A 2,6,9-hetero-trisubstituted purine inhibitor exhibits potent biological effects against multiple myeloma cells

Article abstract of DOI:10.1016/j.bmc.2013.04.080

A focused library of hetero-trisubstituted purines was developed for improving the cell penetrating and biological efficacy of a series of anti-Stat3 protein inhibitors. From this SAR study, lead agent 22e was identified as being a promising inhibitor of MM tumour cells (IC₅₀'s <5 μM). Surprisingly, biophysical and biochemical characterization proved that 22e was not a Stat3 inhibitor. Initial screening against the kinome, prompted by the purine scaffold's history for targeting ATP binding pockets, suggests possible targeting of the JAK family kinases, as well for ABL1 (nonphosphorylated F317L) and AAK1.

Full text of DOI:10.1016/j.bmc.2013.04.080

Bioorganic & Medicinal Chemistry 21 (2013) 5618–5628
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
A 2,6,9-hetero-trisubstituted purine inhibitor exhibits potent
biological effects against multiple myeloma cells
Vijay M. Shahani a, , Daniel P. Ball a, , Allan V. Ramos a, Zhihua Li b, Paul A. Spagnuolo c, Sina Haftchenary a,
Aaron D. Schimmer c, Suzanne Trudel b, Patrick T. Gunning a,
⇑
a Department of Chemistry, University of Toronto, 3359 Mississauga Road North, Mississauga, ON, L5L 1C6, Canada
b Princess Margaret Hospital, McLaughlin Centre for Molecular Medicine, 620 University Avenue, Toronto, ON, M5G 2C1, Canada
c Ontario Cancer Institute, Princess Margaret Hospital, 610 University Avenue, Toronto, ON, M5G 2M9, Canada
a r t i c l e i n f o
a b s t r a c t
Article history:
A focused library of hetero-trisubstituted purines was developed for improving the cell penetrating and
Received 4 February 2013
Revised 18 April 2013
Accepted 26 April 2013
Available online 9 May 2013
biological efficacy of a series of anti-Stat3 protein inhibitors. From this SAR study, lead agent 22e was
identified as being a promising inhibitor of MM tumour cells (IC50’s <5 lM). Surprisingly, biophysical
and biochemical characterization proved that 22e was not a Stat3 inhibitor. Initial screening against
the kinome, prompted by the purine scaffold’s history for targeting ATP binding pockets, suggests possi-
ble targeting of the JAK family kinases, as well for ABL1 (nonphosphorylated F317L) and AAK1.
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
Purine scaffold
Multiple myeloma
Stat3
Kinome
Cancer therapeutics
1. Introduction
resistance in a multitude of human cancer cells.9,10 Many types
of cancer cells exhibit Stat3 ‘addiction’ and are widely
Purines feature in biological molecules that are critically in-
volved in many essential cellular processes. Considered a privi-
leged structure in biological systems, the purine heterocycle
represents an intriguing molecular starting point for new pharma-
ceutical agents.1 Purines have been incorporated into therapeutic
structures for applications in nucleotide anti-metabolites and play
a key structural role in inhibitors of cancer-promoting dysregu-
lated proteins in cancer.2–6 For example, hetero-tri-substituted
purines have been used successfully to inhibit cell cycle initiators,
cyclin-dependant kinases which have been implicated in cancer.7
In addition, purine inhibitors have successfully suppressed the
activity of Heat Shock Protein 90, a protein critical for tumour
survival.8
We have demonstrated that purine scaffolds could be function-
alized as part of a pharmacophore model to bind and inhibit the Src
Homology 2 (SH2) domain of the signal transducer and activator of
transcription 3 (Stat3) protein. Stat3 has been shown to play a key
role in regulating cancer cell growth and differentiation. Hyper-
activation of Stat3 protein levels has been shown to be transforma-
tive, leading to uncontrolled cell proliferation and apoptotic
acknowledged to be hypersensitive to Stat3 inhibition that leads
to programmed cancer cell death. Thus, potent Stat3 inhibitors
have tremendous potential as novel therapeutics.11,12
In previous work, a quantitative structure activity relationship
(QSAR) of a 2,6,9-hetero-trisubstituted purine core was conducted
for targeting the Src Homology 2 domain ‘hot spot’ of Stat3
(Fig. 1).13 Computational docking studies suggested that trisubsti-
tuted purine inhibitors may best access the sub-pockets of Stat3’s
SH2 domain and thus prevent Stat3 cellular function. Specifically,
inhibitors were prepared to access two predominantly hydropho-
bic pockets (various appendages at R1 and the cyclohexylbenzyl
substituent at the N2 position), as well as the polar phosphotyro-
sine (pTyr) binding site with an N9-carboxylate substituent. The
purines prepared showed potent Stat3 protein binding in vitro, as
assessed by surface plasmon resonance (SPR: Biacore 3000), with
Kd values ranging from 1 to 10 lM. However, lead Stat3 binders
8a–g (Fig. 1) displayed only modest anti-proliferative effects in
cancer cell lines. To determine the origins of the lower than
expected IC50 values, experiments were conducted to assess the
cell penetrating properties of lead purine inhibitors. In particular,
Caco2 cell penetrating studies using a Waters Xevo quadrupole
time-of-flight (QTof) mass spectrometer and an ACQUITY UPLC
system revealed that inhibitors poorly traversed the cell
membrane.
⇑
Corresponding author. Tel.: +1 647 669 2969.
E-mail address: patrick.gunning@utoronto.ca (P.T. Gunning).
These authors contributed equally to this work.
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.04.080

V. M. Shahani et al. / Bioorg. Med. Chem. 21 (2013) 5618–5628
5619
Figure 1. 1st generation 2,6,9-heterotrisubstituted purine inhibitors of Stat3.
Herein, synthetic efforts to prepare second-generation anti-
Stat3, purine-based inhibitors, with improved cell permeability
and enhanced cytotoxicity toward cancer cells are presented. Spe-
cifically, synthetic efforts were undertaken to mask the anionic N9-
carboxylic acid substituent, present on lead Stat3 inhibitors pre-
pared in the previous study. Prodrug and bioisostere strategies
were employed to furnish a family of purine-based small mole-
cules that retained the top ranked R1 and R2 substituents from
the previous studies. However, while non-carboxylate containing
analogs did not exhibit Stat3 binding affinity, compound 22e,
substituted with a sulfamate group, exhibited potent cytoxicity
in multiple myeloma (MM) whole cell tumour studies. Efforts to
delineate the intracellular targets are described.
4 was subjected to a successive round of Mitsunobu conditions,
this time with cyclohexyl benzyl alcohol, to furnish the N2 alkyl-
ated product 5. Using microwave mediated nucleophilic aromatic
substitution, 5 was treated with a select series of previously iden-
tified alkylamines,13 to give 6a–g in good yields. Quantitative BOC
deprotection of 6a–g was achieved using standard trifluoroacetic
acid/CH2Cl2 conditions to give the ethyl ester prodrug final mole-
cules, 7a–g. To access acyloxymethyl ester classes of carboxylic
acid prodrug, the ethyl ester of compounds, 7a–g was hydrolysed
using LiOH mediated saponification to give carboxylic acids 8a–g,
then intermediates subjected to treatment with either acetoxy-
methyl bromide to yield the acyloxymethyl ester prodrugs
(9a–g), or pivaloyloxymethyl iodide to yield the pivaloyloxymethyl
ester prodrugs (10a–g). Due to stability issues, all prodrug analogs
were used immediately after HPLC and lyophilisation. Purity levels
were confirmed by analytical HPLC prior to in vitro or biological
testing.
2. Results and discussion
2.1. Inhibitor design
Preparation of N9-sulfamate-containing purine inhibitors were
prepared by Mitsunobu-mediated installation of the sulfamate
substituent (Scheme 2). Briefly, compound 3 was selectively alkyl-
ated on N9 with freshly prepared monosilyated ethylene glycol
using Mitsunobu conditions as previously reported to acquire 17
in good yields.18 Next, 17 was subjected to a subsequent Mitsun-
obu reaction with cyclohexyl benzyl alcohol to yield the N2 alkyl-
ated product 18. Analogous to the prodrug approach, nucleophilic
aromatic substitution was employed to incorporate alkylamines at
C6 to yield compounds 19a–g. TBDMS deprotection using TBAF in
THF afforded the free alcohol 20a–g in excellent yields. Finally, pri-
mary alcohols 20a–g were subjected to NaH and treated with sul-
fonamide chloride (as prepared from chlorosulfonyl isocyante
23)19 to yield the sulfamate products 21a–g in good yields (35–
66%).20 Finally, BOC deprotection using trifluoroacetic acid pro-
vided final molecules 22a–g in excellent yields (73–89%).
Derivatives featuring the lipophilic tetrazole bioisostere utilized
a modified alkylation procedure. Precursor 3, was alkyated with
trityl protected tetrazole 30, proceeding through an SN2 reaction
after Mitsunobu conditions proved poor yielding (Scheme 3). The
resulting mixture of N7 and N9 alkylated products were separated
by chromatography, with the N9 product isolated in good yields
providing 31. Formation of 32 follows conventional N2 alkylation
using Mitsunobu chemistry, and 33a–g were formed using an aro-
matic substitution diversification step. Final molecules 34a–g was
obtained in good yields following a global de-protection of BOC
and trityl groups using trifluoroacetic acid.
To overcome the poor cell penetrating properties of first gener-
ation purines, two design strategies were adopted. First, the N9
carboxylic acid substituent was masked using prodrug strategies.14
Since lead purine compounds were considered relatively non-polar
(clogP = ꢀ0.2–4.1), and that masking the charged appendage
might significantly reduce water solubility, we prepared a range
of prodrugs with varying polarity to circumvent possible inhibitor
aggregation. We reasoned that a successful prodrug approach will
facilitate inhibitor cell membrane penetration, improve inhibitor
half-life, and increase cellular potency.
Second, bioisosteres of the carboxylic acid were introduced for
purposes of increasing inhibitor lipophilicity, reducing anionic
character and improving binding potency through potentially
more favorable and increased intermolecular interactions with
the protein surface. In this study, the N9-carboxylic acid append-
age was replaced with either a tetrazole or sulfamate appendage.
Tetrazole, while possessing a similarly acidic proton to the carbox-
ylate, possesses significantly greater lipophilicity, and potentially
improved cell penetrative properties.15 In addition, a neutral,
hydrogen bonding sulfamate group was selected for making con-
tacts with the pTyr binding pocket of Stat3’s SH2 domain.
