding and diazaspirocyclic groups to produce compounds more
structurally distinct to 1 also produced potentially useful variation in the inhibition profiles.
Altering the position of the basic nitrogen in the diazaspirocycles was expected to change the
interactions to the proteins. This was confirmed by X-ray crystallography in the case of purine-
substituted examples, and was associated with changes in the activity profiles. Interaction of the
basic amine with a non-conserved glutamate residue at the gatekeeper + 7 position in the ribose
pocket conferred activity to purines substituted with diazaspirocycles containing an embedded 4-
aminopiperidine motif.
Ring-closing metathesis was used to enable versatile syntheses of 1,9-
diazaspiro[5.5]undecanes substituted at two positions with polar and hydrophobic groups to
target the P-loop of the kinase active site within a series of purine derivatives. These routes
should be readily extendable to prepare other substituted diazaspirocycles. The introduction of
aryl substituents at C-4 of the N-purinyl 1,9-diazaspiro[5.5]undecane scaffold led to the greatest
increases in activity, which was consistent with modeling that proposed this substitution pattern
would most closely resemble the original PKB inhibitor 1. Importantly, substitution at C-3 with
corresponding aryl groups led to different activity profiles, suggesting that the addition of the P-
loop targeting elements could change the intrinsic activity profile of the purine-diazaspirocyclic
scaffolds. The C-4 phenyl substituted analogue 41 in PKA showed a change in the conformation
of the core spirocycle from chair-chair to twist boat-chair to enable binding of the P-loop
1
9

targeting group, showing that a range of conformations are accessible to the diazaspirocyclic
parts of the scaffolds.
Overall these results show that diazaspirocyclic motifs can be usefully incorporated into the
core binding scaffolds of kinase inhibitors and that these motifs adopt well-defined
conformations within the ATP-site leading to productive interactions. Different kinase inhibitory
profiles can be associated with specific changes to the substitution pattern of the spirocycle and
its orientation with respect to the hinge-binding heteroaryl group. The straightforward synthetic
routes we have demonstrated to make novel substituted diazaspirocycles increase the potential
application of these scaffolds for future inhibitor discovery.
5
5
.
Experimental
.1
General Synthetic Chemistry
Reactions were carried out under N2. Organic solutions were dried over MgSO4 or Na2SO4.
Starting materials and solvents were purchased from commercial suppliers and were used
without further purification. Microwave reactions were carried out using a Biotage Initiator
microwave reactor. Flash column chromatography was performed using Merck silica gel 60
(
0.040-0.063 mm). Gradient silica column chromatography was performed with a Biotage SP1
medium pressure chromatography system, using prepacked silica gel cartridges. Preparative
HPLC was performed using a Gilson GX-281 Liquid Handler system and Gilson 322 HPLC
pump, with a 15 minute gradient elution of 10-90% MeOH/0.1% aqueous formic acid at a flow
rate of 20 mL min-1. Ion exchange chromatography was performed using Isolute Flash SCX-II
acidic resin cartridges. Melting points were determined on a Leica Gallen III or a SRS EZ-Melt
Automated melting point apparatus. 1H NMR spectra were recorded on a Bruker AMX500
instrument at 500 MHz using internal deuterium locks. Chemical shifts ( ) are reported relative
2
0

to TMS ( =0) and referenced to the solvent in which they were measured. 13C-nuclear magnetic
resonance spectra were recorded at 126 MHz on a Bruker AMX500 spectrometer using an
internal deuterium lock. Combined HPLC-MS analyses were performed on a Waters Alliance
2
795 separations module and a Waters 2487 dual wavelength absorbance detector coupled to a
Waters/Micromass LCt time of flight mass spectrometer with ESI source, or on an Agilent 1200
series HPLC and diode array detector coupled to a 6210 time of flight mass spectrometer with
dual multimode APCI/ESI source. HPLC was performed using Supelco DISCOVERY C18, 50
mm x 4.6 mm or 30 mm x 4.6 mm i.d. columns, or using an Agilent 6210 TOF HPLC-MS with a
Phenomenex Gemini 3 μm C18 (3 cm x 4.6 mm i.d.) column. Both HPLC systems were run at a
temperature of 22 °C with gradient elution of 10-90% MeOH/0.1% aqueous formic acid at a
flow rate of 1 mL min-1. UV detection was at 254 nm and ionisation was by positive or negative
ion electrospray as indicated. The molecular weight scan range was 50-1000 amu. All tested
compounds gave >95% purity as determined by these methods. All purified synthetic
intermediates gave >95% purity as determined by these methods except where indicated in the
text.
