6-Substituted purines as ROCK inhibitors with anti-metastatic activity

Article abstract of DOI:10.1016/j.bioorg.2019.103005

Rho-associated serine/threonine kinases (ROCKs) are principal regulators of the actin cytoskeleton that regulate the contractility, shape, motility, and invasion of cells. We explored the relationships between structure and anti-ROCK2 activity in a group

Full text of DOI:10.1016/j.bioorg.2019.103005

Accepted Manuscript
6
-Substituted purines as ROCK inhibitors with anti-metastatic activity
Jiř í Voller, Lenka Zahajská, Lucie Plíhalová, Jana Jeř ábková, David Burget,
Andreea Csilla Pataki, Vladimír Kryštof, Marek Zatloukal, Jan Brábek, Daniel
Rösel, Václav Mik, Martin Tká č, Tomá š Pospí šil, Tomá š Gucký, Karel
Doležal, Miroslav Strnad
PII:
S0045-2068(18)31575-X
https://doi.org/10.1016/j.bioorg.2019.103005
103005
DOI:
Article Number:
Reference:
YBIOO 103005
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
31 December 2018
31 March 2019
19 May 2019
Please cite this article as: J. Voller, L. Zahajská, L. Plíhalová, J. Jeř ábková, D. Burget, A. Csilla Pataki, V.
Kryštof, M. Zatloukal, J. Brábek, D. Rösel, V. Mik, M. Tká č, T. Pospí šil, T. Gucký, K. Doležal, M. Strnad, 6-
Substituted purines as ROCK inhibitors with anti-metastatic activity, Bioorganic Chemistry (2019), doi: https://
doi.org/10.1016/j.bioorg.2019.103005
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6
-Substituted purines as ROCK inhibitors with anti-metastatic activity
a,b *
Jiří Voller
Lenka Zahajská c
Lucie Plíhalová a,d
Jana Jeřábková a
David Burget a
Andreea Csilla Pataki e
Vladimír Kryštof a
Marek Zatloukal d
Jan Brábek e
Daniel Rösel e
Václav Mik d
Martin Tkáč a
Tomáš Pospíšil d
Tomáš Gucký d
Karel Doležal a,d
Miroslav Strnad a
a
Laboratory of Growth Regulators, The Czech Academy of Sciences, Institute of Experimental Botany
&
Palacký University, Šlechtitelů 27, CZ-78371 Olomouc, Czech Republic
b
Department of Clinical and Molecular Pathology, Institute of Molecular and Translational Medicine,
Faculty of Medicine and Dentistry, Palacký University, Hněvotínská 3, 775 15 Olomouc, Czech
Republic
c
Isotope Laboratory, The Czech Academy of Sciences, Institute of Experimental Botany, Vídeňská
1
083, 142 00 Prague 4, Czech Republic
d
Department of Chemical Biology and Genetics, Centre of the Region Haná for Biotechnological and
Agricultural Research, Palacký University, Šlechtitelů 27, CZ-78371 Olomouc, Czech Republic
e
Department of Cell Biology, Faculty of Science, Charles University in Prague, Viničná 7, 12843,
Prague 2, Czech Republic
*
Correspondence: jiri.voller@upol.cz

Keywords:
protein kinase inhibitor - ROCK - anti-metastatic activity - melanoma
Abstract:
Rho-associated serine/threonine kinases (ROCKs) are principal regulators of the actin cytoskeleton that
regulate the contractility, shape, motility, and invasion of cells. We explored the relationships between
structure and anti-ROCK2 activity in a group of purine derivatives substituted at the C6 atom by
piperidin-1-yl or azepan-1-yl groups. Structure-activity relationship (SAR) analyses suggested that
anti-ROCK activity is retained, and may be further increased, by substitution of the parent compounds
at the C2 atom or by expansion of the C6 side chain. These inhibitors of ROCK can reach effective
concentrations within cells, as demonstrated by a decrease in phosphorylation of the ROCK target
MLC, and by inhibition of the ROCK-dependent invasion of melanoma cells in the collagen matrix.
Our study may be useful for further optimization of C6-substituted purine inhibitors of ROCKs and of
other sensitive kinases identified by the screening of a broad panel of protein kinases.
1
. Introduction
Rho-associated serine/threonine kinases (ROCKs) are members of the AGC (cAMP-dependent protein
kinase/protein kinase G/protein kinase C) protein kinase family. The human genome contains two
ROCK genes: ROCK1 and ROCK2. The isoforms share about 60% of amino acid sequences and about
9
0% homology in the kinase domain. The amino acids in the ATP binding sites of both isoforms are
identical, making design of selective inhibitors difficult (reviewed in (Feng et al. 2016)).
ROCKs are principal regulators of the actin cytoskeleton. Through changes to actomyosin,
ROCKs regulate cell contractility, cell shape, cell motility, cell invasiveness, and other cell functions.
They also play an important role in the regulation of cellular metabolism, growth, division, and
apoptosis. ROCKs also modulate neurite outgrowth, cell-cell adhesion, and angiogenesis (for a recent
review see Loirand, 2015).
ROCKs are activated by the small GTPase RhoA and phosphorylate various substrates,
including myosin light chain phosphatase (MLCP). MLCP subsequently dephosphorylates the myosin
light chain (MLC), resulting in the contraction of the actin cytoskeleton. ROCKs can also
phosphorylate MLC directly. Other ROCK substrates involved in the regulation of the actin filament
states are LIM kinases 1 and 2, adducin, and ezrin-radixin-moesin (for a review see Micuda et al.
2
010).

Both ROCK isoforms are expressed ubiquitously in mice. However, the levels of expression
of the two isoforms differ in some tissues (Noma et al. 2006). Intracellular distribution of ROCK
isoforms is cell-type dependent and can differ between ROCK1 and ROCK2 (Iizuka et al. 2012).
Knockout mice for each ROCK isoform demonstrated that the isoforms cannot fully compensate for
each other. In some contexts, they may even play opposing roles (Iizuka et al. 2012).
Both ROCK isoforms are mutated in cancers. The constitutively-active ROCK1 was found
in breast and lung carcinomas (Greenman et al. 2007; Lochhead et al. 2010). Polymorphism in ROCK1
was also associated with the development and progression of prostate cancer (Liu et al. 2017). ROCK2
mutations were observed in gastric cancer and melanoma (see review by Wei et al. 2016). Some
mutations are located in the homologous sequences of both isoforms. Increased ROCK expression
and/or activity has been correlated with tumour aggressiveness (Lane et al. 2008; Wong et al. 2009; Liu
et al. 2011).
The actin cytoskeleton functions in a range of processes that contribute to the development
and progression of malignancies: cell adhesion, migration, and invasion; epithelial-mesenchymal
transition; metastasis; neoangiogenesis; and immune cell infiltration (Morgan-Fisher et al. 2013).
Moreover, reorganization of the actin cytoskeleton modulates gene expression, cell cycle progression,
vesicular transport, and extracellular matrix metabolism (Ridley 2006; Huet et al. 2012).
The Rho-ROCK pathway regulates tumour invasion and metastasis. Its association with
distant metastases has been observed in breast (Tang et al. 2008), ovarian (Cheng et al. 2009), gastric
(Matsuoka and Yashiro 2014), ⁠ and liver cancers (Grise et al. 2009). Tumour cells invade either by
amoeboid or mesenchymal movement depending on the extracellular matrix proteolysis. A change in
motility mode is accompanied by cell morphology change – mesenchymal cells are protracted and
amoeboid cells are spherical. The Rho-ROCK pathway regulates the ameboid migration (Panková et al.
2
010). However its role in the mesenchymal movement appears to be limited.
The anti-angiogenic and anti-metastatic activities of ROCK inhibition have been
demonstrated in vitro on numerous occasions (see review by Rodriguez-Hernandez et al. 2016). The
administration of ROCK inhibitors fasudil, Y-27632, Wf-536, RKI-1447, and several other inhibitors
reduced the growth of breast, hepatocellular or lung carcinomas, myeloma, and neuroblastoma in
xenograft experiments (Itoh et al. 1999; Takamura et al. 2001; Nakajima et al. 2003; Ying et al. 2006;
Patel et al. 2012; Dyberg et al. 2017). Invasiveness and dissemination were reduced by these inhibitors
in melanoma, fibrosarcoma, and carcinomas of breast, kidney, liver, and prostate (review by Feng et al.
2
016).

In vitro and in vivo experiments suggest that ROCK inhibitors may be useful for treating a
variety of other pathological conditions, including glaucoma, hypertension, asthma, erectile
dysfunction, neurodegeneration, kidney failure, and osteoporosis. Since identification of their
therapeutic potential, ROCK inhibitors with a wide variety of scaffolds have been reported (Feng and
LoGrasso 2014; Wang et al. 2014; Defert and Boland 2017). Until now, only a few examples of
ROCK2-selective compounds have been described (WO2015054317; Shah and Savjani 2016).
Many ROCK inhibitors also show only limited selectivity toward other kinases, especially
other members of the AGC family. This is also true for the isoquinoline fasudil and the pyridine
carboxamide Y-27632, the two "classical" ROCK inhibitors most widely used in in vitro and animal
studies. Fasudil and related ripasudil have been approved in Japan for treating cerebral vasospasm
during subarachnoid haemorrhage and glaucoma, respectively. Netarsudil, another isoquinoline
derivative was approved for glaucoma treatment by FDA in 2017.
In this study we explored the relationship between the structure of purine derivatives substituted
at the C6 atom by the piperidin-1-yl group or the azepan-1-yl group and ROCK2 inhibitory activity.
We started with parent compounds (1, 22) that have been identified as micromolar ROCK2 inhibitors
in a high-throughput screening campaign (Pubchem AID:644). The screening set included also several
less active derivatives with a substitution on the piperidine ring (25, 26, 29, 31). Yet, to our knowledge,
no follow-up studies (structure optimization, cell-based experiments) of these compounds have been
conducted.
In the C6-azepanyl series, we evaluated the effects of substitutions in the C2, N3, N7, C8, and
N9 positions of the purine moiety on anti-ROCK2 activity. In the series of C6-piperidinyl derivatives,
substitutions at position 4 of the piperidine ring were studied. The piperidinyl derivatives included
various spirocycles resulting from the reactions of the 4-oxopiperidine moiety with small diols and
dithiols. We also explored anti-ROCK2 activity of several C6-pyrrolidin-1-yl derivatives with the
substitutions that were promissing in the other series.
Furthermore, we studied the effects of new inhibitors on phosphorylation of downstream
effector MLC2, as well as their influence on cellular morphology and invasiveness in a 3D collagen
matrix using human melanoma (A2058 and A375m2) and fibrosarcoma (HT1080) cell lines as
representatives of amoeboid or mesenchymal phenotypes, respectively.
2
2
. Results
.1 Synthesis

The straightforward syntheses of 6-monosubstituted and 2,6-disubstituted purines (Figure 1, Route A)
were carried out by the conventional method (see Allen et al. 2013). Commercially available 6-
chloropurine or 2,6-dichloropurine were heated with azepane, pyrrolidine, piperidine, or piperidine
derivatives preferably in n-propanol or 2-propanol in the presence of triethylamine. Well-known
differences in the reactivity of 6-chloropurine, 2,6-dichloropurine, and 2-chloro-6-substituted purines
towards nucleophilic substitution (Montgomery and Holum 1957; Breshears et al. 1959)⁠ were
reflected in the times and temperatures needed for completion of reactions. Whereas 2,6-dichloropurine
converted into 6-substituted product within 1.5 - 2 h at 65 °C (2, 3, 27 and 33), 6-chloropurine
underwent substitution over 20 h at 90 °C (1, 22, 25, 26, 29-31). The subsequent substitution of the
chlorine atom in 2-chloro-6-substituted purines requires harsher conditions. Thus, for the synthesis of
compounds 4-9, 32, and 38, the solvent of choice was the used amine itself (in the case of
dimethylamine it was its 50% solution in methanol). The reaction with secondary amines was usually
completed by heating overnight at 90 °C, while reactions with primary amines required heating at 160
°
C for durations ranging from 4 h to overnight.
The 8-substituted purines of the azepanyl series were prepared by four-step synthesis (Figure 1,
Route B). We selected 6-chloro-9-(tetrahydropyran-2-yl)purine as a starting material due to the ability
of the THP group to enhance the reactivity of the chlorine in position 6 in the first step (16, 23, 28, 34)
as well as the reactivity of the halogen in position 8 in the third step of the synthesis. The THP group
also serves as a protecting group in the second step of the procedure. Finally, it can be easily removed
by acidic hydrolysis to produce free purine base in the last step of the synthesis (10-14). A halogen
atom or methyl group was introduced into position 8 of 6-(azepan-1-yl)-9-(tetrahydropyran-2-yl)purine
by lithiation and subsequent addition of a halogen donor or iodomethane (1. LDA in THF, -70 °C,
argon atmosphere; 2. e.g. hexachloroethane, 1 h; 18-20; Nolsøe et al., 1998). The halogen atom in
position 8 was subsequently substituted with benzyl alcoholate and methanolate to produce the 8-
hydroxy derivative 21 and the 8-methoxy derivative 14 (after acidic hydrolysis of the THP group),
respectively.
The presence of the keto group in 26 inspired us to synthesize derivatives with acetal-like
spirocycles (35, 36, 39-41; Figure 1, Route C). We employed conditions generally recommended for
protection of carbonyl groups (Kocieński 2005). The reactions were carried out in toluene and
catalysed by p-toluenesufonic acid. Dehydration was accomplished by continuous azeotropic
distillation. Acceptable product yields (60%–83%; apart from 40 with a yield of 24%) were obtained
despite the low solubility of the starting material in toluene.