2.2. Preparation of 2,6,9-tri-heterosubstituted purines
Access to final molecules was achieved through four synthetic
routes each starting from 2-amino-6-chloropurine (Fig. 2). Near
quantitative BOC protection of the N9 position was achieved using
BOC anhydride and catalytic dimethylamino-pyridine to give 2
(Scheme 1).16 As previously reported by our group,17 treatment
with NaH efficiently mediated BOC group transfer from N9 to N2
(3). Mitsunobu reaction conditions using ethyl glycolate afforded
the N9 alkylated product 4, over the less reactive N7 position. Next,
Several lead derivatives of the past library possessed a cyclo-
hexyl carbonyl moiety in the N2 position To successfully acylate
these molecules, the N2 nitrogen was liberated of protecting
groups using trifluoroacetic acid or a Lewis acid (AlCl3). The
remaining steps follow logically from the methods just described
and are detailed in Supplementary data.

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V. M. Shahani et al. / Bioorg. Med. Chem. 21 (2013) 5618–5628
O
R2
N
N
N
N
N
N
R1
N
O
N
N
H
N
R1
N
H
R1
=
R1
=
R2
=
Code
Code (x)
a
Code (#)
7
O
O
O
O
13
HN
HN
O
O
O
b
c
O
O
O
O
9
15a
15b
O
O
O
O
O
O
HN
HN
O
d
e
O
O
10
O
O
F
HN
O
O
NH2
NH2
22
34
28
37
S
S
O
O
O
O
f
N
O
H
N
NO2
H
N
N
g
N
N
N
N
N
Figure 2. Summary and coding system for the 2nd generation library of purine inhibitors of Stat3.
Cl
N
Cl
N
N
N
N
Cl
N
Cl
N
Cl
N
3
N
a
N
b
N
c
d
NBoc
e
N
N
N
O
N
N
N
NHBoc
O
N
Boc
N
H
NH2
N
H
NH2
NHBoc
O
O
1
2
4
5
X
X
N
X
X
N
N
N
N
N
N
N
N
N
N
N
N
f
g
h
N
N
NBoc
NH
N
NH
NH
O
O
O
O
O
O
HO
O
O
Z
O
6a-g
7a-g
8a-g
Z=
Z=
9a-g
O
10a-g
Scheme 1. Reagents and conditions: (a) BOC anhydride, DMSO, DMAP (cat), 0 °C to rt, 30 min, 75%; (b) NaH, THF, rt, 30 min, 95%; (c) (i) ethyl 2-hydroxyacetate, PPh3, THF, rt,
2 min, (ii) DIAD, rt, 15 min, 83%; (d) (i) 4-cyclohexylbenzyl alcohol, PPh3, THF, rt, 2 min, (ii) DIAD, rt, 15 min, 82–74%; (e) X (HNR0R00), DIPEA, DMSO, 105 °C, 40 min, microwave
assisted, 65–97%; (f) TFA:CH2Cl2 (1:1), rt, 1 h, 63–95%; (g) LiOH, THF:H2O (3:1), rt, 15 min, 75–93%; (h) acetoxymethyl bromide, DIPEA, DMF, rt, 6 h; or for 10a–g; (h)
pivoyloxymethyl iodide, DIPEA, DMF, rt, 6 h.
2.3. IC50 determination in tumour cell studies
mia 2 (OCI-AML2)23 were subjected to purine analogs and assessed
for inhibitor-induced cytotoxicity using an MTS colorimetric as-
say.24 Significantly, no biological activity was observed amongst
the library members, with the exception of 22e, substituted at
N9 with a sulphamate group. In MDA-MB-468 cells, 22e showed
To quantify improved cellular efficacy, cell lines known to har-
bor elevated Stat3 levels including, breast cancer (MDA-MB-468),21
human prostate carcinoma (DU 145),22 and acute myeloid leuke-

V. M. Shahani et al. / Bioorg. Med. Chem. 21 (2013) 5618–5628
5621
Cl
X
N
Cl
N
Cl
N
N
N
N
N
N
N
N
N
N
a
b
c
N
N
NBoc
N
NBoc
NHBoc
N
H
OH
NHBoc
O
Si
O
Si
O
Si
O
Si
3
16
17
18
19a-g
X
N
N
X
N
X
N
N
N
N
N
N
N
d
e
f
N
NH
NBoc
N
NBoc
O
S
NH2
O
S
NH2
O
HO
O
O
O
O
S
O
NH2
Cl
20a-g
21a-g
22a-g
23
g
Cl
O
O
S
N
C O
Scheme 2. Reagents and conditions: (a) (i) monosilyated ethylene glycol, PPh3, THF, rt, 2 min, (ii) DIAD, rt, 15 min, 83%; (b) (i) 4-cyclohexylbenzyl alcohol, PPh3, THF, rt,
2 min, (ii) DIAD, rt, 15 min, 82–74%; (c) X (HNR0R00), DIPEA, DMSO, 105 °C, 40 min, microwave assisted, 65–97%; (d) TBAF, THF, rt, 20 min, 80–88%; (e) sulfamoyl chloride, NaH,
THF, rt, 30 min, 75–89%; (f) TFA:CH2Cl2 (1:1), rt, 1 h, 63–95%; (g) formic acid, N2, 0 °C–rt, 81%.
OH
OH
OTs
a
b
N
N
N
N
N
N
NH
N
N
N
Trt
N
Trt
N
Cl
N
29
30
N
N
N
Cl
N
c
d
N
NHBoc
N
N
H
NHBoc
N
N
N
Trt
N
3
31
X
N
X
N
Cl
N
N
N
N
N
N
N
e
N
N
f
NBoc
NH
N
NBoc
N
N
N
N
N
N
N
NH
Trt
N
Trt
N
N
N
33a-g
34a-g
32
Scheme 3. Reagents and conditions: (a) trityl chloride, DBU, DCM, rt, 16 h, 96%; (b) tosylchloride, DMAP, DIPEA, CH2Cl2, 0 ? rt, 16 h, 85%; (c) OTs-trt-tetrazole, DMF, K2CO3,
60 °C, 62%; (d) (i) cyclohexylbenzyl alcohol, PPh3, THF, rt, 2 min, (ii) DIAD, rt, 3 h, 68%; (e) X (HNR0R00), DIPEA, DMSO, 65 °C, 40 min, 32–86 %; (f) TFA:CH2Cl2 (1:1), rt, 1 h, 67–
79%.
promising cell cytotoxicity (EC50 = 19.9 0.9
l
M). Lower potency
MTT assays directly measure levels of cellular metabolism with
decreased levels indicating cell death. However, it is possible that
compound 22e functions as a metabolism suppressor. To evaluate
for compound induced apoptosis, we next conducted an Annexin V
apoptosis assay in the previously indicated cell lines, XG6 and JJN3.
Cells were treated with 22e for 48 h and directly compared to the
activity of the anti-tumor mustard drug, mephalan. Results from
this assay paralleled the results of the MTT, with XG6 cells experi-
encing complete late stage apoptosis after 48 h exposure to 22e at
was observed in the other cell lines treated. For further cellular
evaluation, 22e was incubated with a panel of eleven multiple
myeloma (MM) primary cancer cell lines for 48 h and assessed
for induced cell death using an MTT cytotoxicity assay. Encourag-
ingly, 22e demonstrated potent, dose-dependant cytotoxic effects
in a number of cell lines, whilst being ineffective to others
(Fig. 3). The most sensitive cell line to 22e, XG6, possessing high
levels of activated Stat3, was shown to be completely inhibited
at 10
lM of inhibitor. A surprising result was the success of 22e
a concentration of 15 lM (Fig. 4). Moreover, 22e appears to have
against SKMM2 cells, which do not harbor pStat3. Similarly, JJN3
cells, possessing elevated pStat3 levels were not sensitive to 22e.
Thus, we postulated that 22e’s biological activity was likely a result
of off-target effects and not due to Stat3 inhibition.
significantly more potent effects than mephalan. Biological effects
in JJN3 cells treated with 22e were significantly less than in XG6, as
was previously observed in the MTT assay. With promising IC50
values of sub-10 lM against lethal multiple myeloma cancer cell

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V. M. Shahani et al. / Bioorg. Med. Chem. 21 (2013) 5618–5628
Figure 3. Western blot and the differential cellular response observed in the MTT
assay when multiple myeloma cells are treated with 22e.
lines, we elected to try and identify the intracellular target respon-
sible for 22e’s cytotoxicity.
2.4. Fluoresence polarization (FP) Stat3 binding assay
Figure 4. Annexin assay output developed using Flowo software following 48 h
treatment with 22e alongside positive control, mephalan. Bottom left quandrant
indicates healthy cells, bottom right quandrant visualizes early stage apoptosis, and
top quandrants display late stage apoptosis. Signal strength increases from blue to
red.
To assess for Stat3 SH2 domain binding affinities, purines were
subjected to a competitive Stat SH2 domain FP assay.25 Non-
phosphorylated, full-length Stat3 monomers were incubated with
fluorescently labeled Stat3 SH2 domain binding peptide,
FAM-GpYLPQTV-NH2, and treated with inhibitors and positive con-
trol pYLPQTV. Dose-dependant inhibitor-mediated displacement
of the fluorescent peptide and resultant decreased polarization
signal, facilitates IC50 determination (Fig. 5). Of the 38 inhibi-
tors synthesized, we observed negligible disruption of the
Stat3-phosphopeptide complex at inhibitor concentrations of up
to 400
by MTS assay), failed to displace the fluorescent peptide even at
100 M, suggesting that the Stat3 SH2 domain was not the cellular
lM. Notably, lead anti-cancer compound, 22e (determined
l
target in MM cells.
2.5. Stat3 phosphorylation measured through flow cytometry
While 22e may not inhibit Stat3 SH2 domain complexation
events, it feasibly could be inhibiting de novo Stat3 phosphoryla-
tion in vitro by binding Stat3’s SH2 domain and/or binding else-
where on Stat3 and prevent recruitment to the target kinase and
subsequent phosphorylation. Thus, we conducted phospho-flow
experiments with 22e to rule out a Stat3-related mode of inhibi-
tory action. Thus, MM tumour cells, XG6 and OPM2 were starved
overnight and then treated with various concentrations of 22e
(Fig. 6).26 Starved OPM2 cells were then treated with 22e and stim-
ulated with human interleukin-6. After 30 min, cells were fixed
and stained with mouse anti-pStat3-PE(BD). Samples were run
on FACSCalibur(BD) flow cytometer and analyzed using FlowJo
software. As expected, and in support of our in vitro FP data, 22e
failed to inhibit Stat3 phosphorylation at concentrations above
cytotoxic IC50 values.
Figure 5. Representative purine agents performed poorly in a competitive fluores-
cence polarization assay.
2.6. Selected panel kinase screen and complete kinome screen
Given 22e’s limited in vitro activity against Stat3, we elected to
explore other likely cellular targets to help delineate the observed
potency in myeloma cells. Traditionally, purine therapeutics have
been utilized to great effect for inhibiting protein kinases through
ATP binding site occupation. Since select kinases activate many of

V. M. Shahani et al. / Bioorg. Med. Chem. 21 (2013) 5618–5628
5623
levels compared to control samples. In this screen, hits are classi-
fied as compounds where captured kinase levels fall below a 30%
threshold. Images were generated using TREEspot software tool
and reprinted with permission from KINOMEscan, a division of Dis-
coveRx Corporation, Discoverx Corporation 2010.