5
.1.1 Benzyl 1,8-diazaspiro[4.5]decane-1-carboxylate (9)
1
7
A solution of Et N (1.1 mL, 0.80 mmol), 5 (50 mg, 0.20 mmol) and CbzCl (37 µL, 0.26
3
mmol) in CH Cl (2 mL) was stirred at r.t. for 48 h. Additional CbzCl (15 µL, 0.10 mmol) and
2
2
Et N (0.2 mL, 0.20 mmol) were added and the mixture was stirred for a further 24 h. The mixture
3
was washed with sat. aq. NaHCO (3 x 5 mL), dried and concentrated. Flash column
3
chromatography, eluting with 30% EtOAc/hexane, gave 1-benzyl 8-tert-butyl 1,8-
1
diazaspiro[4.5]decane-1,8-dicarboxylate as colourless oil (61 mg, 0.15 mmol, 82%). H NMR
(
500 MHz, CDCl3) 1.23-1.36 (2H, m), 1.46 (9H, s), 1.76-1.83 (2H, m), 1.90-1.99 (2H, m),
1
3
2
.66-2.81 (4H, m), 3.49-3.58 (2H, m), 4.02-4.22 (2H, m), 5.09 (2H, s), 7.20-7.37 (5H, m); C
2
1

NMR (126 MHz, CDCl ) 22.1, 28.5, 33.0, 35.0, 41.3, 42.3, 47.7, 62.8, 66.1, 79.4, 127.7, 127.8,
3
1
28.4, 137.2, 153.6, 154.6; LC-MS (ESI+) m/z 397 (M+Na); t = 2.77 min, purity = 90%; HRMS
R
m/z calcd. for C H N O Na (M+Na) 397.2098, found 397.2104. A solution of 4 M HCl in
2
1
30
2
4
dioxane (2.37 mL, 9.50 mmol) and 1-benzyl 8-tert-butyl 1,8-diazaspiro[4.5]decane-1,8-
dicarboxylate (358 mg, 0.95 mmol) in MeOH (5 mL) was stirred at 0 °C for 1 h, then at 4 °C for
1
6 h. The reaction mixture was concentrated and purified by ion exchange chromatography on
acidic resin, eluting with 2M NH in MeOH, to give 9 as a colourless oil (261 mg, 0.95 mmol,
3
1
1
00%); H NMR (500 MHz, CDCl ) 1.31-1.38 (2H, m), 1.75-1.81 (2H, m), 1.92-2.01 (2H, m),
3
2.60-2.69 (4H, m), 3.02-3.13 (2H, m), 3.49-3.57 (2H, m), 5.04-5.25 (2H, m), 7.29-7.45 (5H, m);
1
3
C NMR (126 MHz, CDCl ) 22.3, 34.7, 35.8, 44.7, 47.6, 62.9, 66.0, 127.7, 127.7, 128.4, 137.3,
3
1
2
5
53.6; LC-MS (ESI+) m/z 275 (M+H); HRMS m/z calcd. for C H N O (M+H) 275.1754, found
1
6
23
2
2
75.1752.
