The discovery of purine-based agents targeting triple-negative breast cancer and the αB-crystallin/VEGF protein–protein interaction

Article abstract of DOI:10.1007/s00044-018-2275-9

Oestrogen receptor-negative breast cancer, particularly subtypes such as triple-negative breast cancer (TNBC, around 10–15% of cases), are characterised by poor long-term survival, poor response to therapy and early progression to metastasis. Purine-based compounds represent a privileged scaffold in anticancer drug design, with several clinically approved and experimental agents in clinical development comprising a purine core structure. In this study, a series of new purine-based compounds were synthesised; seven of the new analogues were found to significantly reduce the in vitro viability of TNBC cell lines (MDA-MB-231 and MDA-MB-436) with IC₅₀ values of ≤50 μM. In previous work, we have proposed a new concept for targeting angiogenesis driving TNBC progression, by disrupting the protein–protein interaction between the molecular chaperone αB-crystallin (CRYAB) and VEGF. Since previous clinical studies applying anti-VEGF therapy to TNBC patients have met with limited success, we were interested to test our most promising purine analogues against CRYAB/VEGF, using a custom-designed cell-based CRYAB/VEGF₁₆₅ interaction assay platform. Analogues 4e and 4f significantly reduced the interaction between CRYAB/VEGF₁₆₅, and compound 4e (100 μM) was also found to decrease the levels of soluble VEGF expressed by MDA-MB-231 cells by 40%. In conclusion, these promising early activity profiles warrant further investigation to validate this concept.

Full text of DOI:10.1007/s00044-018-2275-9

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-018-2275-9
ORIGINAL RESEARCH
The discovery of purine-based agents targeting triple-negative
breast cancer and the αB-crystallin/VEGF protein–protein interaction
1
Nelly A. Fosu-Mensah1,2 Wen Jiang2 Andrea Brancale1 Jun Cai2 Andrew D. Westwell
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Received: 22 June 2018 / Accepted: 8 December 2018
© The Author(s) 2018
Abstract
Oestrogen receptor-negative breast cancer, particularly subtypes such as triple-negative breast cancer (TNBC, around 10–
15% of cases), are characterised by poor long-term survival, poor response to therapy and early progression to metastasis.
Purine-based compounds represent a privileged scaffold in anticancer drug design, with several clinically approved and
experimental agents in clinical development comprising a purine core structure. In this study, a series of new purine-based
compounds were synthesised; seven of the new analogues were found to significantly reduce the in vitro viability of TNBC
cell lines (MDA-MB-231 and MDA-MB-436) with IC50 values of ≤50 μM. In previous work, we have proposed a new
concept for targeting angiogenesis driving TNBC progression, by disrupting the protein–protein interaction between the
molecular chaperone αB-crystallin (CRYAB) and VEGF. Since previous clinical studies applying anti-VEGF therapy to
TNBC patients have met with limited success, we were interested to test our most promising purine analogues against
CRYAB/VEGF, using a custom-designed cell-based CRYAB/VEGF165 interaction assay platform. Analogues 4e and 4f
significantly reduced the interaction between CRYAB/VEGF165, and compound 4e (100 μM) was also found to decrease the
levels of soluble VEGF expressed by MDA-MB-231 cells by 40%. In conclusion, these promising early activity profiles
warrant further investigation to validate this concept.
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●
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Keywords Triple-negative breast cancer Anticancer Purines αB-crystallin VEGFR
Introduction
breast cancer characterised by the lack of expression of
the oestrogen receptor (ER), progesterone receptor (PR)
and human epidermal growth factor receptor 2 (HER2)
(Fosu-Mensah et al. 2015; Lehmann et al. 2011; Mayer
et al. 2014). As a subset of breast cancer cases, TNBC is
also a “disease of the young”, as it frequently affects pre-
menopausal women of minority background, including
black and Hispanic women. It is an aggressive disease
associated with high histological grade, increased risk of
recurrence and metastatic spread and poor prognosis.
Currently, the standard of care treatment for TNBC
patients involves the use of cytotoxic chemotherapy (e.g.,
taxanes). However, this treatment lacks long-term
efficacy.
Angiogenesis, the development of new blood vessels
from existing vasculature, is essential for early stage
tumourigenesis and metastasis. This process is regulated by
several pro-angiogenic factors, including vascular endo-
thelial growth factor (VEGF). TNBC is highly vascularised
as a result of upregulation of intra-tumoural VEGF levels,
which in turn is associated with poor prognosis. In this
regard, anti-angiogenic therapies were evaluated clinically
Triple-negative breast cancer (TNBC) represents an
important and unmet clinical challenge, accounting for
around 10–15% of all newly diagnosed breast cancer
cases. TNBC is classified as a subpopulation of invasive
Supplementary information The online version of this article (https://
doi.org/10.1007/s00044-018-2275-9) contains supplementary
material, which is available to authorized users.
* Jun Cai
Juncai.284@gmail.com
* Andrew D. Westwell
WestwellA@cf.ac.uk
1
Cardiff School of Pharmacy and Pharmaceutical Science, Cardiff
University, Redwood Building, King Edward VII Avenue,
Cardiff CF10 3NB Wales, UK
2
Cardiff China Medical Research Collaborative, Cardiff University
School of Medicine, Henry Wellcome Building, Heath Park,
Cardiff CF14 4XN Wales, UK