Surprisingly, of the 456 kinases treated with 22e, only mutant
abelson murine leukemia viral oncogene homolog
1 (ABL1
(F317L)) and Adapter-associated protein kinase 1 (AAK1) were
substantially inhibited at 50% and 52%, respectively. Inhibitor 22e
did not, with the remaining kinases inhibit the remaining kinases
below the critical range required for designation as a hit compound
(Fig. 8). JAK families kinases, as expected, were minimally inhibited
at the testing concentrations (Supporting Information). ABL1 is one
half of the chimeric protein, BCR-ABL, which results from a recipro-
cal translocation between chromosome 9 and 22.30 The discovery
that fusion oncoprotein BCR-ABL drives the formation and prolifer-
ation of chronic myeloid leukemia (CML) produced potent tyrosine
kinase inhibitors (TKI), specifically the blockbuster drug imatinib.31
Unfortunately, 20% of patients treated with imatinib develop
Figure 6. Phosphorylated Stat3 levels measured by phospho-flow cytometry.
the intracellular factors that promote the cancer phenotype, we ex-
plored 22e’s potency toward kinases via a kinase filtration binding
assay.27 Briefly, a panel of ten cancer related kinases, ABL1, AKT1,
c-Src, CDK1/cyclin B, CDK2/cyclin A, ERK2/MAPK1, FLT3, JAK2,
JAK3, and LCK were tested at a single concentration (50
experiment measures performance of the target kinase in the pres-
ence of 32P- -ATP or 33P-
-ATP. Of the ten kinases screened, JAK2
lM). The
c
c
and JAK3 were 50% and 85% inhibited by 22e, respectively. The
JAK tyrosine kinases are often implicated in the phosphorylation
of oncogenic proteins.28,29 Multi-dose experiments were per-
formed on the four JAK kinases to establish IC50 concentrations.
As shown in Figure 7, 22e exhibited modest activity against
TYK1, JAK2 and JAK3 kinases with IC50’s of 10.6, 16.6, and
10.5
(100
l
l
M, respectively. JAK1 was negligibly inhibited by 22e
M). Given the potency in whole cells, the levels of JAK inhi-
bition exhibited may contribute to the observed biological effect
but are likely not the only target.
Given the structural homology, and the promising activity
against the JAK family of kinases, we reasoned that other kinases
in the greater kinome family may be more strongly inhibited by
22e. Thus, to screen 22e’s inhibitory activity against other cellular
kinases, we employed competitive qPCR screening to identify
activity against a comprehensive ‘DiscoveRx KINOMEscan’ library
of 456 human kinases. In this assay, kinases labeled with DNA were
treated with 22e (2.5 lM single concentration) and incubated with
an immobilized ligand designed to capture its target kinase. Ultra-
sensitive quantitative PCR (qPCR) is employed to measure levels of
immobilized kinases when treated with 22e and relative kinase
Figure 8. A complete kinome scan highlights insensitivity to treatment with 22e.
Highlighted in red are ABL1and AAK1. Images were generated using TREEspot
software tool and reprinted with permission from KINOMEscan, a division of
DiscoveRx Corporation, Discoverx Corporation 2010.
Figure 7. Compound 22e inhibition of JAK family members kinase activity.

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V. M. Shahani et al. / Bioorg. Med. Chem. 21 (2013) 5618–5628
protein mutations leading to drug-resistance.32 One such mutant is
ABL1 (F317L), a variant which demonstrated sensitivity to treat-
ment with 22e. Agents that bind ABL1 mutants and prevent their
activity are potential useful for treatment of CML patients.33 Third
generation TKIs currently in development boast IC50’s 100-fold
lower than that of 22e.34 In addition, aberrant ABL activity is unre-
lated to the progression of multiple myeloma and thus unlikely to
be our biological target in MM cells. Investigation of 22e’s potency
in treating imatinib resistant CML tumour cells may be an interest-
ing line of research.
1.53–1.95 (m, 6H, CH2CH(CH3)2 and 5H (cyclohexyl)), 2.40–2.47
(m, 1H, CH(CH3)2), 3.38 (br s, 2H, CH2CH(CH3)2), 4.24 (q,
J = 7.1 Hz, 2H, CO2CH2CH3), 4.58 (d, J = 5.8 Hz, 2H, CH2Ar), 4.78 (s,
2H, CH2CO2Et), 5.07 (br s, 1H, NH), 5.56 (br s, 1H, NH), 7.15 (d,
J = 7.9 Hz, 2H, 2CH (Ar)), 7.29 (d, J = 7.8 Hz, 2H, 2CH (Ar)), 7.50 (s,
1H, CH (H-8)); dC (100 MHz, CDCl3) 14.0, 20.1, 26.0, 26.8, 28.5,
29.6, 34.4, 43.6, 44.1, 45.5, 61.8, 113.4, 126.7, 127.5, 136.8, 137.4,
146.6, 151.1, 155.0, 159.5, 167.6; HRMS (MS-ES), calcd for
C26H37N6O2 [M+H] m/z = 465.2978, found: 465.2977; rpHPLC tR:
condition (I) 18.274 (II) 38.507 min, purity 96.8% and 92.7%.
AAK1 plays an up-regulatory role in the Notch pathway.35 Stud-
ies show that dysregulation of Notch signaling can lead to various
human diseases, including cancer.36 A recent study demonstrated
that Dll1/Notch interaction increases MM-cell proliferation. Thus,
we postulate that 22e may be acting as a Notch signaling inhibitor
in MM cells investigated with additional off-target effects against
JAK kinases.37 However, the lack of Stat3 knockdown suggests that
the inhibition of upstream JAK kinases is minimal, as greater inhi-
bition of Stat3 phosphorylation would have been expected. Further
investigation into AAK1’s role in multiple myeloma, and 22e’s
inhibitory effect upon this kinase in MM cells is required before
we can more accurately assign the origin of 22e’s biological
activity.
4.1.3. Ethyl 2-(2-((4-cyclohexylbenzyl)amino)-6-
(isopentylamino)-9H-purin-9-yl)acetate (7c)
IR (KBr, cmꢀ1) 3265, 2959, 2926, 2852, 1737, 1681, 1650, 1610,
1522, 1429, 1384, 1261, 1231, 1202, 1127, 1098, 1021; dH
(400 MHz, CDCl3) 0.92 (d, J = 6.5 Hz, 6H, (CH2)2CH(CH3)2), 1.25–
1.39 (m, 9H, 5H (cyclohexyl), CO2CH2CH3, and CH2CH2CH), 1.49–
1.65 (m, 2H, CH2CH2CH(CH3)2), 1.65–1.89 (m, 7H, CH2CH2CH(CH3)2
and 5H (cyclohexyl)), 2.40–2.47 (m, 1H, CH), 3.58 (br s, 1H, NH),
4.02 (s, 1H, NH), 4.24 (q, J = 7.1 Hz, 2H, CO2CH2CH3), 4.58 (s, 2H,
CH2Ar), 4.59 (s, 2H, CH2CO2Et), 7.15 (d, J = 8.1 Hz, 2H, 2CH (Ar)),
7.26 (d, J = 8.1 Hz, 2H, 2CH (Ar)), 7.61 (s, 1H, CH (H-8)); dC
(100 MHz, CDCl3) 14.0, 22.4, 25.6, 26.0, 26.8, 29.6, 34.4, 38.6,
43.6, 44.1, 45.5, 61.8, 113.4, 126.6, 127.5, 136.7, 137.6, 146.6,
151.2, 155.0, 159.8, 167.6; HRMS (MS-ES), calcd for C27H39N6O2
[M+H] m/z = 479.3134, found: 479.3140; rpHPLC tR: condition (I)
20.092 (II) 39.919 min, purity 94.4% and 93.1%.
3. Conclusions
A focused library of hetero-trisubstituted purines was devel-
oped for improving the cell penetrating and biological efficacy of
a series of anti-Stat3 protein inhibitors. From this SAR study, lead
agent 22e was identified as being a promising inhibitor of MM tu-
4.1.4. Ethyl 2-(2-((4-cyclohexylbenzyl)amino)-6-morpholino-
9H-purin-9-yl)acetate (7d)
IR (KBr, cmꢀ1) 3255, 3096, 2925, 2855, 2589, 1787, 1759, 1677,
1608, 1555, 1436, 1215, 1167; dH (400 MHz, CDCl3) 1.27–1.40 (m,
8H, 5H (cyclohexyl) and CO2CH2CH3), 1.67–1.88 (m, 5H (cyclo-
hexyl)), 2.42–2.48 (m, 1H, CH), 3.73–3.80 (m, 4H, 2CH2, (morpho-
line)), 3.94 (br s, 2H, 2 CH2, (morpholine)), 4.27 (q, J = 7.2 Hz, 2H,
CO2CH2CH3), 4.56 (s, 2H, CH2Ar), 4.64 (br s, 2H, 2CH2, (morpho-
line)), 4.97 (s, 2H, CH2CO2Et), 7.13 (d, J = 8.1 Hz, 2H, 2CH (Ar)),
7.23 (d, J = 8.2 Hz, 2H, 2CH (Ar)), 7.40 (s, 1H, CH (H-8)); dC
(100 MHz, CDCl3) 14.1, 26.1, 26.9, 34.5, 43.8, 44.3, 45.7, 62.0,
67.1, 113.4, 126.9, 127.6, 136.1, 137.4, 146.9, 151.3, 155.2, 159.1,
mour cells (IC50’s <5 lM). Surprisingly, biophysical and biochemi-
cal characterization proved that 22e was not a Stat3 inhibitor.
Initial screening against the kinome, prompted by the purine scaf-
fold’s history for targeting ATP binding pockets, suggests possible
targeting of the JAK family kinases, as well for ABL1 (nonphosphor-
ylated F317L) and AAK1. At 2.5 lM the remaining 454 kinases eval-
uated were insensitive to 22e. However, the exact molecular target
responsible for 22e’s promising cytotoxicity in MM remains to be
defined, and is part of an ongoing investigation within our lab.
167.7; HRMS (MS-ES), calcd for
C26H34N6O3 [M+H] m/
4. Experimental section
z = 479.2692, found: 479.2648; rpHPLC tR: condition (I) 15.691
(II) 36.602 min, purity 92.8% and 94.0%.