.1.2 Benzyl 2,9-diazaspiro[5.5]undecane-2-carboxylate (10)
A solution of Et N (0.1 mL, 0.68 mmol), 6 (50 mg, 0.17 mmol) and CbzCl (38 mg, 0.22 mmol)
3
in CH Cl (1.7 mL) was stirred at r.t. for 48 h. Additional CbzCl (29 mg, 0.16 mmol) was added
2
2
and the mixture was stirred for a further 24 h. The mixture was washed with sat. aq. NaHCO (3
3
x 10 mL), dried and concentrated to give 2-benzyl 9-tert-butyl 2,9-diazaspiro[5.5]undecane-2,9-
1
dicarboxylate as a colourless oil (55 mg, 0.13 mmol, 65%). H NMR (500 MHz, CDCl ) 1.30-
3
1.38 (2H, m), 1.38-1.50 (13H, m), 1.54-1.62 (2H, m), 3.16-3.61 (8H, m), 5.15 (2H, s), 7.30-7.41
1
3
(
5H, m); C NMR (126 MHz, CDCl ) 20.8, 21.0, 28.5, 32.1, 33.4, 35.5, 35.8, 39.5, 44.9, 51.4,
3
5
1.8, 67.1, 79.3, 127.0, 127.6, 127.9, 128.1, 128.5, 128.6, 136.9, 154.9, 155.4, 155.5, multiple
peaks due to rotamers; LC-MS (ESI+) m/z 411 (M+Na); t = 2.85 min, purity = 90%; HRMS m/z
R
calcd. for C H N O Na (M+Na) 411.2254, found 411.2252. A solution of 4M HCl in dioxane
2
2
32
2
4
(0.35 mL, 1.41 mmol) and 2-benzyl 9-tert-butyl 2,9-diazaspiro[5.5]undecane-2,9-dicarboxylate
2
2

(
55 mg, 0.14 mmol) in MeOH (3 mL) was mixed at 0 °C then stirred at 4 °C for 16 h. The
mixture was concentrated and purified by ion exchange chromatography on acidic resin, eluting
1
with 2M NH in MeOH, to give 10 as a colourless oil (32 mg, 0.11 mmol, 80%). H NMR (500
3
MHz, CDCl ) 1.29-1.50 (6H, m), 1.51-1.59 (2H, m), 2.25 (1H, s), 2.68-2.89 (4H, m), 3.29-3.32
3
1
3
(
1H, s), 3.32-3.36 (1H, s), 3.41-3.45 (2H, m), 5.12 (2H, s), 7.29-7.37 (5H, m); C NMR (126
MHz, CDCl3) 20.6, 21.0, 32.2, 34.7, 36.2, 36.5, 42.0, 44.9, 51.8, 52.4, 67.0, 127.8, 128.0,
28.5, 136.9, 155.5, multiple peaks due to rotamers; LC-MS (ESI+) m/z 289 (M+H); HRMS m/z
calcd. for C H N O (M+H) 289.1911, found 289.1921.
1
1
7
25
2
2
5.1.3 4-Benzyl-1-(9H-purin-6-yl)piperidin-4-amine (11)
2
3
A solution of Et N (0.3 mL, 2.30 mmol) 3 (89 mg, 0.46 mmol) and 6-chloropurine (72 mg,
3
n
0
.46 mmol) in BuOH (4.6 mL) was heated at 100 °C for 18 h. The reaction mixture was cooled
and concentrated to dryness. The solid was washed with MeOH to give 11 as an off-white solid
1
(
101 mg, 0.35 mmol, 72%). mp 232 °C (dec); H NMR (500 MHz, d -DMSO) 1.31-1.38 (2H,
6
m), 1.53 (2H, ddd, J = 13.5, 12.5, 4.0 Hz), 2.64 (2H, s), 3.14-3.36 (2H, br m), 3.55-3.72 (2H, br
1
3
m), 4.76-5.09 (2H, br m), 7.17-7.22 (3H, m), 7.25-7.29 (2H, m), 8.06 (1H, s), 8.16 (1H, s); C
NMR (126 MHz, d -DMSO) 37.0, 40.8, 49.8, 50.0, 118.6, 126.0, 127.7, 130.6, 137.4, 137.6,
6
1
3
5
51.3, 151.8, 153.0; LC-MS (ESI+) m/z 292 (M-NH ); HRMS m/z calcd. for C H N (M+H)
2
17 21
6
09.1822, found 309.1825.
.1.4 6-(1,9-Diazaspiro[5.5]undecan-9-yl)-9H-purine (12)
1
7
A solution of Et N (0.24 mL, 1.70 mmol), 4 (100 mg, 0.34 mmol) and 6-chloropurine (55 mg,
3
n
0
.34 mmol) in BuOH (3.4 mL) was stirred at 100 °C for 24 h. The reaction mixture was cooled,
concentrated and triturated with MeOH (2.5 mL). The resulting solid was collected and dried to
give tert-butyl 9-(9H-purin-6-yl)-1,9-diazaspiro[5.5]undecane-1-carboxylate as a yellow solid
(107 mg, 0.29 mmol, 84%). A mixture of 4M HCl in dioxane (0.35 mL, 2.80 mmol) and tert-
butyl 9-(9H-purin-6-yl)-1,9-diazaspiro[5.5]undecane-1-carboxylate (107 mg, 0.28 mmol) in
2
3

MeOH (2 mL) was stirred at r.t. for 48 h. The mixture was concentrated and purified by ion
exchange chromatography on acidic resin, eluting with 2M NH in MeOH to give 12 as an off-
3
1
white solid (66 mg, 0.24 mmol, 84%). mp 250-256 °C; H NMR (500 MHz, d -DMSO) 1.34-
6
1
4
3
.48 (6H, m), 1.51-1.57 (2H, m), 1.64-1.77 (2H, m), 2.69-2.71 (2H, m), 3.79-3.91 (2H, br m),
1
3
.49-4.70 (2H, brd m), 8.07 (1H, s), 8.15 (1H, s); C NMR (126 MHz, d -DMSO) 19.9, 26.9,
6
5.1, 36.7, 40.3, 40.7, 48.8, 118.7, 137.7, 151.4, 151.8, 153.1; LC-MS (ESI+) m/z 273 (M+H);
HRMS m/z calcd. for C H N (M+H) 273.1822, found 273.1822.