2
.2 Kinase assay with recombinant ROCK2 and analysis of structure-activity relationships
(SARs)
Inhibition of ROCK2 was evaluated using a biochemical kinase test based on the measurement of the
incorporation of radioactive phosphate from ATP into the oligopeptide substrate that is a part of the
ROCK target sequence. The IC values of the parent compounds 6-(azepan-1-yl)purine (1) and 6-
5
0
(piperidin-1-yl)purine (22) were 5.7 µM and 7.7 µM, respectively.
In the 6-azepanyl series (Table 1), any substitution in positions N9, N7, N3 and C8,
including a methyl group (15, 43, 42, 12) led to a loss of the ROCK2 kinase activity (IC > 100 µM).
5
0
Other explored 8-substituents included hydroxy, methoxy, bromo and chloro functional groups (13, 14,
1, 10). The group of inactive derivatives also included those with larger N9-substituents such as
1
tetrahydropyran-2-yl (16) or β-D-ribofuranosyl (17). A similar loss of inhibitory activity against
ROCK2 after N9-substitution of purine was also observed in the 6-piperidinyl series (23, 24). The
inactivity of the ribosides suggests that the mode of binding of the ROCK inhibitors we investigated
differs from that of ATP. No inhibitory activity was observed among the 8,9-disubstituted purine
derivatives (18-21).
On the other hand, several active derivatives were obtained after substitution of the purine
C2 atom with hydroxyalkylamino groups. While an improvement in potency was observed for the 3-
hydroxypropylamino derivative (6, IC50 = 2.9 µM), compounds substituted with the 2-
hydroxyethylamino (5, IC = 4.5 µM) and (R/S)-1-hydroxymethylpropylamino (7, IC = 5.3 µM)
5
0
50
groups demonstrated activity that was comparable to that of the parent compound. The 2-
aminoethylamino derivative (4) was about three times less potent than the parent compound, and the
C2 substitutions by chloro (3), fluoro (2), dimethylamino (8), and morpholin-4-yl (9) groups abolished
the activity completely.
In the C6-piperidinyl series (Table 1 continued), the C4 substitution of piperidine with an
aminocarbonyl group yielded the derivative (29) with activity (IC = 7.9 µM) comparable to that of the
5
0
parent compound. The 4-hydroxyl (25), 4-methoxycarbonyl (30) and 4-oxo (26) derivatives were less
active, with IC values of 11.0 µM, 12.2 µM, and 15.6 µM, respectively. The reaction of the 4-oxo
5
0
group with diols was used to synthesise ketals. The most active derivative was the one with the 7,12-
dioxa-3-azaspiro[5.6]dodecane moiety at the C6 purine atom (41, IC50 6.8 µM). Its activity was
comparable to that of the unsubstituted 6-piperidin-1-yl-purine (22). The compounds prepared by
reactions with shorter diols, like 31 (8.5 µM) and 39 (12.5 µM), and those including a glycerol
derivative (40, 9.9 µM) were less active, but were more potent than 6-(4-oxopiperidin-1-yl)purine (26).
We also prepared analogues of 6-(1,4-dioxa-8-azaspiro[4.5]decan-8-yl)purine (31) where one oxygen

atom (35, IC50 = 4.7 µM) or both oxygen atoms (36, IC50 = 2.6 µM) were replaced by sulphur.
Although 1,3-dithiolane and 1,3-oxathiolane moieties are unusual in drug design, examples of their
successful use do exist – the ACE inhibitor spirapryl and anti-muscarinic agent cevimeline serve as
examples. Like the 8-substituted compounds from the azepanyl series, the spirocycles were prepared by
acid hydrolysis of the respective 9-tetrahydropyran-2-yl derivatives. None of those precursors
demonstrated inhibitory activity in a ROCK2 assay.
Based on the SAR knowledge originating from the 6-azepanyl series, spirocyclic derivatives
3
6 and 31, with a 2-hydroxyethylamino group at the purine C2 atom, were prepared by nucleophilic
substitution of the respective 2-chloro derivatives (33, 37; both IC > 100 µM) yielding compounds 38
5
0
(
IC 1.5µM) and 32 (IC 18.9 µM).
50 50
Finally, we also explored the effect of the substitutions of 6-(pyrrolidin-1-yl)purine (44,
IC50 = 10.1 µM) at the purine C2 atom with selected hydroxyalkylamino sidechains that improved
activity of 6-(azepan-1-yl)purine. However, both derivatives (46, 47 IC50>25 µM) were significantly
6
less active than the parent compound. This observation suggests that a bulkier N -substituent is
required for an efficient interaction with the binding site or ROCK2.
Overall our SAR study demonstrates that the parent ROCK inhibitors 6-(azepan-1-yl)purine
(1) and 6-(piperidin-1-yl)purine (22) can be modified by a substitution at the purine C2 atom and by an
expansion of the sidechain at the purine C6 atom. On the other hand, all the substitutions on the
imidazole ring or N3 nitrogen of the purine moiety led to loss of inhibitory activity. Although some
derivatives have improved IC values compared to the parent compounds, none was more potent than
5
0
the classical ROCK inhibitors fasudil (IC = 1.1 µM) and Y-27632 (IC = 0.8 µM).
5
0
50
2
.3. Evaluation of kinase selectivity of the selected compounds
A single concentration (50 µM) of the two most active disubstituted derivatives 6 and 38 were tested on
a commercial panel (DiscoverX, USA) of 96 kinases (Supplemental Table S1). The assay uses a DNA-
labelled kinase that is captured on a solid matrix derivatized with its substrate. When the test compound
binds to the matrix, kinases are released into the solution and the kinase concentrations are quantified
by PCR. Compound 6 released ≥99% of the bound ROCK2, JAK3, JNK1-3 (MAPK 8-10), RSK2, and
SNARK. The list of kinases that are highly sensitive to the more hydrophobic 38 includes these same
kinases, except for JNK2 and RSK1, as well as 9 additional kinases (ARK5, AXL, CSF1R, DYRK1B,
ERK8, JAK2, MKNK, TYK2, and ULK2). Lower selectivity is also apparent for other cut-off levels,
e.g. at 90%. Subsequently K values for 38 and ROCK2 (0.5 µM), ROCK1 (1.3 µM), JAK1 (>25 µM),
d
JAK2 (2.2 µM), and JAK3 (1.4 µM) were determined using the same assay. Kinase selection was

based on Discoverx ScanEdge panel, but some kinases were replaced to enrich the panel for known off-
target kinases of other ROCK inhibitors including fasudil, Y-27632, H-7/8, H-89, and H-1152 (Golen,
2
010). Interestingly, some of the kinases inhibited by all or most of those ROCK inhibitors were
insensitive toward our compounds. For example, the residual activities for MKNK1 were 72% and 45%
for 6 and 38, respectively. PKAC-α (100% and 87%) and PKAC-β (98% and 62%) were even less
sensitive.
2
.4 Effect of the compounds on cell proliferation
A resazurin assay (3-day incubation) was used to evaluate the effect of the compounds on cell
proliferation. IC values were calculated from dose-response curves. For human non-malignant cells
5
0
(
skin fibroblasts BJ, keratinocytes HaCaT, and retinal epithelial cells ARPE-19), IC values were
50
always higher than 50 µM and no effect on proliferation was observed at 12.5 µM (data not shown).
Inhibitor compounds 1, 5, 38, and positive controls fasudil and Y-27632 were also tested on HT1080,
A375m2, and A2058 melanoma cell lines. The IC values for 1 were 70.5 µM, 37.1 µM, and 26.8 µM
5
0
respectively. All IC values for the other two compounds tested were above 100 µM, the highest
5
0
concentration tested. For compounds 5 and 38, no effects on proliferation were observed after
application of 10 µM, the concentration used in the follow-up cell-based experiments. For 1, the
resazurin signal decrease was lower than 5%. Comparable small negative effects on cell proliferation
were observed for both control compounds fasudil and Y-27632.
2
.5 The effect of new ROCK inhibitors on downstream signalling
One downstream effect of ROCK on the actin cytoskeleton and traction forces of cells occurs via the
phosphorylation of the myosin light chain (MLC; Jay et al. 1995; Lauffenburger and Horwitz 1996).
Therefore, we evaluated MLC2 phosphorylation after a 24-h treatment with ROCK inhibitors using
Western blot. The intensities of MLC2 and pMLC2 (T18/S19) signals were measured by densitometry.
Inhibitors 1, 5, and 38 at concentrations of 10 µM significantly decreased MLC2 phosphorylation in
A375m2, A2058, and HT1080 cells (Figures 2 and S1). Their effects were comparable to equimolar
fasudil but, in the melanoma cells, their activity was markedly lower than that of Y-27632. These
results show that the new purine inhibitors can reach relevant concentrations in the cells.
2
.6 Treatments with new purine ROCK inhibitors alters cellular morphology in 3D collagen and
reduces invasiveness of human melanoma cells

Classical ROCK inhibitors like Y-27632 are proven inhibitors of amoeboid movement of melanoma
cells, and they change the morphology of these cells to a longer mesenchymal-type morphology
(Routhier et al. 2010; Sadok et al. 2015). Therefore, we tested newly-prepared ROCK inhibitors at 10
µM concentrations for 3D morphology assays using A375m2 and A2058 melanoma cells. The HT1080
fibrosarcoma cell line served as a control. Equimolar concentrations of commercially available
inhibitors fasudil and Y-27632 were compared. We observed that amoeboid melanoma cells mainly
changed their morphology to the mesenchymal type upon treatment with either inhibitor (Figure 3).
Treatment of A375m2 cells with the prepared inhibitors 1, 5, and 38 increased the proportion of
mesenchymal cells from less than 10% to 69.6%–77.7%. The effects of fasudil and Y-27632 were
slightly weaker (65.5% and 55.1%, respectively). In A2058 cells, the changes from the amoeboid to the
mesenchymal phenotype after ROCK inhibition were also significant. While 93.9% of the vehicle-
treated cells maintained their ameboid morphology, the treatment with the prepared inhibitors increased
proportion of mesenchymal cells to 48.2-66.0%. For fasudil and Y-27632, the proportions of
mesenchymal cells were 53.3% and 78.0%, respectively. As expected, the inhibitors had no effect on
the morphology of the HT1080 cell line. These cells remained mainly mesenchymal (91%-95%).
Since cell morphology is associated with invasion mode, we also tested the effects of both
prepared and commercially available ROCK inhibitors (at 10 µM concentration) on the invasiveness of
the cell lines. Treatment with either of the compounds significantly inhibited the invasiveness of
melanoma cells into a rat collagen matrix. No significant effects were observed for the mesenchymal
HT1080 cell line (Figure 4).
3
. Discussion
The Rho-associated serine/threonine kinases (ROCKs) are the principal regulators of the actin
cytoskeleton. Through cytoskeleton changes, ROCKs regulate the growth, division, contractility, shape,
motility, and death of cells. Pharmacological ROCK inhibitors may therefore be useful for treating a
host of diseases where these cell processes are dysregulated including cancers.
Here we explored the ROCK2-inhibitory activity of a group of purine derivatives
substituted at the C6 atom by the pyrrolidin-1-yl, piperidin-1-yl or azepan-1-yl group. A set of 47
compounds was prepared for this study, and the syntheses of 39 of these derivatives are described for
the first time. Our SAR study suggests that ROCK2-inhibitory activity in piperidin-1-yl or azepan-1-yl
series is retained and may even increase with substitution at the C2 purine atom on the parent
compounds or with expansion of the side-chain at the C6 purine atom.

Kinase profiling of selected derivatives suggested that the 2,6-disubstituted purines we
investigated may not possess selectivity for ROCK isoforms. However, some off-target kinases known
to be inhibited by other ROCK inhibitors were insensitive to these select derivatives. These newly-
developed purine derivatives may also serve as leads for the synthesis of inhibitors of JAK or JNK
kinases. Development of dual-inhibitors of ROCK/JAK and/or ROCK/JNK may be also an interesting
prospect. For example, Sanz-Moreno et al. (2011) showed that ROCK and JAK1 signalling cooperate
to control actomyosin contractility in tumour and stroma cells. Such dual inhibitory activity might be
beneficial for treating illnesses, like fibrosis and inflammation, in which some of the processes
regulated by ROCK, JNK and JAK1 contribute to the pathology. ROCK itself is known to regulate
these processes (for a recent review, see (Loirand 2015)). A recent report suggested that combined
ROCK/JAK/PDGFR-β inhibitors are active in models of choroidal neovascularization.
Since they regulate cytoskeletal functions, ROCKs play an important role in contractility,
stress fibre formation, focal adhesion complex assembly, and in the cell migration and invasion that
occurs during cancer metastasis (Raftopoulou and Hall 2004; Kosla et al. 2013). The ROCK pathway is
involved in both the main types of cancer cell migration: amoeboid and mesenchymal (Sahai and
Marshall 2003); however, its role in amoeboid migration is critical. First-generation ROCK inhibitors
like the commercially-available Y-27632 have demonstrated inhibition of the blebbing and highly
contractile amoeboid movement and conversion of amoeboid melanoma cells into the more-elongated
mesenchymal phenotype (Sahai and Marshall, 2003). We observed similar effects in melanoma cell
lines A375M2 and A2058 treated with either “classical” ROCK inhibitors or selected purine
derivatives using the collagen matrix 3D cultures. Conversely, HT1080 fibrosarcoma cells exhibiting
mesenchymal migration mode maintained their elongated morphology after the application of the
inhibitors.
Since ROCK signalling is also involved in the invasiveness of melanoma cells (Gadea et al.
2
008; Sanz-Moreno et al. 2008; Routhier et al. 2010; Sanz-Moreno et al. 2011; Pankova et al. 2012;
Sadok et al. 2015), the selected derivatives were tested in a 3D collagen invasion assay. As expected,
considering the previous study by Routhier et al. (2010), Y-27632 inhibited the invasiveness of both
amoeboid melanoma cell lines. Fasudil and the purine derivatives significantly suppressed invasion
into the collagen as well. In contrast, the compounds were unable to suppress migration of
mesenchymal HT1080 fibrosarcoma cells. Overall the observed effects of the compounds on the
morphology and invasiveness of the amoeboid melanoma cell lines, and not mesenchymal
fibroblastoma cell lines, agree with the presumed ROCK inhibition.