4.1. Chemistry: final molecule characterization
4.1.5. Ethyl 2-(2-((4-cyclohexylbenzyl)amino)-6-((4-fluoro
phenyl)amino)-9H-purin-9-yl)acetate (7e)
4.1.1. Ethyl 2-(2-((4-cyclohexylbenzyl)amino)-6-
IR (KBr, cmꢀ1) 3357, 2924, 2851, 1747, 1681, 1629, 1594, 1508,
1384, 1208, 1140; dH (400 MHz, CDCl3) 1.26–1.48 (m, 8H, 5H
(cyclohexyl) and CO2CH2CH3), 1.67–1.91 (m, 5H (cyclohexyl)),
2.43–2.49 (m, 1H, CH), 4.26 (q, J = 7.2 Hz, 2H, CO2CH2CH3), 4.60
(s, 2H, CH2Ar), 4.85 (s, 2H, CH2CO2Et), 6.97–7.01 (m, 2H, 2CH
(Ar)), 7.17 (d, J = 7.9 Hz, 2H, 2CH (Ar)), 7.27 (d, J = 8.0 Hz, 2H, 2CH
(Ar)), 7.45–7.78 (m, 4H, 3CH (Ar) and CH (H-8)); dC (100 MHz,
CDCl3) 14.1, 26.1, 26.9, 34.5, 43.8, 44.3, 45.8, 62.1, 113.7, 115.2,
115.4, 121.6, 121.7, 126.9, 127.5, 135.1, 137.1, 137.8, 147.0,
151.7, 152.2, 157.4, 159.5, 159.8, 167.5; HRMS (MS-ES), calcd for
(cyclopentylamino)-9H-purin-9-yl)acetate (7a)
IR (KBr, cmꢀ1) 3373, 2924, 2851, 1753, 1607, 1488, 1384 1261,
1201; dH (400 MHz, CDCl3) 1.18–1.46 (m, 5H, (cyclohexyl)), 1.28 (t,
J = 7.2 Hz, 3H, CO2CH2CH3), 1.46–1.70 (m, 8H, 5H (cyclohexyl) & 3H
(cyclopentyl)), 1.71–1.85 (m, 5H, (cyclopentyl)), 2.03 (br s, 2H
(cyclopentyl)), 2.43–2.50 (m, 1H, CH), 4.24 (q, J = 7.2 Hz, 2H,
CO2CH2CH3), 4.58 (d, J = 5.8 Hz, 2H, CH2Ar), 4.78 (s, 2H, CH2CO2Et),
5.12 (br s, 1H, NH), 5.47 (br s, 1H, NH), 7.15 (d, J = 8.1 Hz, 2H, 2CH
(Ar)), 7.28 (d, J = 8.1 Hz, 2H, 2CH (Ar)), 7.52 (s, 1H, CH (H-8)); dC
(100 MHz, CDCl3) 14.1, 23.8, 26.2, 26.9, 33.4, 34.5, 43.8, 44.3,
45.7, 62.0, 113.4, 126.7, 127.5, 136.8, 137.4, 146.6, 151.1, 155.0,
159.5, 167.6; HRMS (MS-ES), calcd for C27H37N6O2 [M+H] m/
z = 477.2978, found: 465.2990; rpHPLC tR: condition (I) 14.784
(II) 37.297 min, purity 97.4% and 91.7%.
C28H32N6O2 [M+H] m/z = 503.2571, found: 503.2583; rpHPLC tR:
condition (I) 20.913 (II) 40.496 min, purity 92.7% and 85.0%.
4.1.6. Ethyl 2-(2-((4-cyclohexylbenzyl)amino)-6-((furan-2-
ylmethyl)amino)-9H-purin-9-yl)acetate (7f)
IR (KBr, cmꢀ1) 3263, 2923, 2850, 1739, 1678, 1611, 1384, 1200,
1141; dH (400 MHz, CDCl3) 1.25–1.39 (m, 5H, cyclohexyl), 1.63–
1.82 (m, 5H (cyclohexyl)), 2.41–2.49 (m, 1H, CH), 4.16 (q,
J = 7.1 Hz, 2H, CO2CH2CH3), 4.53 (d, J = 5.7 Hz, 2H, CH2NH), 4.65–
4.75 (m, 4H, CH2 (furfuryl) and CH2Ar), 5.19 (br s, 1H, NH), 6.02
4.1.2. Ethyl 2-(2-((4-cyclohexylbenzyl)amino)-6-
(isobutylamino)-9H-purin-9-yl)acetate (7b)
IR (KBr, cmꢀ1) 3263, 2957, 2923, 2851, 1750, 1627, 1602, 1550,
1384, 1261, 1222, 1126; dH (400 MHz, CDCl3) 0.96 (d, J = 6.6 Hz, 6H,
CH2CH(CH3)2), 1.24–1.47 (m, 8H, 5H (cyclohexyl) and CO2CH2CH3),

V. M. Shahani et al. / Bioorg. Med. Chem. 21 (2013) 5618–5628
5625
(br s, 1H, NH (furfuryl)), 6.14–6.16 (m, 1H, CH (furfuryl)), 6.21–6.22
(m, 1H, CH (furfuryl)), 7.10 (d, J = 8.0 Hz, 2H, 2CH (Ar)), 7.21 (d,
J = 8.0 Hz, 2H, 2CH (Ar)), 7.25–7.26 (m, 1H, CH (furfuryl)), 7.43 (s,
1H, CH (H-8)); dC (100 MHz, CDCl3) 14.1, 26.2, 26.9, 34.5, 43.8,
44.3, 45.7, 62.0, 107.3, 110.4, 126.9, 127.6, 137.3, 137.4, 142.0,
146.9, 152.1, 154.4, 167.6; HRMS (MS-ES), calcd for C27H33N6O3
[M+H] m/z = 489.2614, found: 489.2623; rpHPLC tR: condition (I)
17.405 (II) 37.814 min, purity 92.8% and 93.24%.
[M+H] m/z = 551.3346, found: 551.3346; rpHPLC tR: condition (I)
22.228 (II) 41.547 min, purity 99.3% and 98.5%.
4.1.11. (2-(2-((4-Cyclohexylbenzyl)amino)-6-(isopentylamino)-
9H-purin-9-yl)acetoxy)methyl pivalate (9c)
IR (KBr, cmꢀ1) 3417, 2955, 2917, 2849, 1765, 1691, 1430, 1384,
1261, 1205, 1108, 1029; dH (400 MHz, CDCl3) 0.95 (d, J = 7.2 Hz, 6H,
(CH2)2CH(CH3)2), 1.21 (s, 9H, C(CH3)3), 1.24–1.44 (m, 5H, cyclo-
hexyl), 1.55–1.85 (m, 8H, CH2CH2CH(CH3)2 and 5H (cyclohexyl)),
2.43–2.50 (m, 1H, CH), 4.01 (br s, 2H, CH2CH2CH(CH3)2), 4.57 (s,
2H, CH2Ar), 4.82 (s, 2H, CH2CO2H), 5.80 (s, 2H, OCH3), 7.03 (br s,
1H, NH), 7.16 (d, J = 8.0 Hz, 2H, 2CH (Ar)), 7.24 (d, J = 8.0 Hz, 2H,
2CH (Ar)), 7.61 (s, 1H, CH (H-8)); dC (100 MHz, CDCl3) 19.6, 22.3,
26.0, 26.6, 26.7, 28.9, 34.3, 38.6, 43.6, 44.1, 44.5, 51.2, 80.4, 99.8,
110.4, 126.9, 127.3, 134.8, 139.1, 149.4, 152.1, 169.2, 182.6; HRMS
4.1.7. Ethyl2-(2-((4-cyclohexylbenzyl)amino)-6-(4-nitro
phenoxy)-9H-purin-9-yl)acetate (7g)
IR (KBr, cmꢀ1) 3422, 3231, 3074, 2926, 2853, 1748, 1630, 1581,
1526, 1406, 1384, 1251, 1225; dH (400 MHz, CDCl3) 1.29–1.45 (m,
8H, 5H (cyclohexyl) and CO2CH2CH3), 1.73–1.85 (m, 5H (cyclo-
hexyl)), 2.43–2.49 (m, 1H, CH), 4.27 (q, J = 7.1 Hz, 2H, CO2CH2CH3),
4.44 (s, 2H, CH2Ar), 4.86 (s, 2H, CH2CO2Et), 5.28 (br s, 1H, NH), 7.14
(s, 4H, 4CH (Ar)), 7.36 (d, J = 9.9 Hz, 2H, 2CH (Ar)), 7.74 (s, 1H, CH
(H-8)), 8.23 (d, J = 8.7 Hz, 2H, 2CH (Ar)); HRMS (MS-ES), calcd for
(MS-ES), calcd for
C31H45N6O4 [M+H] m/z = 565.3502, found:
565.3496; rpHPLC tR: condition (I) 24.309 (II) 43.125 min, purity
100% and 99.0%.
C28H31N6O5 [M+H] m/z = 531.2356, found: 531.2357; rpHPLC tR:
condition (I) 25.147 (II) 43.779 min, purity 99.2% and 99.3%.
4.1.12. (2-(2-((4-Cyclohexylbenzyl)amino)-6-morpholino-9H-
purin-9-yl)acetoxy)methyl pivalate (9d)
4.1.8. Ethyl 2-(2-(cyclohexanecarboxamido)-6-morpholino-9H-
purin-9-yl)acetate (13)
IR (KBr, cmꢀ1) 3421, 2924, 2851, 1636, 1448, 1384, 1200; dH
(400 MHz, CDCl3) 1.20 (s, 9H, (CH3)3), 1.32–1.45 (m, 5H, cyclohex-
ane), 1.72–1.83 (m, 5H, cyclohexane), 2.44–2.47 (m, 1H, CH (cyclo-
hexane)), 3.76–3.78 (m, 2H, O(CH2)2), 4.20 (m, 2H, N(CH2)2), 4.54
(d, J = 5.0 Hz, 2H, CH2NH), 4.85 (s, 2H, NCH2CO), 5.80 (s, 1H,
OCH2O), 7.14 (d, J = 8.0 Hz, 1H, CH (Ar)), 7.26 (d, J = 8.0 Hz 1H, CH
(Ar)), 7.48 (s, 1H, CH (H-8)); dC (100 MHz, CDCl3) 26.0, 26.6, 26.7,
34.3, 38.6, 44.1, 45.1, 66.6, 80.8, 126.7, 127.1, 135.4, 136.0, 147.0,
152.1, 163.0, 163.3, 165.4, 176.8; HRMS (MS-ES), calcd for
Mp = 142–147 °C; IR (KBr, cmꢀ1) 3551, 3415, 3238, 2928, 2852,
1755, 1669, 1604, 1585, 1514, 1448, 1407, 1327, 1266; dH
(400 MHz, CDCl3) 1.29 (t, J = 7.2 Hz, 3H, CO2CH2CH3), 1.28–1.32
(m, 3H, CH2 (cyclohexyl)), 1.49–1.58 (m, 2H, (cyclohexyl)), 1.69–
1.71 (m, 1H, (cyclohexyl)), 1.82–1.84 (m, 2H, (cyclohexyl)), 1.96–
1.99 (m, 2H, (cyclohexyl)), 2.86–2.91 (m, 1H, CH), 3.82 (t,
J = 4.9 Hz, 4H, 2CH2 (morpholine)), 4.25 (q, J = 7.2 Hz, 2H,
CO2CH2CH3), 4.27 (br s, 4H, 2CH2 (morpholine)), 4.87 (s, 2H,
CH2CO2Et), 7.69 (br s, 1H, NH), 7.70 (s, 1H, CH (H-8)); dC
(100 MHz, CDCl3) 14.3, 26.0, 29.5, 44.2, 45.3, 45.4, 45.5, 62.4,
67.2, 116.4, 138.5, 152.4, 152.7, 154.1, 167.5, 175.9; HRMS (MS-
C30H41N6O4 [M+H] m/z = 565.3138, found: 565.3124; rpHPLC tR:
condition (I) 20.347 (II) 40.128 min, purity 95.8% and 94.1%.