1
4
21
6
5.1.5 6-(1,8-Diazaspiro[4.5]decan-8-yl)-9H-purine (13)
A mixture of Et N (0.21 mL, 1.55 mmol), 9 (87 mg, 0.31 mmol) and 6-chloropurine (49 mg,
3
n
0
.31 mmol) in BuOH (1 mL) was heated to 100 °C for 16 h. The mixture was cooled to room
temperature and concentrated to dryness. The solid was washed with MeOH (2 mL) to give
benzyl 8-(9H-purin-6-yl)-1,8-diazaspiro[4.5]decane-1-carboxylate as an off-white solid (84 mg,
0
.21 mmol, 69%). A mixture of 6 M HCl (1 mL) and benzyl 8-(9H-purin-6-yl)-1,8-
diazaspiro[4.5]decane-1-carboxylate (40 mg, 0.10 mmol) was heated in a microwave reactor to
o
1
00 C for 15 min. The mixture was concentrated and purified by column chromatography,
eluting with 10% 2 M NH in MeOH/CH Cl to give 13 as an off-white solid (26 mg, 0.10 mmol,
3
2
2
o
1
9
9%). mp 223-235 C; H NMR (500 MHz, d -DMSO) 1.56-1.63 (6H, m), 1.76 (2H, dddd, J =
6
1
3
7
.0, 7.0 Hz), 2.93 (2H, dd, J = 7.0, 7.0 Hz), 4.10-4.29 (4H, br m), 8.09 (1H, s), 8.19 (1H, s); C
NMR (126 MHz, d -DMSO) 24.9, 36.7, 36.8, 42.8, 45.1, 61.2, 119.2, 138.4, 151.9, 152.3,
6
1
2
5
53.5; LC-MS (ESI+) m/z 259 (M+H); HRMS m/z calcd. for C H N (M+H) 259.1666, found
1
3
19
6
59.1658.
.1.6 6-(2,9-Diazaspiro[5.5]undecan-9-yl)-9H-purine (14)
A mixture of Et N (0.19 mL, 1.35 mmol), 10 (78 mg, 0.27 mmol) and 6-chloropurine (42 mg,
3
n
0
.27 mmol) in BuOH (2.7 mL) was heated to 100 °C for 16 h. The mixture was cooled and
concentrated to dryness. Flash column chromatography eluting with 5% MeOH/CH Cl gave
2
2
2
4

benzyl 9-(9H-purin-6-yl)-2,9-diazaspiro[5.5]undecane-2-carboxylate as a white solid (67 mg,
.17 mmol, 61%). A mixture of CF CO H (4 mL) and benzyl 9-(9H-purin-6-yl)-2,9-
0
3
2
o
diazaspiro[5.5]undecane-2-carboxylate (53 mg, 0.13 mmol) was heated to 50 C for 24 h. The
mixture was concentrated and purified by ion exchange chromatography, eluting with 2M NH in
3
o
1
MeOH, to give 14 as a pale yellow oil (35 mg, 0.13 mmol, 100%). mp = 246-247 C; H NMR
(
500 MHz, CDCl3) 1.54-1.58 (4H, m), 1.62-1.67 (4H, m), 2.73 (2H, s), 2.80-2.83 (2H, m),
1
3
4
.15-4.43 (2H, br m), 5.02-5.69 (2H, br m), 7.93 (1H, s), 8.35 (1H, s); C NMR (126 MHz,
CDCl3) 22.2, 31.2, 34.7, 34.7, 41.0, 47.2, 55.8, 119.6, 136.7, 151.2, 151.5, 153.9; LC-MS
(
ESI+) m/z 273.14 (M+H); HRMS m/z calcd. for C H N (M+H) 273.1822, found 273.1828.