ROCKs modulate the actin cytoskeleton through phosphorylation of MLC, cofilin, and LIM
kinases (Aizawa et al. 2001; Ueda et al. 2002). We studied the phosphorylation levels of MLC2 after
treatment with the prepared compounds in all three cell lines to further confirm that the phenotypes
observed resulted from ROCK inhibition. A significant decrease of pMLC was observed, as expected.
We synthesized a set of C6-substituted purines with ROCK-inhibitory activity for this SAR study. The
study suggests that the active compounds can be obtained by select substitutions on the pyrimidine
ring, while changes on the imidazole ring of the purine moiety are not tolerated. The inhibitors can
reach effective concentrations within cells, as demonstrated either by decreased phosphorylation of the
ROCK downstream target MLC, or by inhibition of the ROCK-dependent invasion of melanoma cells
into the collagen matrix. These effects were observed at concentrations having no or marginal effects
on cell proliferation. Our study may help researchers interested in further development of purines, and
possibly their bioisosteres as well, into more potent ROCK inhibitors. The findings of our study could
also inspire development of inhibitors of other kinases identified as sensitive by the kinase panel
screening, like members of the JAK and JNK families.
4
. Materials and methods
4
.1. Chemistry
The chemicals, solvents for synthesis, and spectral solvents were purchased in analytical or HPLC
grade quality from available suppliers and used without further purification. Microwave synthesis was
performed in microwave reactor Discover SP (CEM Corporation) in 10 mL closed vessels using
following set up: temperature 120 °C, power 150 W, ramp time 2 min, hold time 10 min, powermax on.
Thin layer chromatography (TLC) was carried out using silica gel 60 F254 plates (Merck Co.).
CHCl :MeOH:NH OH 95:5:0.5, 9:1:0.1, 8:2:0.2 or 7:3:0.3; CHCl :acetone 4:1 or 3:2 were used as
3
4
3
mobile phases. Crude products were purified either by column chromatography using Silikagel 40-63
(
VWR) or by middle pressure column chromatography (MP CC) using silica gel PharmPrep 60 CC
40-63 µm; Merck) as the stationary phase in a glass column (Kronlab, 15 mm diameter column or
(
Pharmacia Fine Chemicals, 25 mm diameter column). As the mobile phase for MP CC, hexanes:EtOAc
:2 or CHCl :MeOH:NH OH 95:5 0.5, 9:1:0.1 or 75:25:2.5 were applied. A flow rate of the mobile
3
3
4
phase through the system was 7.5 mL/min. The purity of prepared compounds was confirmed by
HPLC. Samples for HPLC (carried on a Beckman Gold system) were dissolved in MeOH, applied to a

LiChroCARD 250 x 4 mm column filled with Purospher RP-18e, 5 µm (Merck) and the separated
constituents were eluted by isocratic elution using a mixture of MeOH (HPLC grade) and
AcOH/AcONH buffer (pH 3.4; 40 mM; with addition of 5% MeOH) at a flow rate of 0.5 mL/min. The
4
mixture contained 50%, 60%, 70% or 80% MeOH. Eluting compounds were detected by scanning the
UV absorbance of the eluate between 200 and 300 nm. Elemental analyses were performed using a
CHN-O analyser, Thermo Finnigan Flash, EA 1112 series; the values (C, H, N) agreed with the
calculated ones within acceptable limits. Mass spectra were measured on a MS Waters/Micromass,
1
ZMD-detector, direct inlet, ESI coin voltage 20 V. H NMR spectra were recorded on a Bruker Avance
1
13
3
5
00 spectrometer operating at 300 K and frequency of 300 MHz ( H), 75 MHz ( C) or a JEOL ECA-
1
13
00 spectrometer operating at 298 K and frequency of 500 MHz ( H) and 125 MHz ( C). Samples
were dissolved in DMSO-d , CDCl or CD OD. The NMR spectra were calibrated on residual solvent
6
3
3
1
13
1
13
1
peak: DMSO-d ( H 2.49 ppm, C 39.52 ppm), CDCl ( H 7.26 ppm, C 77.0 ppm), CD OD ( H 3.31
6
3
3
1
3
ppm, C 49.0 ppm). The note (cv) in the text means that the signal was partly covered by another
signal.
4
.1.1. 6-(Azepan-1-yl)purine (1)
6
-Chloropurine (2 g, 12.9 mmol) was refluxed with azepane (14.8 mL, 131.1 mmol) at 80–90 °C for 3
h. The reaction was monitored via TLC (CHCl :MeOH 4:1), concentrated in vacuo. The residue was
3
dissolved in EtOAc and mixed with distilled H O. The layers were separated. The EtOAc layer was
2
dried over Na SO , filtered, and evaporated in vacuo. Crude product was air-dried. White solid. Yield:
2
4
2
.56 g (91%). M.p.: 285-290 °C dec. HPLC R = 10.3 min (70% MeOH + 30% buffer); UV (80%
t
MeOH + 20% buffer) λmin 235 nm, λmax 279 nm; HPLC purity >99%. MS ESI+ (CV 17) m/z (rel. %):
+
+
+ 1
2
18.4 (100) [M + H] , 240 (27) [M + Na] , 435 (47) [2M + H] . H NMR (500 MHz, DMSO-d ) δ
6
(ppm): 1.48 (bs, 4H, azep), 1.76 (bs, 4H, azep), 3.82 (bs, 2H, azep), 4.33 (bs, 2H, azep), 8.06 (s, 1H,
1
3
pur H8), 8.15 (s, 1H, pur H2), 12.90 (bs, 1H, pur NH). C NMR (125 MHz, DMSO-d ) δ (ppm): 26.3,
6
2
6.7, 28.7, 47.5, 49.0, 118.4, 137.9, 151.1, 151.9, 153.5.
4
.1.2. 6-(Azepan-1-yl)-2-fluoropurine (2)
6
-Chloro-2-fluoro-9H-purine (172 mg, 1 mmol), azepane (113 μL, 1 mmol), and triethylamine (420
μL, 3 mmol) were refluxed in PrOH at 95-98 °C while stirring for 4 hours. The reaction mixture was
then cooled down to room temperature and evaporated to give a yellow solid residue which was treated
with water (20 mL). The precipitated yellow solid was filtered off, washed with water, and dried. The
crude product was crystallized from methanol (10 mL). White powder. Yield: 80 mg (34%). HPLC
+
1
purity >99%. ESI+ (CV 20) m/z (rel. %): 236 [M+H] (100). H NMR (500 MHz, DMSO-d ) δ (ppm):
6

1
1
1
.50 (bs, 4H, azep), 1.77 (bs, 4H, azep), 3.76 (bs, 2H, azep), 4.35 (bs, 2H, azep), 8.06 (s, 1H, pur H8),
1
3
3.06 (bs, 1H, pur NH). C NMR (125 MHz, DMSO-d ) δ (ppm): 26.1, 26.2, 26.4, 28.5, 47.9, 49.0,
6
16.8, 138.5, 152.5 (d, J = 20.5 Hz), 154.6 (d, J = 20.5 Hz), 158.2 (d, J = 201.0 Hz). Anal.: Calcd. for
C H FN , M = 235.27: C 56.16; H 6.00; N 29.77 %. Found: C 56.33; H 5.98; N 29.38%.
1
1
14
5
r
4
.1.3. 6-(Azepan-1-yl)-2-chloropurine (3)
2
,6-Dichloropurine (2 g, 10.6 mmol) was refluxed with azepane (1.4 mL, 11.6 mmol) and Et N (5.9
3
mL, 42.3 mmol) in PrOH (40 mL) at 100 °C for 4 h. The reaction mixture was then cooled to room
temperature and a solid was isolated. The solid was filtered off, washed with cold PrOH and cold H O,
2
and dried at 50 °C. White powder. Yield: 2.1 g (78%). M.p.: 248-249 °C. HPLC R = 15.4 min (80%
t
MeOH + 20% buffer); UV (80% MeOH + 20% buffer) λmin 239 nm, λmax 280 nm; HPLC purity 100%.
+
+ 1
MS ESI+ (CV 30) m/z (rel. %): 252 (100) [M + H] , 254 (33) [M + H] . H NMR (300 MHz, DMSO-
d6) δ (ppm): 1.45 (bs, 4H, azep), 1.73 (bs, 4H, azep), 3.73 (bs, 2H, azep), 4.29 (bs, 2H, azep), 8.06 (s,
1
H, pur H8), 13.07 (bs, 1H, pur NH).
4
.1.4. 2-(2-Aminoethylamino)-6-(azepan-1-yl)purine (4)
6
-(Azepan-1-yl)-2-chloropurine (3; 0.5 g, 2.0 mmol) was placed into a pressure tube with
ethylenediamine (5.0 mL, 74.3 mmol) at 165 °C for 3 h. The mixture was then cooled to room
temperature and concentrated in vacuo. The arisen precipitate was dissolved in cold H O and
2
1
recrystallized. White powder. Yield: 0.5 g (96%). M.p.: 177–179 °C. H NMR (500 MHz, DMSO-d ) δ
6
(
ppm): 1.47 (bs, 4H, azep), 1.73 (bs, 4H, azep), 2.66 (t, J = 6.4 Hz, 2H, H N-CH -), 3.20 (q, J = 6.2 Hz,
2 2
2
H, -CH -NH-), 3.75 (bs, 2H, azep), 4.24 (bs, 2H, azep), 6.10 (bs, 1H, -(CH ) -NH-), 7.62 (s, 1H, pur
2 2 2
1
3
H8). C NMR (125 MHz, DMSO-d ) δ (ppm): 26.3, 27.0, 29.0, 40.4, 41.3, 41.5, 44.3, 47.2, 48.6,
6
1
12.9, 134.5, 153.4, 153.6, 157.9, 158.8, 159.0.
4
.1.5. 6-(Azepan-1-yl)-2-(2-hydroxyethylamino)purine (5)
6
-(Azepan-1-yl)-2-chloropurine (3; 0.5 g, 2.0 mmol) reacted with ethanolamine (4.5 mL, 74.3 mmol) in
a pressure tube at 165 °C for 3 h. Reaction mixture was cooled to room temperature which produced
the precipitate. The precipitate was isolated and recrystallized in H O. Yield: 326.4 mg (56%). M.p.:
2
1
2
6
4
12 °C. H NMR (500 MHz, DMSO-d ) δ (ppm): 1.46 (bs, 4H, azep), 1.73 (bs, 4H, azep), 3.29 (q, J =
6
.2 Hz, 2H, -NH-CH -), 3.49 (t, J = 6.2 Hz, 2H, -CH -OH), 3.74 (bs, 2H, azep), 4.24 (bs, 2H, azep),
2
2
.66 (bs, 1H, -OH), 5.99 (t, J = 5.7 Hz, 1H, -CH -NH-), 7.63 (s, 1H, pur H8), 12.14 (bs, 1H, pur NH).
2
1
3
C NMR (125 MHz, DMSO-d ) δ (ppm): 26.4, 27.0, 29.1, 44.0, 47.4, 48.7, 60.5, 113.0, 134.6, 153.3,
6
1
53.7, 158.9.

4
.1.6. 6-(Azepan-1-yl)-2-(3-hydroxypropylamino)purine (6)
6
-(Azepan-1-yl)-2-chloropurine (3; 217 mg, 0.86 mmol) was dissolved in 3-aminopropan-1-ol (2 mL).
The solution was heated at 115 °C for 26 h and at 160 °C for 4 h. After cooling, the mixture was
refrigerated for 2 weeks. The product crystallized from the mixture in lumps. Crystals (first portion)
were filtered off, crushed on sintered glass, and washed thoroughly with H O (15 mL). The second
2
portion of product was obtained from the filtrate which was diluted with H O while washing the
2
crystals and then refrigerated for 1 d. The overall yield after drying both portions of product in a
desiccator with P O was 225 mg (90%). The raw product was crystallized from EtOH (20 mL). White
2
5
wadding substance. Yield: 153 mg (61%). M.p.: 226-230 °C (˃200 °C subl.). HPLC R = 5.1 min (75%
t
MeOH + 25% buffer), R = 6.4 min (60% MeOH + 40% buffer); UV (75% MeOH + 25% buffer) λmax
t
2
39 nm, λmin 251 nm, λmax 259 nm, λmin 275 nm, λmax 290 nm, HPLC purity >99%. MS ESI+ (CV 18)
+
-
-
1
m/z (rel. %): 291 [M+H] (100); ESI (CV 22) m/z (rel. %): 289 [M] (100). H NMR (500 MHz,
DMSO-d ) δ (ppm): 1.47 (bs, 4H, azep H4´, 5´), 1.65 (pent, J = 6.4 Hz, 2H, -CH -CH -CH -), 1.73 (bs,
6
2
2
2
4
2
1
2
H, azep H3´, 6´), 3.25 (q, J = 6.5 Hz, 2H, -HN-CH -), 3.44 (t, J = 5.7 Hz, 2H, HO-CH -), 3.75 (bs,
2 2
2
H, azep H2´), 4.24 (bs, 2H, azep H7´), 4.48 (bs, 1H, -OH), 6.07 (t, J = 5.8 Hz, 1H, -N -H), 7.61 (s,
1
3
H, pur H8), 12.14 (bs, 1H, pur NH). C NMR (125 MHz, DMSO-d ) δ (ppm): 26.3 (azep C4´, 5´),
6
6.9 (azep C3´), 29.0 (azep C6´), 32.7 (-CH -CH -CH -), 38.3 (-HN-CH -), 47.3 (azep C2´), 48.7 (azep
2
2
2
2
C7´), 58.9 (HO-CH -), 112.8 (pur C5), 134.4 (pur C8), 153.3 (pur C4), 153.6 (pur C6), 159.0 (pur C2).
2
4
.1.7. 6-(Azepan-1-yl)-2-(1-hydroxymethylprop-1-ylamino)purine (7)
6
-(Azepan-1-yl)-2-chloropurine (3; 216 mg, 0.86 mmol) was dissolved in 2-aminobutan-1-ol (2 mL).
The solution was heated at 115 °C for 20 h and at 160 °C for 19 h. After cooling, the product was
precipitated from the reaction mixture with H O (40 mL). The suspension was refrigerated overnight.
2
The product crystallized from the mixture as a plaster-like substance. The milk-like precipitate was
filtered off, washed thoroughly with H O (20 mL), and dried in a desiccator with P O . White plaster-
2
2
5
like substance. Yield: 251 mg (96%). M.p.: 185-188 °C. HPLC R = 7.8 min (65% MeOH + 35%
t
buffer); UV (65% MeOH + 35% buffer) λmax 240 nm, λmin 250 nm, λmax 261 nm, λmin 276 nm, λmax 290
+
-
nm; HPLC purity 98%. MS ESI+ (CV 18) m/z (rel. %): 305 [M+H] (100); ESI (CV 23) m/z (rel. %):
-
1
3
03 [M] (100). H NMR (500 MHz, DMSO-d ) δ (ppm): 0.86 (t, J = 7.3 Hz, 3H, -CH ), 1.43 (quint
6 3
(
cv), J = 7.3 Hz, 1H, CH -CH -), 1.46 (bs (cv), 4H, azep H4´, 5´), 1.57 (quint, J = 6.8 Hz, 1H, CH -
3
2
3
CH -), 1.73 (bs, 4H, azep H3´, 6´), 3.36 (bs, 1H, HO-CH - overlap with H O), 3.44 – 3.47 (m, 1H, HO-
2
2
2
CH -), 3.74 (bs (cv), 3H, -HN-CH< and azep H2´), 4.24 (bs, 2H, azep H7´), 4.57 (bs, 1H, HO-), 5.63
2
2
13
(
d, J = 8.5 Hz, 1H, -N -H), 7.61 (s, 1H, pur H8), 12.04 (bs, 1H, pur NH). C NMR (125 MHz, DMSO-