4.1.13. (2-(2-((4-Cyclohexylbenzyl)amino)-6-((4-fluorophenyl)
amino)-9H-purin-9-yl)acetoxy)methyl pivalate (9e)
ES), calcd for
C20H29N6O4 [M+H] m/z = 417.2250, found:
417.2258; rpHPLC tR: condition (I) 4.012 (II) 26.231 min, purity
97.4% and 95.5%.
IR (KBr, cmꢀ1) 3415, 2924, 2851, 1757, 1681, 1594, 1508, 1384,
1203, 1131; dH (400 MHz, CDCl3) 1.22 (s, 9H, C(CH3)3), 1.31–1.45
(m, 5H, cyclohexyl), 1.72–1.88 (m, 5H, cyclohexyl), 2.44–2.53 (m,
1H, CH), 4.60 (s, 2H, CH2Ar), 4.95 (br s, 2H, CH2), 5.83 (s, 2H,
CH2), 7.02–7.08 (m, 2H, C6H4F), 7.17 (d, J = 8.0 Hz, 2H, C6H4), 7.23
(d, J = 7.8 Hz, 2H, C6H4), 7.45–7.51 (m, 2H, C6H4F), 7.61 (br s, 1H,
CH (H-8)); dC (100 MHz, CDCl3) 26.0, 26.6, 26.7, 34.3, 38.6, 44.1,
115.5, 115.7, 126.9, 165.2; HRMS (MS-ES), calcd for C32H37FN6O4
[M+H] m/z = 589.2933, found: 589.2960; rpHPLC tR: condition (I)
25.875 (II) 38.964 min, purity 95.2% and 98.6%.
4.1.9. (2-(2-((4-Cyclohexylbenzyl)amino)-6-(cyclopentyl
amino)-9H-purin-9-yl)acetoxy)methyl pivalate (9a)
IR (KBr, cmꢀ1) 3404, 2927, 2853, 1762, 1686, 1637, 1437, 1384,
1201.7, 1138, 1110; dH (400 MHz, CDCl3) 1.20 (s, 9H, C(CH3)3),
1.22–1.43 (m, 9H, 5H (cyclohexyl) and 4H (cyclopentyl)), 1.67–
1.75 (m, 5H, cyclohexyl), 2.00–2.15 (m, 3H, cyclopentyl), 2.43–
2.48 (m, 1H, CH), 4.56 (s, 2H, CH2Ar), 4.83 (s, 2H, CH2CO2H), 5.80
(s, 2H, OCH2), 6.99 (br s, 1H, NH), 7.15 (d, J = 8.1 Hz, 2H, 2CH
(Ar)), 7.22 (d, J = 7.9 Hz, 2H, 2CH (Ar)), 7.65 (s, 1H, CH (H-8)); dC
(100 MHz, CDCl3) 23.9, 26.3, 27.0, 27.1, 33.7, 34.7, 39.0, 44.0,
44.5, 44.8, 56.2, 80.7, 111.2, 127.3, 127.7, 135.2, 139.5, 147.7,
152.5, 153.6, 163.6, 165.9, 177.1; HRMS (MS-ES), calcd for
4.1.14. (2-(2-((4-Cyclohexylbenzyl)amino)-6-((furan-2-
ylmethyl)amino)-9H-purin-9-yl)acetoxy)methyl pivalate (9f)
IR (KBr, cmꢀ1) 3424, 2961, 2924, 1762, 1685, 1648, 1612, 1429,
1384, 1261; dH (400 MHz, CDCl3) 1.20 (s, 9H, (CH3)3), 1.37–1.45 (m,
5H, cyclohexane), 1.72–1.83 (m, 5H, cyclohexane), 2.45–2.48 (m,
1H, CH (cyclohexane)), 4.77 (m, 2H, CH2 (furan), 4.83 (s, 2H,
CH2CO), 5.80 (s, 2H, OCH2O),), 6.22 (m, 1H, CH (furan)), 6.28–
6.29 (m, 1H, CH (furan)), 7.15 (d, J = 8.0 Hz, 1H, CH (Ar)), 7.26 (d,
J = 8.1 Hz 1H, CH (Ar)), 7.33 (m, 1H, CH (furan)), 7.50 (s, 1H, CH
(H-8)); dC (100 MHz, CDCl3) 26.0, 26.6, 26.7, 34.3, 38.6, 40.4, 44.1,
45.0, 80.3, 99.5, 108.4, 110.3, 123.6, 126.9, 127.3, 139.3, 142.7,
159.1, 177.7; HRMS (MS-ES), calcd for C31H39N6O5 [M+H] m/
z = 575.2982, found: 575.2979; rpHPLC tR: condition (I) 21.615
(II) 41.022 min, purity 96.1% and 94.8%.
C31H43N6O4 [M+H] m/z = 563.3346, found: 563.3351; rpHPLC tR:
condition (I) 22.558 (II) 41.740 min, purity 88.2% and 85.4%.
4.1.10. (2-(2-((4-Cyclohexylbenzyl)amino)-6-(isobutylamino)-
9H-purin-9-yl)acetoxy)methyl pivalate (9b)
IR (KBr, cmꢀ1) 3282, 2961, 2927, 2852, 1761, 1686, 1641, 1433,
1384, 1202, 1109; dH (400 MHz, CDCl3) 1.01 (d, J = 6.0 Hz, 6H,
CH2CH(CH3)2), 1.21 (s, 9H, C(CH3)3), 1.24–1.47 (m, 5H, cyclohexyl),
1.53–1.95 (m, 5H, cyclohexyl), 2.02 (heptet, J = 6.6 Hz, CH(CH3)2),
2.43–2.50 (m, 1H, CH(CH3)2), 3.84–3.87 (m, 2H, CH2CH(CH3)2),
4.58 (s, 2H, CH2Ar), 4.82 (s, 2H, CH2CO2H), 5.81 (s, 2H, OCH2),
7.05 (br s, 1H, NH), 7.16 (d, J = 7.9 Hz, 2H, 2CH (Ar)), 7.26 (d,
J = 7.8 Hz, 2H, 2CH (Ar)), 7.45 (br s, 1H, NH), 7.61 (s, 1H, CH (H-
8)); dC (100 MHz, CDCl3) 19.6, 26.0, 26.6, 26.7, 28.9, 34.3, 38.6,
43.6, 44.1, 44.5, 51.2, 80.3, 110.9, 126.9, 127.3, 134.8, 138.9,
147.4, 152.1, 165.5, 176.8; HRMS (MS-ES), calcd for C30H43N6O4
4.1.15. (2-(2-((4-Cyclohexylbenzyl)amino)-6-(4-nitrophenoxy)-
9H-purin-9-yl)acetoxy)methyl pivalate (9g)
IR (KBr, cmꢀ1) 3441, 2925, 2851, 1770, 1751, 1627, 1581, 1407,
1384, 1348, 1258, 1235, 1114; dH (400 MHz, CDCl3) 1.22 (s, 9H,
C(CH3)3), 1.33–1.45 (m, 5H, cyclohexyl), 1.73–1.85 (m, 5H,

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V. M. Shahani et al. / Bioorg. Med. Chem. 21 (2013) 5618–5628
cyclohexyl), 2.46–2.50 (m, 1H, CH), 4.42 (s, 2H, CH2), 4.94 (s, 2H,
CH2Ar), 5.84 (s, 2H, CH2), 7.09–7.16 (m, 4H, CH (Ar)), 7.36 (d,
J = 8.8 Hz, 2H, CH (Ar)), 7.77 (s, 1H, CH (H-8)), 8.22–8.25 (d,
J = 8.8 Hz, 2H, CH2 (Ar)); dC (100 MHz, CDCl3) 26.3, 27.0, 27.1,
34.7, 39.0, 44.2, 44.4, 45.9, 80.6, 114.34, 122.7, 125.3, 127.2,
127.6, 127.6, 136.2, 140.5. 145.1, 147.6, 157.6, 158.8, 159.5,
166.2, 177.2; HRMS (MS-ES), calcd for C32H37N6O7 [M+H] m/
z = 617.2724, found: 617.2726; rpHPLC tR: condition (I) 29.638
(II) 47.065 min, purity 87.4% and 91.0%.
4.1.20. Acetoxymethyl 2-(2-((4-cyclohexylbenzyl)amino)-6-
morpholino-9H-purin-9-yl)acetate (10d)
IR (KBr, cmꢀ1) 3424, 2924, 2852, 1766, 1606, 1583, 1546, 1450,
1384, 1220, 1164; dH (400 MHz, CDCl3) 1.21–1.44 (m, 5H, cyclo-
hexyl), 1.72–1.83 (m, 5H, cyclohexyl), 2.91 (s, 3H, COCH3), 2.45–
2.50 (m, 1H, CH), 3.77 (t, J = 4.8 Hz, 4H, 2 CH2 (morpholine)), 4.19
(br s, 4H, 2 CH2, (morpholine)), 4.55 (d, J = 5.8 Hz, 2H, CH2Ar),
4.85 (s, 2H, CH2CO2), 5.03 (br s, 1H, NH), 5.79 (s, 2H, OCH2), 7.15
(d, J = 8.0 Hz, 2H, 2 CH (Ar)), 7.27 (d, J = 8.0 Hz, 2H, 2 CH (Ar)),
7.50 (s, 1H, CH, (H-8)); HRMS (MS-ES), calcd for C27H35N6O3
[M+H] m/z = 523.2669, found: 523.2681; rpHPLC tR: condition (I)
13.798 (II) 35.975 min, purity 91.4% and 92.1%.