1
4
21
6
5.1.7 6-(2,8-Diazaspiro[4.5]decan-8-yl)-9H-purine (15)
Prepared as described for 12 starting from 8 and 6-chloropurine to give 15 as a colourless oil
1
(
53% over 2 steps); H NMR (500 MHz, CDCl ) 1.47-1.63 (6H, m), 2.66 (2H, s), 2.87 (2H, dd,
3
1
3
J = 7.5, 7.5 Hz), 4.00-4.43 (4H, br m), 8.09 (1H, s), 8.19 (1H, s); C NMR (126 MHz, CDCl )
3
36.1, 37.7, 42.0, 43.2, 45.6, 57.7, 119.2, 138.4, 151.9, 152.2, 153.6; LC-MS (ESI+) m/z 259
(
M+H); HRMS m/z calcd. for C H N (M+H) 259.1666, found 259.1669.
1
3
19
6
5.1.8 6-(2,9-Diazaspiro[5.5]undecan-2-yl)-9H-purine (16)
Prepared as described for 12 starting from 6 and 6-chloropurine to give 16 as a white solid
1
(
60% over 2 steps). mp 183-187 °C; H NMR (500 MHz, CDCl ) 1.49 (2H, ddd, J = 13.5, 9.5,
3
4
.0 Hz, C7/10-Hax), 1.61-1.68 (4H, m, C7/10-H, C4-H), 1.74-1.80 (2H, m, C3-H), 2.90-2.96
2H, m, C8/9-H), 3.06-3.13 (2H, m, C8/9-H), 4.14-4.44 (4H, brd m, C2/6-H), 7.95 (1H, s, C14-
H), 8.34 (1H, m, C12-H); 13C NMR (126 MHz, CDCl3) 21.2 (C3), 33.2 (C5), 33.9 (C7/10),
6.8 (C4), 41.4 (C8/9), 119.1 (CAr), 136.6 (C14), 151.2 (CAr), 151.7 (C12), 154.24 (CAr), (C2
and C6 Missing); HRMS [M+H+] calcd for C H N 274.1848; found 274.1849.
(
3
1
4
21
6
5.1.9 6-(2,8-Diazaspiro[5.5]undecan-2-yl)-9H-purine (17)
2
5

Prepared as described for 12 starting from 7 and 6-chloropurine to give 17 as a yellow oil
1
(
48% over 2 steps). H NMR (500 MHz, CDCl ) 1.40-1.76 (8H, m), 2.46 (1H, d, J = 13.0 Hz),
3
2
.67 (1H, dd, J = 11.5, 11.5 Hz), 2.98-3.10 (1H, br s), 3.11-3.14 (1H, m), 3.41 (1H, d, J = 13.0
1
3
Hz), 4.77-5.10 (2H, br m), 5.65-6.07 (2H, m), 7.88 (1H, s), 8.33 (1H, s); C NMR (126 MHz,
CDCl ) 21.1, 21.8, 33.5, 34.2, 35.7, 45.0 (br), 46.1, 51.6, 117.0, 137.3, 151.4, 152.2, 153.4; LC-
3
MS (ESI+) m/z 273 (M+H); HRMS m/z calcd. for C H N (M+H) 273.1822, found 273.1835.
1
4
21
6
5.1.10 6-(1,8-Diazaspiro[4.5]decan-1-yl)-9H-purine (18)
Prepared as described for 12 starting from 5 and 6-chloropurine to give 18 as a colourless oil
1
(
7% over 2 steps). H NMR (500 MHz, d -DMSO) 1.45-1.52 (2H, m), 1.88-1.96 (2H, m), 2.06-
6
2.12 (2H, m), 2.87-2.99 (2H, m), 3.19-3.25 (2H, m), 3.44-3.56 (2H, m), 4.22-4.27 (2H, m), 8.07
1
3
(
1H, s), 8.19 (1H, s); C NMR (126 MHz, d -DMSO) 22.5, 29.7, 30.5, 36.1, 40.0, 51.2; 150.9,
6
4
quaternary carbons not observed. LC-MS (ESI+) m/z 260 (M+H); HRMS m/z calcd. for
C H N 259.1666, found 259.1663.