d ) δ (ppm): 10.7 (-CH ), 23.9 (CH -CH -), 26.4 (azep C4´, 5´), 26.9 (azep C3´), 29.0 (azep C6´), 47.2
6
3
3
2
(
azep C2´), 48.6 (azep C7´), 54.0 (-HN-CH<), 63.1 (HO-CH -), 112.9 (pur C5), 134.5 (pur C8), 153.3
2
(pur C4), 153.6 (pur C6), 158.8 (pur C2).
4
.1.8. 6-(Azepan-1-yl)-2-dimethylaminopurine (8)
6
-(Azepan-1-yl)-2-chloropurine (3; 210 mg, 0.83 mmol) was dissolved in 50% solution of
dimethylamine and MeOH (3 mL). The solution was heated at 60 °C for 16 h and at 115 °C for 4 h.
After cooling, the mixture was refrigerated for 3 h. The product crystallized from the mixture as
needles. Crystals were filtered off, washed thoroughly with MeOH (13 mL) and H O (30 mL), and
2
dried in a desiccator with P O for 2 d. White crystals. Yield: 113 mg (52%). M.p.: 237-238 °C (˃200
2
5
°
C subl.). HPLC R = 11.2 min (80% MeOH + 20% buffer); UV (80% MeOH + 20% buffer) λ 245
t max
nm, λshoulder 257-264 nm, λmin 277 nm, λmax 291 nm; HPLC purity >99%. MS ESI+ (CV 17) m/z (rel.
+
-
-
1
%
): 261 [M+H] (100), ESI (CV 23) m/z (rel. %): 259 [M] (100). H NMR (500 MHz, DMSO-d ) δ
6
(
ppm): 1.46 (bs, 4H, azep H4´, 5´), 1.75 (bs, 4H, azep H3´, 6´), 3.03 (s, 6H, -N-(CH ) ), 3.76 (bs, 2H,
3 2
1
3
azep H2´), 4.26 (bs, 2H, azep H7´), 7.65 (s, 1H, pur H8), 12.21 (bs, 1H, pur NH). C NMR (125 MHz,
DMSO-d ) δ (ppm): 26.2 (azep C4´, 5´), 26.4 (azep C3´), 29.2 (azep C6´), 37.0 (-N-(CH ) ), 47.5 (azep
6
3 2
C2´), 48.8 (azep C7´), 112.2 (pur C5), 134.7 (pur C8), 153.1 (pur C6), 153.5 (pur C4), 158.9 (pur C2).
4
.1.9. 6-(Azepan-1-yl)-2-(morpholin-4-yl)purine (9)
6
-(Azepan-1-yl)-2-chloropurine (3; 209 mg, 0.83 mmol) was dissolved in morpholine (2 mL). The
solution was heated at 60 °C for 14 h and at 115 °C for 5 h. After cooling, the mixture was refrigerated
overnight. The product crystallized from the mixture as a plaster-like substance. The product was
filtered off, washed thoroughly with MeOH (10 mL) and dried in a desiccator with P O . White plaster-
2
5
like substance. Yield: 221 mg (88%). M.p.: 242-244 °C (˃200 °C subl.). HPLC R = 13.3 min (75%
t
MeOH + 25% buffer), R = 10.6 min (80% MeOH + 20% buffer); UV (80% MeOH + 20% buffer) λmax
t
2
45 nm, λmin 275 nm, λmax 290 nm; HPLC purity >97%. MS ESI+ (CV 18) m/z (rel. %): 303 [M+H]+
-
-
1
(
100); ESI (CV 23) m/z (rel. %): 301 [M] (100). H NMR (500 MHz, DMSO-d ) δ (ppm): 1.47 (bs,
6
4
6
H, azep H4´, 5´), 1.75 (bs, 4H, azep H3´, 6´), 3.57 (bs, 4H, morph H3´, 5´), 3.62 (bs, 4H, morph H2´,
1
3
´), 3.74 (bs, 2H, azep H2´), 4.28 (bs, 2H, azep H7´), 7.71 (s, 1H, pur H8), 12.31 (bs, 1H, pur NH). C
NMR (125 MHz, DMSO-d ) δ (ppm): 26.1 (azep C4´, 5´), 26.4 (azep C3´), 29.1 (azep C6´), 44.8
6
(morph C3´,5´), 47.6 (azep C2´), 48.8 (azep C7´), 66.1 (morph H2´,6´), 113.0 (pur C5), 135.4 (pur C8),
1
53.1 (pur C6), 153.2 (pur C4), 158.3 (pur C2).
4
.1.10. 6-(Azepan-1-yl)-8-chloropurine (10)

6
-(Azepan-1-yl)-8-chloro-9-(tetrahydropyran-2-yl)purine (18; 150 mg, 0.45 mmol) was dissolved in
MeOH (6.5 mL). H O (0.7 mL) and 10% HCl (6 drops) were added. The solution was stirred at room
2
temperature overnight. The product precipitated after addition of H O (5 mL) and conc. NH OH (2
2
4
drops). The suspension was refrigerated overnight. The product was filtered off, washed thoroughly
with H O, and briefly with MeOH (2 mL) and diethyl ether. The product was dried at room
2
temperature. White plaster-like substance. Yield: 97 mg (88%). M.p.: 227-228 °C (˃200 °C subl.).
HPLC R = 17.6 min (70% MeOH + 30% buffer); UV (70% MeOH + 30% buffer) λmin 247 nm, λmax
t
+
+
2
85 nm; HPLC purity >98%. MS ESI+ (CV 17) m/z (rel. %): 252 [M+H] (100), 254 [M+H] (33);
-
-
+
1
ESI (CV 22) m/z (rel. %): 250 [M] (100), 252 [M+H] (33). H NMR (300 MHz, DMSO-d ) δ (ppm):
6
1
.47 (bs, 4H, azep H4´, 5´), 1.75 (bs, 4H, azep H3´, 6´), 3.82 (bs, 2H, azep H2´), 4.24 (bs, 2H, azep
1
3
H7´), 8.20 (s, 1H, pur H2), 13.77 (bs, 1H, pur NH). C NMR (75 MHz, DMSO-d ) δ (ppm): 26.1 (azep
6
C4´, 5´), 26.6 (azep C3´), 28.3 (azep C6´), 47.7 (azep C2´), 49.0 (azep C7´), 119.3 (pur C5), 140.3 (pur
C8), 147.8 (pur), 151.0 (pur), 151.9 (pur).
4
.1.11. 6-(Azepan-1-yl)-8-bromopurine (11)
6
-(Azepan-1-yl)-8-bromo-9-(tetrahydropyran-2-yl)purine (19; 150 mg, 0.39 mmol) was dissolved in
MeOH (5 mL). H O (0.5 mL) and 10% HCl (5 drops) were added. The solution was stirred at room
2
temperature overnight. The product precipitated after addition of H O (16 mL) and conc. NH OH (2
2
4
drops). The suspension was refrigerated overnight. The product was filtered off, washed thoroughly
with H O and dried in a desiccator with P O .Yellowish powder. Yield: 113 mg (98%). M.p.: 215-218
2
2
5
°
C (˃170 °C subl.). HPLC R = 11.8 min (80% MeOH + 20% buffer); UV (80% MeOH + 20% buffer)
t
+
λmin 247 nm, λmax 285 nm; HPLC purity >99%. MS ESI+ (CV 15) m/z (rel. %): 296 [M+H] (100), 298
+
-
-
+
1
[
M+H] (100); ESI (CV 8) m/z (rel. %): 294 [M] (98), 296 [M+H] (100). H NMR (300 MHz,
DMSO-d ) δ (ppm): 1.47 (bs, 4H, azep H4´, 5´), 1.75 (bs, 4H, azep H3´, 6´), 3.81 (bs, 2H, azep H2´),
6
1
3
4
.22 (bs, 2H, azep H7´), 8.16 (s, 1H, pur H2), 13.74 (bs, 1H, pur NH). C NMR (75 MHz, DMSO-d )
6
δ (ppm): 26.1 (azep C4´, 5´), 26.6 (azep C3´), 28.3 (azep C6´), 47.7 (azep C2´), 49.1 (azep C7´), 120.2
pur C5), 126.8 (pur C8), 149.1 (pur), 151.9 (pur), 152.0 (pur).
(
4
.1.12. 6-(Azepan-1-yl)-8-methylpurine (12)
6
-(Azepan-1-yl)-8-methyl-9-(tetrahydropyran-2-yl)purine (20; 260 mg, 0.82 mmol) was dissolved in
MeOH (8.2 mL). H O (0.8 mL) and 10% HCl (11 drops) were added. The opalescent solution was kept
2
at room temperature for 10 days. H O (15 mL) was added and pH adjusted to 7 with conc. NH OH.
2
4
The slightly opalescent yellowish reaction mixture was refrigerated overnight. The white jelly like
substance (first portion of the product) was filtered off, washed with H O, then briefly with cold MeOH
2

and diethyl ether. The second portion of the product was obtained by evaporation of ¾ of total volume
of the filtrate and by precipitation of the product. The third portion was obtained after evaporation of
nd
the 2 filtrate to dryness. All portions of the product were dried in a desiccator with P O . White
2
5
crystals. Yield: 153 mg (80%). M.p.: 198-200 °C (˃180 °C subl.). HPLC R = 18.2 min (60% MeOH +
t
4
0% buffer); UV (60% MeOH + 40% buffer) λmin 239 nm, λmax 280 nm; HPLC purity >99%. MS ESI+
+
-
-
1
(
CV 17) m/z (rel. %): 232 [M+H] (100); ESI (CV 20) m/z (rel. %): 230 [M] (100). H NMR (300
MHz, DMSO-d ) δ (ppm): 1.46 (bs, 4H, azep H4´, 5´), 1.74 (bs, 4H, azep H3´, 6´), 2.40 (s, 3H, -CH ),
6
3
1
3
3
.86 (bs, 2H, azep H2´), 4.22 (bs, 2H, azep H7´), 8.08 (s, 1H, pur H2), 12.61 (bs, 1H, pur NH). C
NMR (75 MHz, DMSO-d ) δ (ppm): 14.6 (-CH ), 26.3 (azep C4´, 5´), 27.3 (azep C3´), 28.5 (azep C6´),
6
3
4
8.1 (azep C2´, 7´), 118.5 (pur C5), 146.7 (pur), 151.1 (pur), 152.0 (pur), 152.6 (pur).
4
.1.13. 6-(Azepan-1-yl)-7,9-dihydro-8H-purin-8-one (13)
6
-(Azepan-1-yl)-8-benzyloxy-9-(tetrahydropyran-2-yl)purine (synthesis 4.1.21.1.; 200 mg, 0.49 mmol)
was dissolved in MeOH (10 mL). 1M HCl (2 mL) was added and the mixture heated at 45 °C for 3
days. The mixture was diluted with H O (5 mL) and pH adjusted to 5 with conc. NH OH (10 drops).
2
4
The stiff white suspension was refrigerated overnight. The product was filtered off, washed thoroughly
with H O, then briefly with MeOH. The product was dried at room temperature. White crystals. Yield:
2
1
14 mg (99%). M.p.: 303-310 °C (decomp.). HPLC R = 10.7 min (60% MeOH + 40% buffer); UV
t
(70% MeOH + 30% buffer) λmin 242 nm, λmax 284 nm; HPLC purity >98%. MS ESI+ (CV 38) m/z (rel.
+
+
+
-
-
%
): 234 [M+H] (100), 256 [M+Na] (27), 272 [M+K] (18); ESI (CV 20) m/z (rel. %): 232 [M]
1
(
100). H NMR (300 MHz, DMSO-d ) δ (ppm): 1.44 (bs, 4H, azep H4´, 5´), 1.68 (bs, 4H, azep H3´,
6
1
3
6
´), 3.66 (t, J = 5.8 Hz, 4H, azep H2´, 7´), 7.99 (s, 1H, pur H2), 10.83 (bs, 2H, pur N7H, N9H). C
NMR (75 MHz, DMSO-d ) δ (ppm): 26.3 (azep C4´, 5´), 28.0 (azep C3´, 6´), 47.8 (azep C2´, 7´), 103.7
6
(pur C5), 146.4 (pur), 149.0 (pur), 150.3 (pur), 153.5 (pur).
4
4
.1.14. 6-(Azepan-1-yl)-8-methoxypurine (14)
.1.14.1. 6-(Azepan-1-yl)-8-methoxy-9-(tetrahydropyran-2-yl)purine
Na (40 mg, 1.74 mmol) was dissolved in MeOH (6 mL). 6-(Azepan-1-yl)-8-chloro-9-(tetrahydropyran-
-yl)purine (18) (0.30 g, 0.89 mmol) was added. The solution was stirred under Ar at 60 °C for 32 h.
After cooling, the mixture was diluted with MeOH (15 mL) and H O (5 mL) and pH adjusted to 6 with
2
2
1
M HCl. The mixture was concentrated in vacuo to total volume of 5 mL and extracted with EtOAc (2
×
20 mL). The EtOAc layer was dried with Na SO and evaporated. The residuum was dissolved in
2
4
hexane (10 mL). Undissolved particles were filtered off. The filtrate was concentrated in vacuo and
dried in a desiccator with P O . Viscous white opalescent substance. Yield: 280 mg (94%). HPLC R =
2
5
t