4.1.16. (2-(2-(cyclohexanecarboxamido)-6-morpholino-9H-
purin-9-yl)acetoxy)methyl pivalate (15a)
IR (KBr, cmꢀ1) 3441, 3229, 2928, 2851, 1774, 1751, 1665, 1604,
1586, 1515, 1384, 1315, 1262, 1113; dH (400 MHz, CDCl3) 1.24 (s,
9H, C(CH3)3), 1.27–1.33 (m, 3H, CH2 (cyclohexyl)), 1.48–1.56 (m,
2H, (cyclohexyl)), 1.68–1.70 (m, 1H, (cyclohexyl)), 1.81–1.83 (m,
2H, (cyclohexyl)), 1.97–2.00 (m, 2H, (cyclohexyl)), 2.88–2.93 (m,
1H, CH), 3.83 (t, J = 4.9 Hz, 4H, 2 CH2 (morpholine)), 4.28 (br s,
4H, 2CH2 (morpholine)), 4.88 (s, 2H, CH2), 5.85 (s, 2H, CH2), 7.68
(br s, 1H, NH), 7.76 (s, 1H, CH (H-8)); HRMS (MS-ES), calcd for
4.1.21. Acetoxymethyl 2-(2-((4-cyclohexylbenzyl)amino)-6-((4-
fluorophenyl)amino)-9H-purin-9-yl)acetate (10e)
IR (KBr, cmꢀ1) 3416, 2925, 2852, 1770, 1682, 1626, 1508, 1203;
dH (400 MHz, CDCl3) 1.26–1.45 (m, 5H, cyclohexyl), 1.72–1.88 (m,
5H, cyclohexyl), 2.12 (s, 3H, COCH3), 2.42–2.48 (m, 1H, CH),
3.45–3.50 (m, 2H, cyclohexyl), 4.60 (s, 2H, CH2Ar), 4.97 (s, 2H,
CH2CO2), 5.82 (s, 2H, OCH2O), 7.03 (t, J = 8.3 Hz, 2H, C6H4F), 7.17
(d, J = 8.0 Hz, 2H, C6H4), 7.25 (d, J = 8.0 Hz, 2H, C6H4), 7.49–7.53
(m, 2H, C6H4F), 7.58 (s, 1H, CH (H-8)); dC (100 MHz, CDCl3) 20.8,
26.3, 27.1, 34.7, 44.5, 45.5, 80.35, 115.8, 116.0, 117.9, 124.5,
127.3, 127.6, 135.5, 147.6, 147.7, 163.9, 164.25, 165.8, 169.6;
HRMS (MS-ES), calcd for C29H31FN6O4 [M+H] m/z = 547.2469,
found: 547.2478; rpHPLC tR: condition (I) 20.286 (II) 40.006 min,
purity 97.4% and 93.8%.
C24H35N6O6 [M+H] m/z = 503.2618, found: 503.2615; rpHPLC tR:
condition (I) 3.745 (II) 24.977 min, purity 80.8% and 80.1%.
4.1.17. Acetoxymethyl 2-(2-((4-cyclohexylbenzyl)amino)-6-
(cyclopentylamino)-9H-purin-9-yl)acetate (10a)
dH (400 MHz, CDCl3) 1.22–1.43 (m, 9H, 5H (cyclohexyl) and 4H
(cyclopentyl)), 1.55 (s, 3H, COCH3), 1.67–1.75 (m, 5H, cyclohexyl),
2.00–2.15 (m, 3H, cyclopentyl), 2.43–2.48 (m, 1H, CH), 4.56 (s,
2H, CH2Ar), 4.83 (s, 2H, CH2CO2H), 5.80 (s, 2H, OCH2), 6.99 (br s,
1H, NH), 7.15 (d, J = 8.1 Hz, 2H, 2CH (Ar)), 7.22 (d, J = 7.9 Hz, 2H,
2CH (Ar)), 7.65 (s, 1H, CH (H-8)); dC (100 MHz, CDCl3) 13.8, 21.7,
27.8, 27.9, 28.3, 43.0, 43.7, 47.3, 79.3, 115.7, 126.5, 127.0, 127.1,
128.0, 140.0, 141.3, 149.8, 153.9, 155.2, 169.2; HRMS (MS-ES),
calcd for C27H37N6O2 [M+H] m/z = 477.2978, found: 477.2991;
rpHPLC tR: condition (I) 17.105 (II) 37.622 min, purity 52.54% and
53.85%.
4.1.22. Acetoxymethyl 2-(2-((4-cyclohexylbenzyl)amino)-6-
((furan-2-ylmethyl)amino)-9H-purin-9-yl)acetate (10f)
IR (KBr, cmꢀ1) 3416, 2924, 2851, 1764, 1685, 1612, 1384, 1261,
1201, 1127; dH (400 MHz, CDCl3) 1.27–1.41 (m, 5H, 5H (cyclo-
hexyl)), 1.69–1.89 (m, 5H (cyclohexyl)), 2.12 (s, 3H, CH3), 2.45–
2.50 (m, 1H, CH), 4.60 (br s, 2H, CH2), 4.76 (s, 1H, NH), 4.88 (br s,
2H, CH2Ar), 5.24 (br s, 2H, CH2), 5.80 (s, 2H, OCH2), 6.29–6.33 (m,
1H, CH (furfuryl)), 6.35 (br s, 1H, CH (furfuryl)), 7.16 (d,
J = 8.1 Hz, 2H, C6H4), 7.25 (d, J = 7.9 Hz, 2H, C6H4), 7.26 (br s, 1H,
CH), 7.96 (s, 1H, CH (H-8)); dC (100 MHz, CDCl3) 13.9, 20.5, 22.5,
26.0, 26.8, 29.6, 34.4, 36.6, 43.4, 44.1, 45.5, 63.5, 79.7. 107.1,
110.2, 126.8, 127.5, 136.7, 141.8, 146.8, 152.0, 166.4, 169.2; HRMS
4.1.18. Acetoxymethyl 2-(2-((4-cyclohexylbenzyl)amino)-6-
(isobutylamino)-9H-purin-9-yl)acetate (10b)
IR (KBr, cmꢀ1) 3386, 2927, 2853, 1774, 1685, 1642, 1515,
1433.53, 1367, 1202, 1139; dH (400 MHz, CDCl3) 1.19–1.28 (m,
2H, CH2CH), 1.29–1.33 (d, J = 6.7 Hz, 6H, CH(CH3)2), 1.32–1.43 (m,
5H, cyclohexyl), 1.70–1.90 (m, 5H, cyclohexyl), 1.96–2.08 (m, 1H,
CH(CH3)2), 2.11 (s, 3H, COCH3), 2.43–2.49 (m, 1H, CH), 3.84 (t,
J = 6.6 Hz, 2H, NHBn), 4.56 (s, 1H, NHCH2CH), 4.57 (s, 2H, CH2),
4.83 (s, 2H, CH2CO2), 5.80 (s, 2H, OCH2O), 7.16 (d, J = 7.7 Hz, 2H,
C6H4), 7.24 (d, J = 7.9 Hz, 2H, C6H4), 7.49–7.53 7.63 (s, 1H, CH (H-
8)); dC (100 MHz, CDCl3) 19.7, 20.4, 26.0, 26.7, 28.9, 34.3, 43.5,
44.1, 44.5, 51.2, 79.9, 110.9, 126.9, 127.4, 134.9, 138.9, 147.4,
149.6, 152.1, 165.6, 169.2; HRMS (MS-ES), calcd for C27H37N6O4
[M+H] m/z = 509.2876, found: 509.2887; rpHPLC tR: condition (I)
15.851 (II) 36.589 min, purity 97.2% and 95.6%.
(MS-ES), calcd for
C28H32N6O5 [M+H] m/z = 533.2512, found:
533.2521; rpHPLC tR: condition (I) 15.849 (II) 36.522 min, purity
96.8% and 96.7%.
4.1.23. Acetoxymethyl 2-(2-((4-cyclohexylbenzyl)amino)-6-(4-
nitrophenoxy)-9H-purin-9-yl)acetate (10g)
IR (KBr, cmꢀ1) 3423, 2926, 2851, 1769, 1627, 1577, 1524, 1384,
1346, 1237; dH (400 MHz, CDCl3) 1.33–1.43 (m, 5H, cyclohexyl),
1.73–1.84 (m, 5H, cyclohexyl), 2.13 (s, 3H, CH3) 2.46–2.50 (m,
2H, CH), 4.42 (s, 2H, CH2), 4.96 (s, 2H, CH2) 5.70 (s, 1H, NH), 5.83
(s, 2H, CH2), 7.02–7.12 (m, 4H, CH (Ar)), 7.35 (d, J = 8.6 Hz, 2H,
CH (Ar)), 7.86 (s, 1H, CH (H-8)), 8.23 (d, J = 8.6 Hz, 2H, CH (Ar));
dC (100 MHz, CDCl3) 20.8, 26.3, 27.1, 34.7, 44.4, 45.8, 80.2, 122.7,
125.3, 127.0, 127.2, 127.6, 136.0, 140.6, 145.2, 147.6, 157.4,
158.8, 159.5, 166.1, 169.6; HRMS (MS-ES), calcd for C29H31N6O7
[M+H] m/z = 575.2254, found: 575.2253; rpHPLC tR: condition (I)
23.736 (II) 42.657 min, purity 87.0% and 83.6%.
4.1.19. Acetoxymethyl 2-(2-((4-cyclohexylbenzyl)amino)-6-
(isopentylamino)-9H-purin-9-yl)acetate (10c)
dH (400 MHz, CDCl3) 0.91 (d, J = 7.1 Hz, 6H, (CH2)2CH(CH3)2),
1.23–1.27 (m, 9H, 5H (cyclohexyl), 1.47–1.53 (m, 2H,
CH2CH2CH(CH3)2), 1.64–1.83 (m, 7H, CH2CH2CH(CH3)2 and 5H
(cyclohexyl)), 2.10 (s, 3H, COCH3), 2.40–2.52 (m, 1H, CH), 3.56 (br
s, 1H, NH), 4.02 (s, 1H, NH), 4.58 (s, 2H, CH2Ar), 4.83 (s, 2H,
CH2CO2), 5.79 (s, 2H, OCH2), 7.14 (d, J = 8.2 Hz, 2H, 2CH (Ar)),
7.26 (d, J = 8.1 Hz, 2H, 2CH (Ar)), 7.48 (s, 1H, CH (H-8)); dC
(100 MHz, CDCl3) 14.1, 20.5, 20.9, 22.4, 22.6, 26.0, 26.8, 34.3
38.5, 43.3, 44.1, 45.4, 60.6, 76.6, 76.9, 77.3, 79.7, 126.7, 127.5;
HRMS (MS-ES), calcd for C31H45N6O4 [M+H] m/z = 565.3502, found:
565.3496.