1
3
19
6
5.1.11 4-Benzyl-1-(isoquinolin-5-ylsulfonyl)piperidin-4-amine (19)
2
3
A solution of Et N (0.3 mL, 2.19 mmol), 3 (100 mg, 0.53 mmol) and 5-isoquinoline
3
sulfonylchloride (120 mg, 0.44 mmol) in CH Cl (5 mL) was stirred at r.t. for 16 h. The mixture
2
2
was diluted with H O (5 mL) and washed with H O (3 x 10 mL). The organic layer was dried and
2
2
concentrated. Flash column chromatography eluting with 1-5% MeOH/CH Cl gave 19 as a pale
2
2
1
yellow oil (65 mg, 0.17 mmol, 33%). H NMR (500 MHz, CDCl ) 1.35-1.41 (2H, m), 1.73
3
(
2H, ddd, J = 12.0, 12.0, 4.0 Hz), 2.61 (2H, s), 2.99 (2H, ddd, J = 12.0, 12.0, 4.0 Hz), 3.63 (2H,
ddd, J = 12.0, 4.0, 4.0 Hz), 7.08-7.10 (2H, m), 7.31-7.22 (3H, m), 7.71 (1H, dd, J = 7.5, 7.5 Hz),
.21 (1H, dd, J = 7.5, 1.0 Hz), 8.38 (1H, dd, J = 7.5, 1.0 Hz), 8.52 (1H, d, J = 6.0 Hz), 8.67 (1H,
8
1
3
d, J = 6.0 Hz), 9.35 (1H, s); C NMR (126 MHz, CDCl ) 37.0, 41.8, 49.3, 51.5, 117.8, 125.9,
3
1
26.7, 128.2, 129.1, 130.5, 132.0, 133.0, 133.6, 133.9, 136.2, 145.0, 153.2; LC-MS (ESI+) m/z
82 (M+H); HRMS m/z calcd. for C H N O S (M+H) 382.1584, found 382.1601.
3
2
1
24
3
2
2
6

5.1.12 5-(1,9-Diazaspiro[5.5]undecan-9-ylsulfonyl)isoquinoline (20)
1
7
A mixture of Et N (0.12 mL, 0.85 mmol), 4 (50 mg, 0.17 mmol) and 5-isoquinoline sulfonyl
3
chloride (89 mg, 0.34 mmol) in CH Cl (1.7 mL) was stirred at r.t. for 16 h. The reaction was
2
2
quenched with H O (5 mL) and washed with H O (3 x 5 mL). The organic layer was dried and
2
2
concentrated. Flash column chromatography eluting with 0-5% MeOH/CH Cl (0.1% NEt3) gave
2
2
tert-butyl 9-(isoquinolin-5-ylsulfonyl)-1,9-diazaspiro[5.5]undecane-1-carboxylate as a light
brown oil (50 mg, 0.11 mmol, 66%). A mixture of 4M HCl in dioxane (0.26 mL, 1.05 mmol) and
tert-butyl 9-(isoquinolin-5-ylsulfonyl)-1,9-diazaspiro[5.5]undecane-1-carboxylate (47 mg, 0.11
mmol) in MeOH (1 mL) was stirred at r.t. for 16 h. The mixture was concentrated and purified
by ion exchange chromatography on acidic resin eluting with 2M NH in MeOH to give 20 as a
3
dark orange oil (33 mg, 0.10 mmol, 91%). 1H NMR (500 MHz, CDCl ) 1.34-1.38 (2H, m),
3
1.40-1.47 (2H, m), 1.52-1.58 (2H, m), 1.62 (2H, ddd, J = 10.0, 12.0, 4.0 Hz), 1.67-1.74 (2H, m),
2.69 (2H, t, J = 7.0 Hz), 3.11-3.20 (2H, m), 3.35-3.43 (2H, m), 7.72 (1H, dd, J = 7.5, 7.5 Hz),
8.22 (1H, d, J = 7.5 Hz), 8.40 (1H, d, J = 7.5 Hz), 8.54 (1H, d, J = 6.0 Hz), 8.70 (1H, d, J = 6.0
1
3
Hz), 9.36 (1H, s); C NMR (126 MHz, CDCl ) 19.8, 26.6, 35.0, 36.8, 40.3, 41.4, 48.0, 117.7,
3
125.9, 129.1, 132.0, 126.9, 133.1, 133.8, 145.1, 153.3; LC-MS (ESI+) m/z 346 (M+H); HRMS
m/z calcd. for C H N O S (M+H) 346.1584, found 346.1587.