1
5.1 min (85% MeOH + 15% buffer); UV (85% MeOH + 15% buffer) λmin 238 nm, λmax 278 nm;
+
HPLC purity 97%. MS ESI+ (CV 17) m/z (rel. %): 332 [M+H] (100); ESI+ (CV 40) m/z (rel. %): 332
+
+
1
[
M+H] (100), 248 [M−THP+H] (62). H NMR (500 MHz, DMSO-d ) δ (ppm): 1.41-1.71 (m, 12H),
6
1
.88-1.91 (m, 1H), 2.62-2.70 (m, 1H), 3.54 (bt, J = 11.3 Hz, 3.3 Hz, 1H), 3.86-4.16 (bm, 8H), 5.44 (dd,
1
3
J = 11.1 Hz, 2.4 Hz, 1H), 8.06 (s, 1H, pur H2). C NMR (125 MHz, DMSO-d ) δ (ppm): 23.3, 25.0,
6
2
6.7, 28.5, 48.7, 57.3, 68.3, 81.2, 114.7, 150.4, 150.9, 152.3, 153.7.
4
.1.14.2. 6-(Azepan-1-yl)-8-methoxypurine (14)
6
-(Azepan-1-yl)-8-methoxy-9-(tetrahydropyran-2-yl)purine (synthesis 4.1.14.1; 220 mg, 0.66 mmol)
was dissolved in MeOH (6 mL) and 10% HCl (9 drops). H O (0.6 mL) was added and the mixture
2
heated at 60 °C for 21 h. The mixture was diluted with H O (5 mL) and pH adjusted to 6 with conc.
2
NH OH (1 drop). H O (10 mL) was added to enhance the precipitation of the product. The suspension
4
2
was refrigerated overnight. The product was filtered off, washed thoroughly with H O and in a
2
desiccator with P O . White plaster like substance. Yield: 61 mg (37%). M.p.: 213-216 °C (˃ 170 °C
2
5
subl.). HPLC R = 11.5 min (80% MeOH + 20% buffer); UV (80% MeOH + 20% buffer) λmin 240 nm,
t
+
-
λmax 280 nm; HPLC purity 97%. MS ESI+ (CV 47) m/z (rel. %): 248 [M+H] (100); ESI (CV 9) m/z
-
1
(
rel. %): 246 [M] (100). H NMR (300 MHz, DMSO-d ) δ (ppm): 1.46 (bs, 4H, azep H4´, 5´), 1.74 (bs,
6
4
H, azep H3´, 6´), 3.98 (bs, 7H, azep H2´, 7´, -OCH ), 8.04 (s, 1H, pur H2), 12.47 (bs, 1H, pur NH).
3
1
3
C NMR (75 MHz, DMSO-d ) δ (ppm): 26.2 (azep C4´, 5´), 28.0 (azep C3´, C6´), 48.1 (azep C2´, 7´),
6
5
6.0 (-OCH ), 115.3 (pur C5), 150.2 (pur), 150.7 (pur), 151.5 (pur), 154.7 (pur).
3
4
.1.15. 6-(Azepan-1-yl)-9-methylpurine (15) and 6-(azepan-1-yl)-7-methylpurine (43)
4
.1.15.1. 6-Chloro-9-methylpurine and 6-chloro-7-methylpurine
6
-Chloropurine (5 g, 32.35 mmol) was dissolved in DMF (80 mL) and finely pulverized anhydrous
K CO (8.96 g, 64.83 mmol) was added. The heterogeneous mixture was mixed at room temperature
2
3
for 3 h while CH I (3 × 3.5 mL, 168.66 mmol) was slowly added. The reaction was monitored by TLC
3
(
CHCl :MeOH 9:1). After this time, the reaction mixture was filtered and a small amount of DMF was
3
added. Subsequently, the mixture was concentrated in vacuo, crude product was dissolved in EtOAc,
and a small portion of distilled H O was added. The EtOAc portion was separated, dried over
2
anhydrous Na SO and concentrated in vacuo. After 12 h, the product was dissolved in 2-PrOH at 50
2
4
°
C and purified with pulverized coal. The final product was concentrated in vacuo and a mixture of two
1
isoforms was obtained. Yield: 3.86 g (71%), HPLC: 89.30% (6Cl9MP) and 10.70% (6Cl7MP). H
NMR (300 MHz, DMSO-d ) δ (ppm): 3.86 (s, N9-CH ), 4.07 (s, N7-CH ), 8.65 (s, pur H86Cl9MP), 8.74
6
3
3
(s ,pur H86Cl7MP), 8.77 (s, pur H2).

4
.1.15.2. 6-(Azepan-1-yl)-9-methylpurine (15) and 6-(azepan-1-yl)-7-methylpurine (43)
The crude mixture of 6-chloro-9-methylpurine and 6-chloro-7-methylpurine (synthesis 4.1.15.1; 0.55 g,
.26 mmol) was dissolved in PrOH (10 mL) together with azepane (0.3 g, 3.02 mmol) and Et N (2.0
3
3
mL, 14.35 mmol). The mixture was then refluxed at 100 °C. Azepane (0.44 g, 4.44 mmol) was added
after 1 h. The mixture was heated for 3 h and then evaporated. The residuum was dissolved in EtOAc,
mixed with distilled H O and shaken properly. The EtOAc phase was then concentrated in vacuo. The
2
mixture of two arisen isoforms (N9 and N7 methylated derivatives) was dissolved in the mobile phase
(
CHCl :MeOH 9:1) (10 mL) and separated on column.
3
4
.1.15.2.1. 6-(Azepan-1-yl)-9-methylpurine (15)
+
1
Yield: 0.16 g (24%). HPLC purity >99%. MS ESI+ (CV 20) m/z (rel. %): 232.4 (100) [M + H] . H
NMR (300 MHz, DMSO-d ) δ (ppm): 1.47 (bs, 4H, azep), 1.76 (bs, 4H, azep), 3.70 (s, 3H, N9-CH ),
6
3
1
3
3
.82 (bs, 2H, azep); 4.31 (bs, 2H, azep), 8.07 (s, 1H, pur); 8.19 (s, 1H, pur). C NMR (125 MHz,
DMSO-d ) δ (ppm): 26.2, 26.6, 28.7, 29.3, 39.5, 47.6, 48.9, 118.6, 140.4, 150.6, 151.9, 153.5.
6
4
.1.15.2.2. 6-(Azepan-1-yl)-7-methylpurine (43)
1
H NMR (300 MHz, DMSO-d ) δ (ppm): 1.54 (bs, 4H, azep), 1.81 (bs, 4H, azep), 3.86–3.71 (m, 4H,
6
1
3
azep), 3.95 (s, 3H, N7-CH ), 8.27 (s, 2H, pur H2 and H8). C NMR (125 MHz, DMSO-d ) δ (ppm):
3
6
2
7.2, 27.4, 36.1, 50.5, 113.7, 147.7, 150.5, 153.7, 161.1.
4
6
2
.1.16. 6-(Azepan-1-yl)-9-(tetrahydropyran-2-yl)purine (16)
-Chloro-9-(tetrahydropyran-2-yl)purine (prepared from 6-chloropurine according to Taddei et al.
004; 5.55 g, 23 mmol) was dissolved in 2-PrOH (60 mL). Et N (3.24 mL, 28 mmol) and azepane (3.6
3
ml, 32 mmol) were added. The solution was heated at 45 °C overnight. The reaction mixture was a
suspension of needle crystals and light beige powder. After cooling, the precipitate was filtered off,
washed thoroughly with 2-PrOH and H O (10 ml) and dried in a desiccator with P O . Fine white
2
2
5
powder. Yield: 5.15 g (74%). M.p.: 163-165 °C. HPLC R = 13.6 min (80% CH OH + 20% buffer); UV
t
3
(80% CH OH + 20% buffer) λ 236 nm, λmax 280 nm; HPLC purity >99%. MS ESI+ (CV 18) m/z
3 min
+
1
(
rel. %): 302 [M+H] (100). H NMR (300 MHz, DMSO-d ) δ (ppm): 1.48 (s, 4H, azep H4´, 5´), 1.55-
6
1
.57 (m, 2H, THP H4´, 5´), 1.70-1.77 (m, 5H, azep H3´, 6´, THP 5´), 1.90-1.97 (m, 2H, THP H3´,
H4´), 2.17–2.24 (m, 1H, THP H3´), 3.63-3.69 (m, 1H, THP H6´), 3.83 (bs, 2H, azep H2´, 7´), 3.97-4.00
m, 1H, THP H6´), 4.31 (bs, 2H, azep H2´, 7´), 5.63 (dd, J = 11.1, 2.1 Hz, 1H, THP H2´), 8.20 (s, 1H,
(
1
3
pur H8), 8.32 (s, 1H, pur H2). C NMR (125 MHz, DMSO-d ) δ (ppm): 22.5, 24.5, 26.2, 26.6, 28.6,
6
3
0.1, 47.6, 49.0, 67.7, 80.6, 118.6, 137.7, 149.7, 152.1, 153.5.

4
.1.17. 6-(Azepan-1-yl)-9-(β-D-ribofuranosyl)purine (17)
6
-Chloropurine riboside (2 g, 6.98 mmol) was dissolved in PrOH (30 mL). Azepane (0.7 g, 7.06 mmol)
and Et N (4.9 mL, 35.16 mmol) were then added. The mixture was refluxed at 100 °C and monitored
3
by TLC (CHCl :MeOH 4:1) for 3 h. The reaction mixture was concentrated in vacuo. At the end of
3
concentration, distilled H O (3 mL) was added to form an azeotropic mixture with PrOH. The
2
evaporated mixture was dissolved in EtOAc and extracted with distilled H O. The EtOAc phase was
2
dried over anhydrous Na SO and the solvent was evaporated. The residue was washed with Et O and
2
4
2
placed in a desiccator. Yield: 2.01 g (82%). HPLC purity 99%. MS ESI+ (CV 20) m/z (rel. %): 350
+
1
(
100) [M + H] . H NMR (300 MHz, DMSO-d ) δ (ppm): 1.48 (bs, 4H, azep), 1.77 (bs, 4H, azep),
6
3
.51–3.56 (m, 1H), 3.62–3.68 (m, 1H), 3.84 (bs, 2H, azep), 3.95 (s, 1H), 4.14 (s, 1H), 4.31 (bs, 2H,
azep), 4.60 (q, J = 5.6 Hz, 1H), 5.19 (d, J = 4.4 Hz, 1H), 5.40 (t, J = 5.2 Hz, 1H), 5.46 (d, J = 6.0 Hz,
1
H), 5.89 (d, J = 6.0 Hz, 1H), 8.19 (s, 1H, pur), 8.35 (s, 1H, pur).
4
.1.18. 6-(Azepan-1-yl)-8-chloro-9-(tetrahydropyran-2-yl)purine (18)
6
-(Azepan-1-yl)-9-(tetrahydropyran-2-yl)purine (16) (2.5 g, 8.29 mmol) was dissolved under Ar in dry
THF (50 mL). The solution was cooled to -75 °C and a suspension formed. The suspension was treated
dropwise with LDA (1M soln. in hexanes/THF) (13.5 mL, 13.5 mmol) over 10 min. The reaction
mixture was further stirred at -75 °C for 1 h 10 min. After this time, a solution of C Cl (2 g, 8.45
2
6
mmol) in dry THF (10 mL) was added dropwise over 13 min. The reaction mixture was then stirred for
h. Then, 20% NH Cl (60 mL) was added dropwise. After spontaneous warming to room temperature,
1
4
the layers were separated. The organic layer was extracted with H O (2 × 50 mL), dried with Na SO ,
2
2
4
filtered, and evaporated with silica gel (4.31 g). The resulting powder was poured onto a column and
purified by column chromatography (mobile phase hexanes:EtOAc 9:1), flow rate 11 mL/min). The
product was crystallized from the mobile phase. White crystals. Yield: 2.18 g (78%). M.p.: 87-90 °C.
HPLC R = 19.3 min (85% MeOH + 15% buffer); UV (85% MeOH + 15% buffer) λmin 241 nm, λmax
t
+
2
2
81 nm; HPLC purity >99%. MS ESI+ (CV 17) m/z (rel. %): 336 (100) + 338 (37) [M+H] , ESI+ (CV
+
+ 1
7) m/z (rel. %): 252 (98) + 254 (33) [M-THP+H] , 336 (100) + 338 (40) [M+H] . H NMR (300
MHz, DMSO-d ) δ (ppm): 1.47–1.87 (m, 12H, azep H3´, 4´, 5´, 6´,THP H3´, 4´, 5´), 1.97 (d, J = 10.6
6
Hz, 1H, THP H4´), 2.84 (qd, J = 12.4, 4.1 Hz, 1H, THP H3´), 3.64 (td, J = 11.1, 4.8 Hz, 1H, THP H6´),
3
.82 (bs, 2H, azep H2´, 7´), 4.02 (d, J = 11.4 Hz, 1H, THP H6´), 4.16 (bs, 2H, azep H2´, 7´), 5.66 (dd, J
1
3
=
11.3, 2.4 Hz, 1H, THP H2´), 8.21 (s, 1H, pur H2). C NMR (75 MHz, DMSO-d ) δ (ppm): 22.5,
6
2
4.4, 26.1, 26.5, 28.0, 28.3, 47.7, 48.9, 68.1, 82.8, 117.3, 134.9, 151.1, 152.2, 152.3.