4.1.24. Acetoxymethyl 2-(2-(cyclohexanecarboxamido)-6-
morpholino-9H-purin-9-yl)acetate (15b)
dH (400 MHz, CDCl3) 1.18–1.38 (m, 3H, cyclohexyl), 1.44–1.55
(m, 2H, cyclohexyl), 1.66–1.72 (m, 1H, cyclohexyl), 1.77–1.84 (m,
2H, cyclohexyl), 1.92–1.99 (m, 2H, cyclohexyl), 2.13 (s, 3H, COCH3),
2.62–2.71 (m, 1H, cyclohexyl), 3.84–3.89 (m, 4H, morpholine), 4.34
(br s, 4H, morpholine), 5.03 (s, 2H, CH2CO2), 5.81 (s, 2H, OCH2), 7.71
(s, 1H, CH (H-8)); dC (100 MHz, CDCl3) 20.4, 25.3, 25.5, 29.0, 44.6,

V. M. Shahani et al. / Bioorg. Med. Chem. 21 (2013) 5618–5628
5627
45.4, 66.6, 79.9, 115.7, 138.7, 148.4, 149.6, 152.1, 165.7, 169.3,
176.7; HRMS (MS-ES), calcd for 21H29N6O6 [M+H] m/
z = 461.2143, found: 461.2134; rpHPLC tR: condition (I) 4.491 (II)
7.27 (d, J = 7.7 Hz, 2H (Ar)), 7.68 (dd, J = 8.9 and 4.8 Hz, 2H, 2 CH
(Ar))), 8.15 (s, 1H, CH (H-8)); dc NMR (100 MHz, MeOD) 27.3,
28.0, 28.4, 35.8, 44.1, 45.7, 48.4, 49.6, 52.3, 68.3, 83.5, 101.4,
116.2, 116.4, 123.9, 127.7, 128.0, 128.2, 137.3, 143.6, 148.1,
C
27.159 min, purity 94.8% and 88.1%.
156.2; HRMS (MS-ES), calcd for
C26H31FN7O3S [M+H] m/
4.1.25. 2-(2-((4-Cyclohexylbenzyl)amino)-6-(cyclopentyl-
amino)-9H-purin-9-yl)ethyl sulfamate (22a)
z = 540.2193, found: 540.2205; rpHPLC tR: condition (I) 15.449
(II) 36.313 min, purity 97.3% and 96.8%.
IR (KBr, cmꢀ1) 3384, 2924, 2851, 1609, 1541, 1513, 1489, 1351,
1179, 1017, 912, 787, 551; dH (400 MHz, DMSO) 1.12–1.25 (m, 2H,
cyclohexyl), 1.25–1.41 (m, 4H, cyclohexyl), 1.41–1.57 (m, 4H,
cyclopentyl), 1.58–1.68 (m, 9H, cyclopentyl and cyclohexyl),
2.35–2.46 (m, 1H, cyclohexyl), 4.27 (t, J = 4.8 Hz, 2H,
H2NSO3CH2CH2N), 4.33 (t, J = 4.9 Hz, H2NSO3CH2CH2N), 4.39 (d,
J = 6.1 Hz, Ar-CH2), 6.85 (br s, 1H, cyclopentyl-NH), 7.05 (br s, 1H,
cyclohexylbenzyl-NH), 7.09 (d, J = 7.9 Hz, 2H, ArCH), 7.23 (d,
J = 7.9 Hz, 2H, ArCH), 7.55 (s, 2H, H2NSO3), 7.64 (s, 1H, ArCH); dC
(100 MHz, DMSO) 23.4, 25.6, 26.3, 32.1, 34, 41.6, 43.5, 44.3, 66.6,
126.2, 127.4, 137.0, 138.9, 145.5, 159.1; HRMS (MS-ES), calcd for
4.1.30. 2-(2-((4-Cyclohexylbenzyl)amino)-6-((furan-2-yl
methyl)amino)-9H-purin-9-yl)ethyl sulfamate (22f)
dH (400 MHz, DMSO-d6) 1.03–1.48 (m, 5H, cyclohexyl), 1.59–
1.88 (m, 5H, cyclohexyl), 2.43–2.49 (m, 1H, cyclohexyl), 4.29 (t,
J = 5.2 Hz, 2H, OCH2CH2N), 4.35 (t, J = 5.2 Hz, 2H, OCH2CH2N),
4.42 (d, J = 5.4 Hz, 2H, CH2Ar), 4.58 (s, 1H, ArNH), 6.15 (s, 1H,
ArNH), 6.31 (t, J = 2.6 Hz, 1H, furylamine), 7.10 (d, J = 7.8 Hz, 2H,
2CH (Ar)), 7.24 (d, J = 7.7 Hz, 2H, 2CH (Ar)), 7.50 (d, J = 1.8 Hz, 1H,
furylamine), 7.56 (s, 1H, furylamine), 7.69 (s, 1H, CH (H-8)); dC
(100 MHz, DMSO-d6) 25.6, 26.3, 34.0, 38.8, 39.0, 41.6, 43.4, 44.1,
47.3, 47.5, 47.7, 66.5, 106.5, 110.3, 126.2, 127.5, 137.5, 141.5;
HRMS (MS-ES), calcd for C26H38N7O3S [M+H] m/z = 526.2237,
found: 526.2239; rpHPLC tR: condition (I) 12.579 (II) 34.259 min,
purity 99.3% and 98.1%.
C25H36N7O3S [M+H] m/z = 514.2600, found: 514.2588; rpHPLC tR:
condition (I) 14.026 (II) 35.274 min, purity 91.7% and 92.2%.
4.1.26. 2-(2-((4-Cyclohexylbenzyl)amino)-6-(isobutylamino)-
9H-purin-9-yl)ethyl sulfamate (22b)
IR (KBr cmꢀ1) 3463, 3426, 3314, 2923, 2851, 1616, 1522, 1384,
1175, 1020, 919, 786, 550; dH (400 MHz, CDCl3) 0.96 (d, J = 6.6 Hz,
6H, CH(CH3)2), 1.06–1.49 (m, 5H, cyclohexyl), 1.70–2.10 (m, 7H,
cyclohexyl and alkyl), 2.48 (s, 1H, cyclohexyl), 3.37 (s, 2H, alkyl),
4.36 (t, J = 4.9 Hz, 2H, NH2SO3CH2CH2)), 4.51 (t, J = 5.0 Hz, 2H,
NH2SO3CH2CH2)), 4.57 (d, J = 5.7 Hz, 2H, NH-CH2), 5.23 (s, 1H,
NH), 5.78 (s, 1H, NH), 7.17 (d, J = 7.8 Hz, 2H, 2 CH (Ar)), 7.29 (s,
2H, 2 CH (Ar)), 7.45 (d, 1H, CH (H-8)); LRMS (MS-ES) calcd for
4.1.31. 2-(2-(Cyclohexanecarboxamido)-6-morpholino-9H-
purin-9-yl)ethyl sulfamate (28)
IR (KBr, cmꢀ1) 3299, 2924, 2854, 1667, 1594, 1578, 1467, 1384,
1306, 1263, 1235, 1179; dH (400 MHz, DMSO-d6) 1.08–1.39 (m, 5H,
(cyclohexyl)), 1.60–1.79 (m, 5H, (cyclohexyl)), 2.62–2.68 (m, 1H,
(cyclohexyl)), 3.69–3.71 (m, 4H, (morpholine)), 4.18 (br s, 2H,
CH2N), 4.38–4.42 (m, 4H, (morpholine)), 6.73 (br s, 2H, NH2),
7.55 (s, 2H, OCH2), 8.04 (s, 1H, CH (H-8)), 9.99 (s, 1H, NH); dC
(100 MHz, DMSO-d6) 25.6, 25.8, 29.3, 42.5, 44.3, 66.5, 66.8, 116.2,
140.0, 151.9, 152.4, 153.1, 158.4, 174.9, 194.8; HRMS (MS-ES),
calcd for C18H27N7O5S [M+H] m/z = 454.1873, found: 454.1886;
rpHPLC tR: condition (I) 3.209 (II) 24.516 min, purity 92.6% and
92.4%.
C24H35N7O3S [M+H] m/z = 502.25, found: 502.42.
4.1.27. 2-(2-((tert-Butoxycarbonyl)(4-cyclohexylbenzyl)amino)-
6-(isopentylamino)-9H-purin-9-yl)ethyl sulfamate (22c)
IR (KBr, cmꢀ1) 3425, 3275, 2980, 2940, 2868, 1761, 1705, 1625,
1495, 1390, 1270, 1208; dH (400 MHz, MeOD) 0.92 (d, J = 6.6 Hz,
6H, CH(CH3)2) 1.18–2.00 (m, 15H, alkyl and cyclopentyl), 2.46
(m, 1H, cyclohexyl), 3.44–3.60 (m, 2H, –NH2), 4.40 (dt, J = 8.3,
4.0 Hz, 4H, SOCH2CH2), 4.56 (s, 2H, ArNH-CH2), 7.13 (d, J = 8.1 Hz,
2H, 2Ar (CH)), 7.26 (d, J = 7.8 Hz, 2H, 2Ar (CH)), 7.69 (s, 1H, Ar
(CH)); dc NMR (100 MHz, MeOD) 20.8, 23.0, 27.0, 27.3, 28.0, 35.8,
39.7, 43.5, 45.7, 46.2, 49.7, 54.8, 68.3, 114.2, 127.7, 128.4, 139.0,
139.38, 147.7, 156.2, 161.2; HRMS (MS-ES), calcd for C25H37N7O3S
[M+H] m/z = 516.2757, found: 516.2758; rpHPLC tR: condition (I)
15.891 (II) 36.656 min, purity 99.5% and 99.6%.
4.1.32. 9-(2-(1H-Tetrazol-5-yl)ethyl)-N2-(4-cyclohexylbenzyl)-
N6-cyclopentyl-9H-purine-2,6-diamine (34a)
IR (KBr, cmꢀ1) 3384, 2924, 2851, 1676, 1636, 1448, 1384, 1351,
1203, 1139; dH (400 MHz, DMSO-d6) 1.28–1.53 (m, 9H, 5H (cyclo-
hexyl) and 4H (cyclopentyl)), 1.65–1.91 (m, 10 H, 5H (cyclohexyl)
and 5H (cyclopentyl)), 2.41–2.45 (m, 1H, CH (cyclohexyl), 3.41–
3.45 (m, 2H, NCH2CH2), 4.39–4.43 (m, 2H, NCH2CH2), 4.41 (br s,
2H, CH2 (benzyl)), 7.09 (br s, 1H, NH), 7.11 (d, J = 7.9 Hz, 2H
C6H4), 7.26 (d, J = 7.9 Hz, 2H, C6H4), 7.65 (s, 1H, CH (H-8)); dC
(100 MHz, DMSO-d6) 23.4, 23.5, 25.6, 26.4, 32.1, 32.2, 34.1, 40.7,
43.5, 44.4, 126.3, 126.3, 127.7, 127.7, 129.4, 137.4, 138.3, 145.9,
145.9, 153.7; HRMS (MS-ES), calcd for C26H35N10 [M+H] m/
z = 487.3046, found: 487.3044; rpHPLC tR: condition (I) 12.326
(II) 34.008, purity 96.6% and 94.6%.