1
8
24
3
2
5.1.13 5-(1,8-Diazaspiro[4.5]decan-8-ylsulfonyl)isoquinoline (21)
1
7
4
M HCl in dioxane (1.77 mL, 7.10 mmol) was added to a solution of 5 (171 mg, 0.71 mmol)
in MeOH (4 mL) and stirred at r.t. for 16 h. The reaction mixture was concentrated to give crude
,8-diazaspiro[4.5]decane dihydrochloride salt (150 mg). Et N (0.79 mL, 5.68 mmol) and 5-
1
3
isoquinoline sulfonyl chloride (188 mg, 0.71mmol) were added to the crude 1,8-
diazaspiro[4.5]decane dihydrochloride salt in CH Cl (6 mL) and the mixture was stirred at r.t.
2
2
for 16 h. The reaction was quenched with H O (5 mL) and washed with H O (3 x 5 mL). The
2
2
organic layer was dried and concentrated. Flash column chromatography eluting with 5%
2
7

1
MeOH/CH Cl gave 21 as an orange oil (41 mg, 0.12 mmol, 17% over 2 steps). H NMR (500
2
2
MHz, CDCl ) 1.71 (2H, dd, J = 7.5, 7.5 Hz), 1.81 (2H, ddd, J = 13.0, 9.0, 3.0 Hz), 1.88-1.96
3
(4H, m), 3.10 (2H, dd, J = 7.0, 7.0 Hz), 3.25-3.32 (2H, m), 3.38-3.46 (2H, m), 7.71 (1H, dd, J =
7
.5, 7.5 Hz), 8.22 (1H, d, J = 7.5 Hz), 8.38 (1H, d, J = 7.5 Hz), 8.45 (1H, d, J = 6.0 Hz), 8.68
1
3
(
1H, d, J = 6.0 Hz), 9.34 (1H, s); C NMR (126 MHz, CDCl ) 23.3, 34.6, 36.1, 42.8, 44.6, 62.9,
3
1
17.6, 126.0, 129.2, 131.9, 132.7, 133.9, 134.0, 145.2, 153.3; LC-MS (ESI+) m/z 332 (M+H);
HRMS m/z calcd. for C H N O S (M+H) 332.1427, found 332.1432.
1
7
22
3
2
5.1.14 5-(2,9-Diazaspiro[5.5]undecan-9-ylsulfonyl)isoquinoline (22)
A solution of Et N (0.16 mL, 1.20 mmol), 10 (70 mg, 0.24 mmol) and 5-isoquinoline sulfonyl
3
chloride (126 mg, 0.48 mmol) in CH Cl (2 mL) was stirred at r.t. for 16 h then quenched with
2
2
H O (5 mL). The organic layer was washed with H O (3 x 5 mL), dried and concentrated. Flash
2
2
column chromatography eluting with 50% EtOAc/hexane gave benzyl 9-(isoquinolin-5-
ylsulfonyl)-2,9-diazaspiro[5.5]undecane-2-carboxylate as a clear oil (68 mg, 0.14 mmol, 59%).
A
mixture of CF CO H (4 mL) and benzyl 9-(isoquinolin-5-ylsulfonyl)-2,9-
3
2
o
diazaspiro[5.5]undecane-2-carboxylate (58 mg, 0.13 mmol) was heated to 45 C for 16 h. The
mixture was cooled and concentrated. Column chromatography on a Biotage SNAP KP-NH
column, eluting with 1% EtOH/CH Cl gave 22 as a light yellow oil (10 mg, 0.03 mmol, 23%).
2
2,
1
H NMR (500 MHz, CDCl ) 1.40-1.71 (6H, m), 1.74-1.79 (2H, m), 2.80 (2H, s), 2.95-3.06 (4H,
3
m), 3.19-3.23 (2H, m), 7.74 (1H, dd, J = 8.0, 7.5 Hz), 8.20-8.25 (1H, m), 8.39-8.43 (1H, m), 8.45
1
3
(
1H, d, J = 6.0 Hz), 8.69 (1H, d, J = 6.0 Hz), 9.38 (1H, s); C NMR (126 MHz, CDCl ) 18.1,
3
3
0.4, 31.9, 33.4, 40.8, 44.1, 51.0, 117.5, 125.9, 129.1, 131.8, 132.5, 134.0, 134.3, 145.1, 153.3;
LC-MS (ESI+) m/z 346 (M+H); HRMS m/z calcd. for C H N O S (M+H) 346.1584, found
1
8
24
3
2
3
46.1593.