4
.1.19. 6-(Azepan-1-yl)-8-bromo-9-(tetrahydropyran-2-yl)purine (19)
6
-(Azepan-1-yl)-9-(tetrahydropyran-2-yl)purine (16; 1.5 g, 4.98 mmol) was dissolved under Ar in dry
THF (35 mL). The solution was cooled to -75 °C and a suspension formed. The suspension was treated
dropwise with LDA (1 M soln. in hexanes/THF; 9 mL, 9 mmol) over 10 min. The reaction mixture was
further stirred at -75 °C for 1 h. After this time, a solution of CBr (1.67 g, 5.04 mmol) in dry THF (8
4
mL) was added dropwise over 10 min to the reaction mixture, which was then stirred for 2.5 h. Then,
2
0% NH Cl (40 mL) was added dropwise. After spontaneous warming to room temperature, the layers
4
were separated. The organic layer was extracted with H O (2 × 40 mL), dried with Na SO , filtered,
2
2
4
and evaporated with silica gel (3 g). The resulting powder was poured onto a column and purified by
column chromatography (mobile phase hexanes:EtOAc 9:1, flow rate 11 mL/min). The product was
crystallized from the mobile phase. White crystals. Yield: 1.51 g (80%). M.p.: 90-94 °C. HPLC R =
t
1
8.7 min (85% MeOH + 15% buffer); UV (85% MeOH + 15% buffer) λmin 241 nm, λmax 283 nm;
+
HPLC purity >99%. MS ESI+ (CV 17) m/z (rel. %): 380 (90) + 382 (100) [M+H] , ESI+ (CV 27) m/z
+
+
1
(
rel. %): 296 (62) + 298 (67) [M-THP+H] , 380 (88) + 382 (100) [M+H] . H NMR (300 MHz,
DMSO-d ) δ (ppm): 1.47–1.83 (m, 12H, azep H3´, 4´, 5´, 6´, THP H3´, 4´, 5´), 1.97–1.99 (m, 1H, THP
6
H4´), 2.97 (qd, J = 12.2, 4.1 Hz, 1H, THP H3´), 3.63 (td, J = 11.0, 3.7 Hz, 1H, THP H6´), 3.81 (bs, 2H,
azep H2´, 7´), 4.03 (d, J = 11.6 Hz, 1H, THP H6´), 4.18 (bs, 2H, azep H2´, 7´), 5.63 (dd, J = 11.3, 2.3
1
3
Hz, 1H, THP H2´), 8.18 (s, 1H, pur H2). C NMR (75 MHz, DMSO-d ) δ (ppm): 22.6, 24.4, 26.1,
6
2
6.6, 28.0, 28.3, 47.7, 48.9, 68.1, 83.9, 119.1, 124.4, 151.5, 152.0, 152.3.
4
.1.20. 6-(Azepan-1-yl)-8-methyl-9-(tetrahydropyran-2-yl)purine (20)
6
-(Azepan-1-yl)-9-(tetrahydropyran-2-yl)purine (16; 0.5 g, 1.66 mmol) was dissolved under Ar in dry
THF (15 mL). The solution was cooled to -75 °C and a suspension formed. The suspension was treated
dropwise with LDA (1 M soln. in hexanes/THF) (3 mL, 2 mmol) over 3 min. The reaction mixture was
further stirred at -75 °C for 1 h. After this time, CH I (0.1 mL, 1.66 mmol) was added. The reaction
3
mixture was then stirred for 3 h 15 min. The reaction was quenched with 20% NH Cl (10 mL) added
4
dropwise. After spontaneous warming to room temperature, the layers were separated. The organic
layer was extracted with H O (2 × 10 mL), dried with Na SO , filtered, and evaporated. The viscous
2
2
4
crude product was dried in a desiccator until it became crystalline. The solid product was crystallized
from hexanes/EtOAc. White crystals. Yield: 427 mg (82%). M.p.: 116-119 °C. HPLC R = 13.7 min
t
(85% MeOH + 15% buffer); UV (80% MeOH + 20% buffer) λmin 239 nm, λmax 279 nm; HPLC purity
+
1
9
1
9%. MS ESI+ (CV 17) m/z (rel. %): 316 (100) [M+H] . H NMR (300 MHz, DMSO-d ) δ (ppm):
6
.46–1.74 (m, 12H, azep H3´, 4´, 5´, 6´, THP H3´, 4´, 5´), 1.92-1.96 (m, 1H, THP H4´), 2.51–2.53 (m

overlapped with DMSO-d , 1H, THP H3´), 2.57 (s, 1H, -CH ), 3.65 (td, J = 11.0, 3.7 Hz, 1H, THP
6
3
H6´), 3.82 (bs, 2H, azep H2´, 7´), 4.03 (d, J = 11.5 Hz, 1H, THP H6´), 4.27 (bs, 2H, azep H2´, 7´), 5.66
1
3
(
dd, J = 11.3, 2.2 Hz, 1H, THP H2´), 8.13 (s, 1H, pur H2). C NMR (75 MHz, DMSO-d ) δ (ppm):
6
1
5.3, 22.7, 24.6, 26.2, 26.6, 28.6, 29.2, 47.9, 48.6, 68.1, 82.0, 117.4, 147.0, 151.0, 151.1, 152.7.
4
.1.21. 6-(Azepan-1-yl)-9-(tetrahydropyran-2-yl)-7,9-dihydro-8H-purin-8-one
Na (0.16 g, 6.96 mmol) was dissolved in benzyl alcohol (6 mL). 6-(Azepan-1-yl)-8-chloro-9-
tetrahydropyran-2-yl)purine (18; 0.45 g, 1.34 mmol) was added. The solution was stirred under Ar at
0 °C overnight. Then, the mixture was evaporated at p = 0.5 mbar, t = 100 °C. The residuum was
(
9
dissolved in MeOH (6.5 mL), H O (11 mL) and acidified with AcOH (0.5 mL). The mixture was
2
evaporated. The residuum was mixed with H O (6 mL) and extracted with EtOAc (3 × 6 mL). The
2
EtOAc portions were dried over Na SO . Na SO was filtered off and the filtrate was evaporated. The
2
4
2
4
residuum was mixed with hexanes:EtOAc 9:1 (3–4 mL) and subjected to column chromatography
mobile phase hexanes:EtOAc 9:1, flow rate 11 mL/min). Column chromatography provided two
products:
(
4
.1.21.1. 6-(Azepan-1-yl)-8-benzyloxy-9-(tetrahydropyran-2-yl)purine
White viscous substance. Yield: 0.2 g (37%). TLC R = 0.16 (hexanes:EtOAc 9:1). MS ESI+ (CV 17)
f
+
+
m/z (rel. %): 408 (100) [M+H] , ESI+ (CV 27) m/z (rel. %): 324 (27) [M-THP+H] , 408 (100)
+
1
[
M+H] . H NMR (500 MHz, DMSO-d ) δ (ppm): 1.42 (s, 4H, azep H4´, 5´), 1.45-1.50 (m overlapped,
6
2
1
H, THF), 1.54–1.65 (m overlapped, 1H, THP), 1.71 (s, 4H, azep H3´, 6´), 1.65-1.76 (m overlapped,
H, THP), 1.85-1.93 (m, 1H, THP H4´), 2.67 (dq, J = 12.3, 4.0 Hz, 1H, THP H3´), 3.55 (m, 1H, THP
H6´), 3.80 (bs, 2H, azep H2´, 7´), 3.94 (d, J = 13.0 Hz, 1H, THP H6´), 4.06 (bbs, 2H, azep H2´, 7´),
5
8
2
1
.44 (m, 3H, THP H2´, -OCH -), 7.29-7.33 (m, 1H, Bn), 7.34-7.39 (m, 2H, Bn), 7.45-7.50 (m, 2H, Bn),
2
1
3
.07 (s, 1H, pur H2). C NMR (75 MHz, DMSO-d ) δ (ppm): 23.3, 25.1, 26.7 (2x), 27.8 (bs), 28.6,
6
9.5 (bs), 48.8, 48.9, 68.3, 71.5, 81.2, 114.7, 128.7 (2x), 128.8, 128.9 (2x), 136.3, 150.3, 151.0, 152.2,
52.9.
4
.1.21.2. 6-(Azepan-1-yl)-9-(tetrahydropyran-2-yl)-7,9-dihydro-8H-purin-8-one (21)
White powder. Yield: 63.2 mg (15%). M.p.: 258-263 °C. HPLC R = 14.1 min (70% MeOH + 30%
t
buffer); UV (70% MeOH + 30% buffer) λmin 242 nm, λmax 284 nm; HPLC purity >99%. MS ESI+ (CV
+
1
1
7) m/z (rel. %): 318 (100) [M+H] . H NMR (300 MHz, DMSO-d ) δ (ppm): 1.44–1.68 (m, 12H, azep
6
H3´,4´, 5´, 6´, THP H3´, 4´, 5´), 1.90–1.93 (m, 1H, THP H4´), 2.85–2.98 (m, 1H, THP H3´), 3.53 (td, J
11.0, 3.7 Hz, 1H, THP H6´), 3.67 (t, J = 6.0 Hz, 4H, azep H2´, 7´), 3.94 (d, J = 11.3 Hz, 1H, THP
=
1
3
H6´), 5.35 (dd, J = 11.4, 2.0 Hz, 1H, THP H2´), 8.07 (s, 1H, pur H2), 10.72 (s, 1H, pur N7H). C

NMR (75 MHz, DMSO-d ) δ (ppm): 23.0, 24.6, 26.3 (2x), 27.2, 27.9 (2x), 47.9 (2x), 67.7, 79.9, 102.7,
6
1
46.8, 147.9, 150.2, 152.0.
4
.1.22. 6-(Piperidin-1-yl)purine (22)
6
-Chloropurine (1 g, 6.47 mmol) was refluxed with piperidine (4.5 mL, 65.77 mmol) in PrOH at 80-90
°
C for 3 hrs. The reaction was monitored via TLC (CHCl :MeOH 4:1). After completion, the reaction
3
mixture was evaporated. The residue was dissolved in EtOAc and mixed with distilled H O. EtOAc
2
was dried over Na SO , filtered, and evaporated. Crude product was air-dried. Yield: 1.2 g (91%).
2
4
+
1
HPLC purity >99%. MS ESI+ (CV 17) m/z (rel. %): 204 (100) [M+H] . H NMR (500 MHz, DMSO-
d6) δ (ppm): 1.53-1.58 (m, 4H), 1.64-1.68 (m, 2H), 4.18 (s, 4H), 8.07 (s, 1H), 8.16 (s, 1H), 12.96 (s,
1
3
1
H). C NMR (125 MHz, DMSO-d ) δ (ppm): 24.3, 25.7, 45.6, 118.6, 137.7, 151.3, 151.8, 153.1.
6
4
6
2
3
.1.23. 6-(Piperidin-1-yl)-9-(tetrahydropyran-2-yl)purine (23)
-Chloro-9-(tetrahydropyran-2-yl)purine (prepared from 6-chloropurine according to Taddei et al.
004; 2.0 g, 8.38 mmol) was dissolved in PrOH. Piperidine (1.0 mL, 10.1 mmol) and Et N (5.0 mL,
3
5.8 mmol) were added. The mixture was refluxed at 90 °C for 3 hrs and monitored by TLC
(
CHCl :MeOH 4:1). The final mixture was evaporated and diethylether was added. Emerging white
3
crystals were collected. Yield: 2.05 g (85%). HPLC purity >99%. MS ESI+ (CV 18) m/z (rel. %): 288
+
1
[
M+H] (100). H NMR (500 MHz, DMSO-d ) δ (ppm): 1.55-1.57 (m, 6H), 1.64-1.76 (m, 3H), 1.89-
6
1
4
.96 (m, 2H), 2.20 (tdd, J = 12.9, 10.9, 3.8 Hz, 1H), 3.63-3.69 (m, 1H), 3.97-4.01 (m, 1H), 4.17 (bs,
1
3
H), 5.64 (dd, J = 11.1, 2.2 Hz, 1H), 8.20 (s, 1H), 8.33 (s, 1H). C NMR (125 MHz, DMSO-d ) δ
6
(ppm): 22.5, 24.2, 24.5, 25.7, 30.1, 67.7, 80.7, 118.7, 137.4, 149.9, 152.0, 153.1.
4
.1.24. 6-(Piperidin-1-yl)-9-(β-D-ribofuranosyl)purine (24)
6
-Chloropurine riboside (2 g, 6.98 mmol) was dissolved in PrOH (30 mL). Piperidine hydrochloride
(
0.9 g, 7.4 mmol) and Et N (6 mL, 43.05 mmol) were then added. The mixture was refluxed at 100 °C
3
for 2 h and then evaporated. The residue was dissolved in EtOAc and distilled H O was added. The
2
emerged precipitate was filtered off, washed with distilled H O, and air-dried. Yield: 0.93 g (40%).
2
+
1
HPLC purity >99%. MS ESI+ (CV 20) m/z (rel. %): 336.4 (100) [M + H] . H NMR (500 MHz,
DMSO-d ) δ (ppm): 1.55–1.58 (m, 4H, piper), 1.65–1.68 (m, 2H, piper), 3.54 (ddd, J = 12.0, 7.0, 3.6
6
Hz, 1H), 3.66 (ddd, J = 12.1, 4.5, 3.6 Hz, 1H), 3.95 (q, J = 3.5 Hz, 1H), 4.13 (td, J = 4.8, 3.5 Hz, 1H),
4
5
.19 (bs, 4H), 4.57 (td, J = 6.1, 4.9 Hz, 1H), 5.18 (d, J = 4.6 Hz, 1H); 5.36 (dd, J = 7.0, 4.5 Hz, 1H),
1
3
.45 (d, J = 6.2 Hz, 1H), 5.89 (d, J = 5.9 Hz, 1H); 8.20 (s, 1H, pur), 8.36 (s, 1H, pur). C NMR (125
MHz, DMSO-d ) δ (ppm): 24.2, 25.7, 61.5, 70.5, 73.4, 85.8, 87.8, 119.5, 138.5, 150.1, 151.8, 153.1.
6