4.1.28. 2-(2-((4-Cyclohexylbenzyl)amino)-6-morpholino-9H-
purin-9-yl)ethyl sulfamate (22d)
dH (400 MHz, DMSO-d6) 0.99–1.49 (m, 5H, cyclohexyl),
1.57–1.87 (m, 5H, cyclohexyl), 2.42–2.45 (m, 1H, cyclohexyl),
3.98–4.03 (m, 6H, morpholine and OCH2CH2N), 4.24–4.53 (m, 6H,
morpholine and OCH2CH2N), 7.11 (d, J = 7.8 Hz, 2H, 2CH (Ar)),
7.24 (d, J = 7.8 Hz, 2H, 2CH (Ar)), 7.74 (s, 1H, CH (H-8)); dC
(100 MHz, DMSO-d6) 25.5, 26.3, 34.0, 38.6, 38.8, 41.8, 43.5, 44.2,
45.1, 47.0, 47.2, 47.5, 47.7, 47.9, 66.1, 66.4, 126.2, 127.4, 137.11,
4.1.33. 9-(2-(1H-Tetrazol-5-yl)ethyl)-N2-(4-cyclohexylbenzyl)-
N6-isobutyl-9H-purine-2,6-diamine (34b)
IR (KBr, cmꢀ1) 3404, 2924, 2851, 1615, 1514, 1447, 1384, 1351,
1262; dH (400 MHz, DMSO-d6) 0.82 (d, J = 6.3 Hz, 6H,
CH2CH(CH3)2), 1.23–1.37 (m, 5H, cyclohexyl), 1.66–1.76 (m, 5H,
cyclohexyl), 1.83–1.87 (m, 1H, CH(CH3)2), 2.43–2.50 (m, 1H,
CH(CH3)2), 3.17 (br s, 2H, CH2CH(CH3)2), 3.39 (t, J = 6.9 Hz, 2H
NCH2CH2), 4.37 (t, J = 7.1 Hz, 2H, NCH2CH2), 4.37 (br s, 2H, CH2
(benzyl)), 6.88 (br s, 1H, NH), 7.09 (d, J = 8.1 Hz, 2H, 2CH (Ar)),
7.25 (d, J = 8.0 Hz, 2H, 2CH (Ar)), 7.23 (br s, 1H, NH), 7.61 (s, 1H,
CH (H-8)); dC (100 MHz, DMSO-d6) 20.1, 23.68, 25.6, 26.3, 34.0,
43.4, 44.3, 78.6, 78.9, 79.2, 126.1, 127.4, 127.5, 127.5, 136.7,
138.9, 145.5, 154.0, 154.8, 159.1; HRMS (MS-ES), calcd for
138.3,; HRMS (MS-ES), calcd for
C24H34N7O4S [M+H]
m/z = 516.2393, found: 516.2396; rpHPLC tR: condition (I) 10.709
(II) 32.895 min, purity 98.4% and 98.6%.
4.1.29. 2-(2-((4-Cyclohexylbenzyl)amino)-6-((4-fluorophenyl)
amino)-9H-purin-9-yl)ethyl sulfamate (22e)
dH (400 MHz, MeOD) 1.30 (m, 5H, cyclohexyl), 1.83 (m, 5H,
cyclohexyl), 2.49 (s, 1H, cyclohexyl), 4.50 (t, J = 5.0 Hz, 2H,
OCH2CH2N), 4.59 (d, J = 5.0 Hz, 2H, OCH2CH2N), 5.11 (s, 2H, CH2Ar),
6.97 (t, J = 8.6 Hz, 2H, CH (Ar)), 7.16 (d, J = 8.0 Hz, 2H, 2 CH (Ar)),

5628
V. M. Shahani et al. / Bioorg. Med. Chem. 21 (2013) 5618–5628
C25H35N10 [M+H] m/z = 475.3046, found: 475.3037; rpHPLC tR:
condition (I) 12.328 (II) 34.028 min, purity 97.0% and 96.8%.
1H, CH (H-8)), 9.88 (s, 1H, CONH); HRMS (MS-ES), calcd for
C19H26N10O2 [M+H] m/z = 427.2318, found: 427.2318; rpHPLC tR:
condition (I) 2.859 (II) 22.922 min, purity 98.2% and 96.1%.
4.1.34. 9-(2-(1H-Tetrazol-5-yl)ethyl)-N2-(4-cyclohexylbenzyl)-
N6-isopentyl-9H-purine-2,6-diamine (34c)
Acknowledgments
IR (KBr, cmꢀ1) 3417, 2924, 2851, 1643, 1603, 1514, 1384, 1261,
1126; dH (400 MHz, DMSO-d6) 0.95 (d, J = 6.6 Hz, 6H, CH(CH3)2),
1.31–1.43 (m, 5H, cyclohexyl), 1.55–1.58 (m, 1H, CH(CH3)2),
1.64–1.78 (m, 7H, 5H (cyclohexyl) and 2H (CH2CH(CH3)2)), 2.39–
2.44 (m, 1H, cyclohexyl), 3.33 (br s, 2H, CH2CH2CH), 3.38–3.42
(m, 2H, NCH2CH2), 4.36–4.41 (m, 4H, 2H (NCH2CH2) and CH2 (ben-
zyl)), 6.86 (br s, 1H, NH), 7.09 (d, J = 8.0 Hz, 2H, C6H4), 7.18 (s, 1H,
NH), 7.25 (d, J = 7.9 Hz, 2H, C6H4), 7.52 (s, 1H, CH (H-8)); HRMS
(MS-ES), calcd for C26H37N10 [M+H] m/z = 489.3203, found:
489.3197; rpHPLC tR: condition (I) 14.342 (II) 35.483 min, purity
98.3% and 98.1%.
This work was supported by NSERC (P.G.), Leukemia and Lym-
phoma Society of Canada (P.G.) and by an Ontario Graduate Schol-
arship (V.M.S.). The authors would like to thank Dr. Rob C. Laister
and the Minden group for generously providing full length purified
Stat3 protein.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.04.080.
4.1.35. 9-(2-(1H-Tetrazol-5-yl)ethyl)-N-(4-cyclohexylbenzyl)-6-
morpholino-9H-purin-2-amine (34d)
References and notes
IR (KBr, cmꢀ1) 3424, 3082, 1924, 2851, 2359, 1682, 1602, 1550,
1439, 1384, 1198, 1184; dH (400 MHz, DMSO-d6) 1.23–1.37 (m, 5H,
cyclohexyl), 1.66–1.76 (m, 5H, cyclohexyl), 2.39–2.44 (m, 1H,
cyclohexyl), 3.50 (t, J = 6.8 Hz, 2H, NCH2CH2), 3.60–3.70 (m, 4H,
morpholine), 4.11 (br s, 4H, morpholine), 4.44–4.48 (m, 4H,
NHCH2CH2 and CH2Ar), 7.12 (d, J = 8.0 Hz, 2H, C6H4), 7.26 (d,
J = 8.0 Hz, 2H, C6H4), 7.75 (s, 1H, CH (H-8)); dC (100 MHz, DMSO-
d6) 23.4, 25.6, 26.3, 34.0, 40.8, 43.4, 44.3, 45.3, 113.0, 114.3,
117.2, 126.4, 137.3, 137.8, 145.9, 158.3, 158.7, 159.1; HRMS (MS-
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C25H33N10
O
[M+H] m/z = 489.2839, found:
489.2849; rpHPLC tR: condition (I) 9.251 (II) 31.975 min, purity
98.5% and 98.2%.
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4.1.36. 9-(2-(1H-tetrazol-5-yl)ethyl)-N2-(4-cyclohexylbenzyl)-
N6-(4-fluorophenyl)-9H-purine-2,6-diamine (34e)
IR (KBr, cmꢀ1) 3444.9, 2962, 2924, 2851, 1629, 1592, 1507,
1384, 1262, 1223, 1098; dH (400 MHz, DMSO-d6) 1.25–1.37 (m,
5H, (cyclohexyl)), 1.63–1.75 (m, 5H, (cyclohexyl)), 2.36–2.45 (m,
1H, (cyclohexyl)), 4.39 (s, 2H, ArCH2), 4.41 (t, J = 6.3 Hz, 2H,
NCH2CH2), 6.99 (t, J = 8.9 Hz, 2H, C6H4F), 7.11 (d, J = 8.0 Hz, 2H,
C6H4), 7.25 (d, J = 7.8 Hz, C6H4), 7.70 (s, 1H, CH (H-8)), 7.79–7.90
(m, 2H, C6H4F); HRMS (MS-ES), calcd for C27H30F1N6 [M+H] m/
z = 513.2639, found: 513.26567; rpHPLC tR: condition (I) 14.524
(II) 35.649 min, purity 93.5% and 92.8%.
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4.1.37. 9-(2-(1H-Tetrazol-5-yl)ethyl)-N-(4-cyclohexylbenzyl)-6-
(4-nitrophenoxy)-9H-purin-2-amine (34g)
IR (KBr, cmꢀ1) 3415, 2924, 2851, 1631, 1576, 1447, 1384, 1231
1162; dH (400 MHz, CDCl3) 1.35–1.40 (m, 5H, cyclohexyl), 1.65–
1.83 (m, 2H, (CH2)3CH2CH3), 2.46–2.50 (m, 2H, CH2(CH2)3CH3),
3.59–3.63 (m, 2H, NCH2CH2), 4.51 (br s, 2H, CH2Ar), 4.59–4.63
(m, 2H, NCH2CH2) = 7.18 (br s, 1H, NH), 7.25–7.33 (m, 6H, 4CH
(benzyl) and 2CH (nitrophenol)), 7.73 (s, 1H, CH (H-8)), 8.24–
8.26 (m, 2H, CH (nitrophenol)); dC (100 MHz, DMSO-d6) 23.4,
25.6, 26.37, 34.0, 41.0, 43.5, 44.4, 80.58, 113.6, 122.7, 125.3,
126.3, 126.7, 127.8, 137.6, 141.4, 144.3, 145.8, 147.8, 157.7,
158.4, 158.4; HRMS (MS-ES), calcd for C27H29N10O3 [M+H] m/
z = 541.2417, found: 541.2418; rpHPLC tR: condition (I) 17.407
(II) 37.851 min, purity 95.2% and 94.1%.
32. Jabbour, E.; Kantarjian, H. M.; Jones, D.; Reddy, N.; O’Brien, S.; Garcia-Manero,
G.; Burger, J.; Cortes, J. Blood 2008, 112, 4839.
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4.1.38. N-(9-(2-(1H-Tetrazol-5-yl)ethyl)-6-morpholino-9H-
purin-2-yl)cyclohexanecarboxamide (37)
IR (KBr, cmꢀ1) 2929, 2853, 1697, 1596, 1450, 1384, 1263, 1195,
1113; dH (400 MHz, DMSO-d6) 1.19–1.26 (m, 5H, cyclohexyl), 1.64–
1.81 (m, 5H, cyclohexyl), 2.68–2.72 (m, 1H, cyclohexyl), 3.45 (t,
J = 7.0 Hz, 2H, NCH2CH2), 3.69 (t, J = 4.6 Hz, 4H, morpholine), 4.18
(br s, 4H, morpholine), 4.50 (t, J = 7.0 Hz, 2H, NCH2CH2), 7.92 (s,