5.1.15 5-(2,8-Diazaspiro[4.5]decan-8-ylsulfonyl)isoquinoline (23)
2
8

Prepared as described for 20 starting from 8 and 5-isoquinoline sulfonyl chloride to give 23 as
1
a colourless oil (29% over 2 steps). H NMR (500 MHz, CDCl ) 1.54 (2H, dd, J = 7.0, 7.0 Hz),
3
1
.61-1.64 (4H, m), 2.12 (1H, br s), 2.69 (2H, s), 2.99 (2H, dd, J = 7.0, 7.0 Hz), 3.15-3.21 (2H,
m), 3.22-3.28 (2H, m), 7.74 (1H, dd, J = 7.5, 7.5 Hz), 8.23-8.26 (1H, m), 8.41 (1H, dd, J = 7.5,
1
3
1
.5 Hz), 8.49-8.52 (1H, m), 8.71 (1H, d, J = 6.5 Hz), 9.38 (1H, d, J = 1.0 Hz); C NMR (126
MHz, CDCl ) 35.5, 37.0, 40.9, 43.5, 45.7, 57.4, 117.7, 125.8, 129.1, 131.9, 132.8, 133.8, 134.1,
3
1
3
5
45.1, 153.3; LC-MS (ESI+) m/z 332 (M+H); HRMS m/z calcd. for C H N O S (M+H)
1
7
22
3
2
32.1427, found 332.1437.
.1.16 5-(2,9-Diazaspiro[5.5]undecan-2-ylsulfonyl)isoquinoline (24)
Prepared as described for 20 starting from 6 and 5-isoquinoline sulfonyl chloride to give 24 as
1
a colourless oil (52% over 2 steps). H NMR (500 MHz, CDCl ) 1.26-1.40 (6H, m), 1.60-1.66
3
(
(
(
2H, m), 2.57-2.63 (2H, ddd, J = 12.5, 8.5, 3.5 Hz), 2.65-2.71 (2H, m), 2.96 (2H, s), 3.14-3.18
2H, m), 7.69 (1H, dd, J = 7.5, 7.5 Hz), 8.19 (1H, d, J = 7.5 Hz), 8.36 (1H, d, J = 7.5 Hz), 8.46
1
3
1H, d, J = 6.0 Hz), 8.65 (1H, d, J = 6.0 Hz), 9.32 (1H, s); C NMR (126 MHz, CDCl ) 20.7,
3
3
2.0, 34.8, 35.3, 41.7, 46.3, 53.8, 117.7, 125.9, 129.1, 131.9, 133.0, 133.6, 133.9, 144.9, 153.2;
LC-MS (ESI+) m/z 346 (M+H); HRMS m/z calcd. for C H N O S (M+H) 346.1584, found
1
8
24
3
2
3
46.1588.
5.1.17 5-(2,8-Diazaspiro[5.5]undecan-2-ylsulfonyl)isoquinoline (25)
Prepared as described for 20 starting from 7 and 5-isoquinoline sulfonyl chloride to give 25 as
1
a colourless oil (71% over 2 steps). H NMR (500 MHz, CDCl ) 1.19-1.26 (2H, m), 1.37-1.41
3
(3H, m), 1.47-1.53 (1H, m), 1.61-1.68 (2H, m), 1.75-1.88 (1H, br s), 2.50 (1H, d, J = 12.5 Hz),
2
.60-2.67 (2H, m), 2.79-2.85 (1H, m), 3.02 (1H, d, J = 12.0 Hz), 3.11 (1H, d, J = 12.0 Hz), 3.13-
.24 (2H, m), 7.64-7.77 (1H, m), 8.20 (1H, d, J = 8.2 Hz), 8.40 (1H, dd, J = 7.5, 1.0 Hz), 8.52
3
1
3
(
1H, d, J = 6.0 Hz), 8.69 (1H, d, J = 6.0 Hz), 9.35 (1H, d, J = 1.0 Hz); C NMR (126 MHz,
CDCl ) 20.8, 22.1, 32.2, 32.8, 33.4, 46.3, 46.5, 52.5, 53.9, 117.8, 126.0, 129.1, 131.9, 133.0,
3
2
9