4
.1.25. 6-(4-Hydroxypiperidin-1-yl)purine (25)
6
-Chloropurine (0.7 g, 4.53 mmol) was dissolved in PrOH (30 mL). 4-Hydroxypiperidine (0.6 g, 5.93
mmol) and Et N (2 mL, 14.35 mmol) were then added. The mixture was refluxed at 100 °C and
3
monitored by TLC (CHCl :MeOH 4:1) for 1.5 h. The emerged crystalline solid was filtered off, washed
3
with distilled H O, and air-dried. Yield: 0.6 g (60%). HPLC purity >99%. MS ESI+ (CV 20) m/z (rel.
2
+
1
%
): 220.4 (100) [M + H] . H NMR (500 MHz, DMSO-d ) δ (ppm): 1.23-1.48 (m, 2H), 1.81 (s, 2H),
6
3
.59 (s, 2H), 3.77 (s, 1H), 4.60-5.04 (m, 3H), 8.09 (s, 1H), 8.18 (s, 1H), 12.99 (s, 1H).
4
.1.26. 1-(Purin-6-yl)piperidin-4-one (26)
6
-Chloropurine (0.2 g; 1.29 mmol) was dissolved in DMF (5 mL). Et N (0.44 mL, 3.15 mmol) and 4-
3
piperidone monohydrate hydrochloride (276 mg, 1.77 mmol) were added. The solution was heated at
0 °C for 2 h. After cooling, the reaction mixture was refrigerated for 2 d. The resulting solid substance
5
was filtered off and washed with distilled H O. Glassy crystals were dissolved and yellow powder
2
remained (portion I of the product, m = 45 mg). The filtrate was evaporated, the residuum was
adsorbed on silica gel (1 g) and purified by column chromatography (mobile phase
CHCl :MeOH:NH OH 95:5:0.5, flow rate 10 mL/min). Combined fractions with the product were
3
4
partly evaporated to a total volume 15-20 mL and refrigerated for 4 d. The emerging crystals were
filtered off and air-dried (portion II of the product, m = 72 mg). The filtrate was evaporated to dryness
(portion III of the product, m = 0.1 g). White powder. Yield: 216 mg (77%). M.p.: 280-285 °C. HPLC
R = 8.8 min (broad peak, R = 6.7–9.9 min) (40% MeOH + 60% buffer); UV (60% MeOH + 40%
t
t
buffer) λmin 234 nm, λmax 277 nm. HPLC purity 98% (portion II). MS ESI+ (CV 13) m/z (rel. %): 218
+
+
-
-
[
M+H] (47), 250 [M+CH OH+H] (100); ESI (CV 20) m/z (rel. %): 216 [M] (100), 248
3
-
1
[
M+CH OH] (32). H NMR (500 MHz, DMSO-d ) δ (ppm): 2.53 (bs, 4H, piper H3, H5), 4.45 (bs, 4H,
3
6
1
3
piper H2, H6), 8.20 (s, 1H, pur H8´), 8.30 (s, 1H, pur H2´), 13.15 (bs, 1H, pur NH). C NMR (125
MHz, DMSO-d ) δ (ppm): 40.7 (piper C3, C5), 43.7 (piper C2, C6), 119.1 (pur C5´), 138.6 (pur C8´),
6
1
51.5 (pur C4´), 151.9 (pur C2´), 152.9 (pur C6´), 207.5 (piper C4).
4
2
4
6
.1.27. 1-(2-Chloropurin-6-yl)piperidin-4-one (27)
,6-Dichloropurine (0.51 g; 2.7 mmol) was dissolved in 2-PrOH (8 mL). Et N (0.84 mL, 6 mmol) and
3
-piperidone monohydrate hydrochloride (502 mg, 3.28 mmol) were added. The solution was heated at
0 °C for 2 h. After cooling, the pH of the reaction mixture was adjusted with acetic acid (15 drops) to
pH = 6. The suspension was refrigerated overnight. The precipitate was filtered off, washed thoroughly
with 2-PrOH (4 mL) and distilled H O (10 mL), and dried in a desiccator with P O . White plaster-like
2
2
5
solid. Yield: 514 mg (76%). M.p.: 283-286 °C. HPLC R = 7.7 min (broad peak, R = 5.6 – 9.0 min)
t
t

(60% MeOH + 40% buffer); UV (60% MeOH + 40% buffer) λmin 238 nm, λmax 278 nm. HPLC purity
+
+
+
>
98%. MS ESI+ (CV 18) m/z (rel. %): 252 [M+H] (44), 254 [M+H] (18), 284 [M+CH OH+H] (96),
3
+
+
+
-
2
86 [M+CH OH+H] (34), 306 [M+CH OH+Na] (100), 308 [M+CH OH+Na] (33); ESI (CV 19)
3 3 3
-
-
-
-
1
m/z (rel. %): 250 [M] (100), 252 [M] (34), 282 [M+CH OH] (42), 284 [M+CH OH] (16). H NMR
3
3
1
3
(
500 MHz, DMSO-d ) δ (ppm): 2.50 (t, J = 6.2 Hz, 4H), 4.45 (bs, 4H), 8.17 (s, 1H), 13.24 (bs, 1H). C
6
NMR (125 MHz, DMSO-d ) δ (ppm): 40.3, 43.5 (bs), 118.0, 139.1, 152.4, 152.6, 153.3, 207.1.
6
4
6
2
.1.28. 1-[9-(Tetrahydropyran-2-yl)purin-6-yl]piperidin-4-one (28)
-Chloro-9-(tetrahydropyran-2-yl)purine (prepared from 6-chloropurine according to Taddei et al.
004; 1.3 g, 5.45 mmol) was dissolved in 2-PrOH (20 mL). Et N (1.66 mL, 11.90 mmol) and 4-
3
piperidone monohydrate hydrochloride (1 g, 6.51 mmol) were added. The mixture was stirred at room
temperature for 6 days. The mixture was evaporated. The residuum was dissolved in distilled H O (30
2
mL) and EtOAc (30 mL). After extraction, the EtOAc layer was dried with Na SO , evaporated and
2
4
dried in a desiccator with P O . The crude product (1.52 g, 93%) was crystallized from EtOAc (8 mL).
2
5
White crystals. Yield: 0.59 g (36%). M.p.: 136-138 °C. HPLC R = 11.0 min (60% MeOH + 40%
t
buffer); UV (70% MeOH + 30% buffer) λmin 235 nm, λmax 278 nm. HPLC purity >99%. MS ESI+ (CV
+
+
1
1
8) m/z (rel. %): 334 [M+H+MeOH] (100), 302 [M+H] (46). H NMR (500 MHz, DMSO-d ) δ
6
(
ppm): 1.57–1.60 (m, 2H, THP H5´), 1.71–1.76 (m, 1H, THP H4´), 1.91–1.98 (m, 2H, THP H3´, H4´),
.20–2.26 (m, 1H, THP H3´), 2.46 (t, J = 6.1 Hz, 4H, piper H3´, H5´), 3.67 (dt, J = 11.0, 4.0 Hz, 1H,
THP H6´), 3.98–4.02 (m, 1H, THP H6´), 4.48 (bs, 4H, piper H2, H6), 5.68 (dd, J = 11.0, 2.0 Hz, 1H,
2
1
3
THP H2´), 8.31 (s, 1H, pur H2´), 8.42 (s, 1H, pur H8´). C NMR (125 MHz, DMSO-d ) δ (ppm): 22.4
6
(THP C4´), 24.5 (THP C5´), 30.1 (THP C3´), 40.6 (piper C3, C5), 43.7 (piper C2, C6), 67.7 (THP
C6´), 80.8 (THP C2´), 119.2 (pur C5´), 138.3 (pur C8´), 150.1 (pur C4´), 152.1 (pur C2´), 153.0 (pur
C6´), 207.4 (piper C4).
4
.1.29. 1-(Purin-6-yl)piperidine-4-carboxamide (29)
6
-Chloropurine (0.1 g, 0.65 mmol) was dissolved in PrOH. 4-Piperidinecarboxamide (0.99 g, 7.72
mmol) and Et N (0.5 mL, 3.58 mmol) was added. The mixture was refluxed at 90 °C for 3 hrs and
3
monitored by TLC (CHCl :MeOH 4:1). The final mixture was concentrated. Et O was added. The
3
2
resulting yellowish crystals were collected. Yield: 136 mg (85%). HPLC purity >99%. MS ESI+ (CV
+
1
1
8) m/z (rel. %): 247 [M+H] (100). H NMR (300 MHz, DMSO-d ) δ (ppm): 1.52 (qd, J = 12.4, 4.2
6
Hz, 2H), 1.79 (dd, J = 13.4, 3.7 Hz, 2H), 2.42–2.47 (m, 1H), 3.12 (m, 2H), 5.32 (bs, 2H), 6.80 (s, 1H),
1
3
7
4
.32 (s, 1H), 8.12 (s, 1H), 8.20 (s, 1H), 13.0 (bs, 1H). C NMR (125 MHz, DMSO-d ) δ (ppm): 28.4,
6
1.7, 44.5, 118.6, 138.3, 151.1, 151.4, 152.9, 176.1.

4
.1.30. 6-(4-Methoxycarbonylpiperidin-1-yl)purine (30)
6
-Chloropurine (0.59 g, 3.82 mmol) was dissolved in PrOH (30 mL). Methyl 4-piperidinecarboxylate
(
0.7 g, 4.89 mmol) and Et N (1.6 mL, 11.48 mmol) was then added. The mixture was refluxed at 100
3
°
C and monitored by TLC (CHCl :MeOH 9:1) for 5.5 h. The resulting mixture was evaporated to half
3
the volume, and the crystals of the final product occurred after the solution was cooled. The crystals
were filtered off, washed with distilled H O, and air-dried. Yield: 0.7 g (70%). HPLC purity 98%. MS
2
+
1
ESI+ (CV 20) m/z (rel. %): 262.4 (100) [M + H] . H NMR (500 MHz, CD OD) δ (ppm): 1.69–1.77
3
(m, 2H), 2.04 (dd, J = 13.3, 3.8 Hz, 2H), 2.77 (tt, J = 11.0, 4.0 Hz, 1H), 3.34–3.40 (m, 2H), 3.69 (s,
1
3
3
4
H), 5.24 (d, J = 12.6 Hz, 2H), 8.00 (s, 1H), 8.20 (s, 1H). C NMR (125 MHz, CD OD) δ (ppm): 29.3,
3
2.2, 45.8, 52.2, 120.3, 139.0, 153.0, 155.0, 176.6.
4
6
1
.1.31. 6-(1,4-Dioxa-8-azaspiro[4.5]decan-8-yl)purine (31)
-Chloropurine (200 mg, 1.29 mmol) was dissolved in DMF (5 mL). Et N (0.22 mL, 1.56 mmol) and
3
,4-dioxa-8-azaspiro[4.5]decane (0.18 mL, 1.42 mmol) were added. The solution was heated at 50 °C
overnight. After cooling, the pH of the mixture was adjusted with acetic acid (10 drops) to pH = 7. The
suspension was refrigerated overnight. The resulting precipitate was filtered off, washed thoroughly
with distilled H O, and dried in a desiccator with P O . White powder. Yield: 207 mg (61%). M.p.:
2
2
5
2
80-283 °C. HPLC R = 8.7 min (60% MeOH + 40% buffer); UV (60% MeOH + 40% buffer) λmin 234
t
+
-
nm, λmax 277 nm. HPLC purity >99%. MS ESI+ (CV 18) m/z (rel. %): 262 [M+H] (100); ESI (CV
-
1
1
9) m/z (rel. %): 260 [M] (100). H NMR (500 MHz, DMSO-d ) δ (ppm): 1.66–1.68 (m, 4H,
6
piperidine H3´, H5´), 3.91 (s, 4H, dioxolane H4´, H5´), 4.27 (bs, 4H, piperidine H2´, H6´), 8.10 (s, 1H,
1
3
pur H8), 8.19 (s, 1H, pur H2), 13.00 (bs, 1H, pur N9H). C NMR (125 MHz, DMSO-d ) δ (ppm): 34.7
6
(
piperidine C3´, C5´), 42.7 (bs, piperidine C2´, C6´), 63.8 (dioxolane C4´, C5´), 106.5 (spiro C), 118.8
pur C5), 138.1 (pur C8), 151.4 (pur C4), 151.8 (pur C2), 152.9 (pur C6).
(
4
.1.32. 2-(2-Hydroxyethylamino)-6-(1,4-dioxa-8-azaspiro[4.5]decan-8-yl)purine (32)
2
-Chloro-6-(1,4-dioxa-8-azaspiro[4.5]decan-8-yl)purine (33; 143 mg, 0.49 mmol) was dissolved in
ethanolamine (1.5 mL). The solution was heated at 147 °C for 25 h. After cooling, the reaction mixture
was refrigerated for one week. During this time, the product solidified. The solid was filtered off,
washed thoroughly with cold 2-PrOH (10 mL) and distilled H O (10 mL), and dried in a desiccator
2
with P O . White plaster-like solid. Yield: 120 mg (78%). M.p.: 246-248 °C. HPLC Rt = 5.4 min (60%
2
5
MeOH + 40% buffer), Rt = 6.9 min (50% MeOH + 50% buffer); UV (50% MeOH + 50% buffer) λmax
38 nm, λmin 251 nm, λmax 255 nm, λmin 273 nm, λmax 291 nm. HPLC purity 100%. MS ESI+ (CV 18)
2
-
-
1
m/z (rel. %): 321 [M+H]+ (100); ESI (CV 19) m/z (rel. %): 319 [M] (100). H NMR (500 MHz,

DMSO-d ) δ (ppm): 1.63–1.65 (m, 4H), 3.28 (q, J = 6.1 Hz, 2H), 3.50 (t, J = 6.1 Hz, 2H), 3.91 (s, 4H),
6
1
3
4
.19 (bs, 4H), 4.63 (bs, 1H), 6.09 (t, J = 5.8 Hz, 1H), 7.66 (s, 1H), 12.27 (bs, 1H). C NMR (125 MHz,
DMSO-d ) δ (ppm): 34.7, 42.4, 43.9, 60.3, 63.8, 106.6, 113.2, 134.6, 153.1, 153.7, 158.8.
6
4
.1.33. 2-Chloro-6-(1,4-dioxa-8-azaspiro[4.5]decan-8-yl)purine (33)
2
,6-Dichloropurine (206 mg, 1.09 mmol) was dissolved in 2-PrOH (3 mL). Et N (0.23 mL, 1.63 mmol)
3
and 1,4-dioxa-8-azaspiro[4.5]decane (0.17 mL, 1.30 mmol) were added. The solution, which soon
became a suspension, was heated at 65 °C for 1.5 h. After cooling, the pH of the mixture was adjusted
with acetic acid (10 drops) to pH = 7. The suspension was refrigerated overnight. The precipitate was
filtered off, washed thoroughly with 2-PrOH and distilled H O, and dried in a desiccator with NaOH.
2
White plaster-like solid. Yield: 280 mg (87%). M.p.: 275-278 °C. HPLC R = 16.9 min (60% MeOH +
t
4
0% buffer); UV (60% MeOH + 40% buffer) λmin 238 nm, λmax 278 nm. HPLC purity 100%. MS ESI+
+
+
+
+
(
CV 18) m/z (rel. %): 296 [M+H] (74), 298 [M+H] (27), 318 [M+Na] (100), 320 [M+Na] (38);
-
-
-
1
ESI (CV 19) m/z (rel. %): 294 [M] (100), 296 [M] (31). H NMR (500 MHz, DMSO-d ) δ (ppm):
6
1
3
1
.70-1.73 (m, 4H), 3.93 (s, 4H), 4.25 (bs, 4H), 8.08 (s, 1H), 13.05 (bs, 1H). C NMR (125 MHz,
DMSO-d ) δ (ppm): 34.6, 42.2 (bs from HMBC), 63.9, 106.3, 117.7, 138.7, 152.4, 152.6, 153.1.
6
4
6
2
.1.34. 6-(1,4-Dioxa-8-azaspiro[4.5]decan-8-yl)-9-(tetrahydropyran-2-yl)purine (34)
-Chloro-9-(tetrahydropyran-2-yl)purine (prepared from 6-chloropurine according to Taddei et al.
004; 0.95 g, 3.98 mmol) was dissolved in 2-PrOH (20 mL), and Et N (0.67 mL, 4.80 mmol) and 1,4-
3
dioxa-8-azaspiro[4.5]decane (0.61 mL, 4.76 mmol) were added. The solution was stirred at room
temperature for 1 d and then evaporated. The residuum was dissolved in distilled H O (20 mL) and
2
EtOAc (50 mL). The mixture was shaken in a separating funnel. The resulting solid particles were
filtered off (portion I of the product, m = 0.15 g). Brine (10 mL) was added to the filtrate. After
shaking, the EtOAc layer was separated and extracted with brine (2 × 20 mL). Brine layers were re-
extracted with EtOAc (20 mL). Combined EtOAc layers were dried with Na SO . Na SO was filtered
2
4
2
4
off, and the filtrate was concentrated to 4 mL volume, inoculated with crystals from portion I, and
refrigerated for 6 d. The resulting crystals were filtered off, washed briefly with cold EtOAc and air-
dried (portion II of the product, m = 0.62 g). Precipitation with hexanes from the filtrate produced
portion III of the product (m = 0.34 g). White crystals. Overall yield: 1.11 g (81%). M.p.: 113-118 °C.
HPLC R = 13.4 min (70% MeOH + 30% buffer); UV (80% MeOH + 20% buffer) λmin 235 nm, λmax
t
+
1
2
78 nm. HPLC purity >99%. MS ESI+ (CV 23) m/z (rel. %): 346 [M+H] (100). H NMR (500 MHz,
DMSO-d ) δ (ppm): 1.54–1.58 (m, 2H, THP H5´), 1.67–1.72 (m, 5H, piperidine H3´, H5´, THP H4´),
6
1
.89–1.96 (m, 2H, THP H3´, H4´), 2.20 (dq, J = 12.0 Hz, 3.3 Hz, 1H, THP H3´), 3.66 (td, J = 11.0 Hz,

3
.7 Hz, 1H, THP H6´), 3.92 (s, 4H, dioxolane H4´, H5´), 3.95–4.01 (m, 1H, THP H6´), 4.27 (bs, 4H,
piper H2´, H6´), 5.64 (dd, J = 10.7, 2.2 Hz, 1H, THP H2´), 8.23 (s, 1H, pur H2), 8.36 (s, 1H, pur H8).
1
3
C NMR (125 MHz, DMSO-d ) δ (ppm): 22.4 (THP C4´), 24.5 (THP C5´), 30.1 (THP C3´), 34.7
6
(
piperidine C3´, C5´), 42.8 (bs, piperidine C2´, C6´), 63.8 (dioxolane C4´, C5´), 67.7 (THP C6´), 80.8
THP C2´), 106.5 (spiro C), 118.9 (pur C5), 137.8 (pur C8), 150.0 (pur C4), 152.0 (pur C2), 152.9 (pur
(
C6).
4
.1.35. 6-(1-Oxa-4-thia-8-azaspiro[4.5]decan-8-yl)purine (35)
-(Purin-6-yl)piperidin-4-one (26; 193 mg, 0.89 mmol) was mixed with toluene (10 mL),
1
mercaptoethanol (81 µL, 1.15 mmol), and p-toluenesulfonic acid monohydrate (165 mg, 0.87 mmol).
The suspension was heated at 104 °C for 4 h. Dehydration was ensured by continuous azeotropic
distillation. The reaction mixture was a suspension of white precipitate and dark solid on the bottom of
the flask in clear liquid. The reaction mixture was mixed with CHCl to dissolve solid particles, then
3
extracted with 0.25% Na CO . The organic layer was dried with Na SO and evaporated. The residuum
2
3
2
4
was dissolved in MeOH, adsorbed on silica gel (1.44 g), and purified by column chromatography
mobile phase CHCl :MeOH:NH OH 92.5:7.5:0.75, flow rate 10 mL/min). White crystals. Yield: 148
(
3
4
mg (60%). M.p.: 283-284 °C. HPLC R = 10.7 min (70% MeOH + 30% buffer); UV (70% MeOH +
t
3
0% buffer) λmin 234 nm, λmax 278 nm. HPLC purity 99%. MS ESI+ (CV 18) m/z (rel. %): 278 [M+H]+
+
-
-
1
(
100), 300 [M+Na] (12); ESI (CV 19) m/z (rel. %): 276 [M] (100). H NMR (300 MHz, DMSO-d ) δ
6
(
ppm): 1.95 (t, J = 5.6 Hz, 4H, piperidine H3´, H5´), 3.07 (t, J = 6.0 Hz, 2H, SCH -), 4.16 (bs, cv, 2H,
2
piperidine H2´, H6´), 4.16 (t, J = 6.0 Hz, cv, 2H, OCH -), 4.40 (bs, 2H, piperidine H2´, H6´), 8.11 (1H,
2
1
3
s, pur H8), 8.20 (s, 1H, pur H2), 13.03 (1H, bs, pur NH). C NMR (75 MHz, DMSO-d ) δ (ppm): 32.5
6
(
piperidine C3´, C5´), 38.9 (oxathiolane C4´, cv with DMSO-d ), 43.2 (bs, piperidine C2´, C6´), 69.4
6
(oxathiolane C5´), 93.6 (spiro C), 118.8 (pur C5), 138.1 (pur C8), 151.4 (pur C4), 151.7 (pur C2),
1
52.8 (pur C6).
4
.1.36. 6-(1,4-Dithia-8-azaspiro[4.5]decan-8-yl)purine (36)
1
-(Purin-6-yl)piperidin-4-one (26) (209 mg, 0.96 mmol) was mixed with toluene (10 mL), 1,2-
ethanedithiol (155 µL, 1.85 mmol), and p-toluenesulfonic acid monohydrate (268 mg, 1.41 mmol). The
suspension was heated at 104 °C for 6 h. Dehydration was ensured by continuous azeotropic
distillation. The reaction mixture was a suspension of dark solid on the bottom of the flask in clear rust-
coloured liquid. The reaction mixture was mixed with CHCl to dissolve the solid, then shaken with
3
0
.25% Na CO . The organic layer was dried with Na SO and evaporated. The residuum was dissolved
2 3 2 4
in MeOH, adsorbed on silica gel (1.57 g), and purified by column chromatography (gradient elution

starting with mobile phase CHCl :MeOH 99.5:0.5, ending with CHCl :MeOH 97.5:2.5, flow rate 10
3
3
mL/min). The post-chromatography product (234 mg, 83%) was crystallized from CHCl :MeOH
3
5
3
5:45. White crystals. Yield: 48 mg (17%). M.p.: 314-320 °C. HPLC R = 17.3 min (70% MeOH +
t
0% buffer); UV (70% MeOH + 30% buffer) λmin 235 nm, λmax 279 nm. HPLC purity 100%. MS ESI+
+
-
-
1
(
CV 18) m/z (rel. %): 294 [M+H] (100); ESI (CV 19) m/z (rel. %): 292 [M] (100). H NMR (300
MHz, DMSO-d ) δ (ppm): 2.07–2.11 (m, 4H, piperidine H3´, H5´), 3.34 (s, 4H, dithiolane H4´, H5´),
6
1
3
4
.29 (bs, 4H, piper H2´, H6´), 8.11 (s, 1H, pur H8), 8.20 (s, 1H, pur H2), 13.02 (bs, 1H, pur NH). C
NMR (75 MHz, DMSO-d ) δ (ppm): 37.9 (piperidine C3´, C5´), 41.4 (dithiolane C4´, C5´), 44.6 (bs,
6
piperidine C2´, C6´), 66.5 (spiro C), 118.8 (pur C5), 138.1 (pur C8), 151.4 (pur C4), 151.7 (pur C2),
1
52.8 (pur C6).
4
1
1
.1.37. 2-Chloro-6-(1,4-dithia-8-azaspiro[4.5]decan-8-yl)purine (37)
-(2-Chloropurin-6-yl)piperidin-4-one (27; 215 mg, 1.00 mmol) was suspended in toluene (10 mL).
,2-Ethanedithiol (0.11 mL, 1.31 mmol) and p-toluenesulfonic acid monohydrate (198 mg, 1.04 mmol)
were added. The reaction mixture was heated at 90 °C for 3 h 40 min. After cooling, the reaction
mixture was refrigerated overnight. A plaster-like product precipitated. The product was filtered off
and washed thoroughly with cold toluene (10 mL), 2-PrOH (2.5 mL) and distilled H O (5 mL). The
2
product was dried in a desiccator with P O . Raw product (207 mg, 63%) was crystallized from
2
5
ethanol. White powder. M.p.: 283-286 °C (sublimation preceded melting; product melted at coincident
decomposition). HPLC R = 16.7 min (80% MeOH + 20% buffer); UV (80% MeOH + 20% buffer) λmin
t
+
2
38 nm, λmax 280 nm. HPLC purity >98%. MS ESI+ (CV 18) m/z (rel. %): 328 [M+H] (100), 330
+
+
+
-
-
[
M+H] (48), 350 [M+Na] (97), 352 [M+Na] (46); ESI (CV 19) m/z (rel. %): 326 [M] (100), 328
-
1
[
M] (42). H NMR (500 MHz, DMSO-d ) δ (ppm): 2.10–2.12 (m, 4H), 3.34 (s, 4H), 4.26 (bs, 4H),
6
1
3
8
1
.12 (s, 1H), 13.18 (bs, 1H). C NMR (125 MHz, DMSO-d ) δ (ppm): 37.9, 41.2, 44.8 (bs), 66.2,
6
17.7, 138.8, 152.3, 152.6, 153.0.
4
.1.38. 2-(2-Hydroxyethylamino)-6-(1,4-dithia-8-azaspiro[4.5]decan-8-yl)purine (38)
2
-Chloro-6-(1,4-dithia-8-azaspiro[4.5]decan-8-yl)purine (37; 137 mg, 0.42 mmol) was dissolved in
ethanolamine (1.3 mL). The solution was heated at 149 °C for 19 h. After cooling, the product was
precipitated with distilled H O (10 mL). The suspension was refrigerated overnight. The milk-like
2
precipitate was filtered off, washed thoroughly with distilled H O (10 mL), and dried in a desiccator
2
with P O . Raw product (134 mg, 91%) was crystallized from ethanol. White plaster-like solid. M.p.:
2
5
2
22-224 °C. HPLC R = 6.3 min (80% MeOH + 20% buffer), R = 8.5 min (70% MeOH + 30% buffer);
t t
UV (80% MeOH + 20% buffer) λmax 239 nm, λmin 272 nm, λmax 294 nm. MS ESI+ (CV 18) m/z (rel.