Medicinal Chemistry Research
as potential targeted therapies in TNBC albeit not suc-
cessfully in terms of long-term survival. Bevacizumab is an
anti-angiogenic antibody that has been used in combination
with chemotherapy and was first investigated as a ther-
apeutic option due to enhanced angiogenesis in TNBC
presentations (Steeg 2006; Kim et al. 2004). This therapy
showed promising results in early clinical trials, however,
meta-analysis of clinical trial data showed no beneficial
effect on overall survival rates (O’Shaughnessy et al. 2009;
von Minckwitz et al. 2012; Bear et al. 2012). Most patients
were non-responsive to the therapy, while patients who
showed initial response eventually developed resistance and
relapsed disease. Moreover, in 2011 the FDA revoked its
approval of bevacizumab for breast cancer therapy due to
reported adverse side effects (Goozner 2011; Ranpura et al.
2011; O’Reilly et al. 2015). There are currently no mole-
cularly targeted therapies that are effective against TNBC in
the clinical setting, and therefore there is a high unmet
medical need for the development of new effective and
efficacious therapies for this poor prognosis patient group
(Fosu-Mensah et al. 2015; Chen et al. 2014).
Given the high priority afforded to the further study and
treatment of this disease, TNBC has been the subject of
intense gene array profiling studies. These studies identified
new molecular targets including but not limited to basal
epithelial proteins, such as cytokeratin 5 and 6, CK14, CK17,
P-cadherin, p53 mutations, epidermal growth factor receptor
(EGFR) and αB-crystallin (Lehmann et al. 2011). Ultimately,
these studies will provide potential therapeutic targets for drug
discovery programmes. Since many of the newly identified
TNBC therapeutic targets have established therapies in other
disease settings, there are presently numerous clinical studies
underway to assess their efficacy in TNBC.
An interesting TNBC-associated target under investigation
within our laboratory is αB-crystallin (CRYAB, HspB5).
CRYAB is a member of the mammalian small heat-shock
protein family that function as molecular chaperones to
modulate cell proteostasis, and consequently, cell survival by
suppressing aggregation of denatured proteins in stress-
induced environments (Tsang et al. 2012; Arrigo et al.
2007; Garrido et al. 2012). CRYAB is ubiquitously expressed
in many tissues with varying functions, and upregulation of
this protein is observed in many disease pathologies, includ-
ing neuropathy, myopathy, ischaemia/reperfusion, cataract
and various solid tumours (gliomas, prostate, ovarian, colon,
liver, head and neck cancer) (Ruan et al. 2011). CRYAB
represents a promising therapeutic target owing to its diverse
functions as a molecular chaperone. Studies in breast cancers
have provided compelling clinical data that showed that
CRYAB is constitutively expressed in the most aggressive
phenotypes of the disease, including TNBC. Accumulating
evidence has revealed that overexpression of CRYAB in
TNBC contributes to tumour progression and correlates with
the poor survival rates observed in this aggressive tumour
type. This can be attributed to its critical role as a metastatic
and anti-apoptotic regulator. Thus, CRYAB has been eval-
uated as a potentially important biomarker in TNBC (Tsang
et al. 2012; Ruan et al. 2011). Our previous structure-based
molecular docking studies identified 3-methylglutamic acid as
a small molecule with the potential to inhibit CRYAB/VEGF
interaction. 3-Methylglutamic acid was shown to disrupt
CRYAB/VEGF165 interaction and elicit both in vitro and
in vivo antitumour, anti-proliferative and anti-angiogenic
effects through downregulation of VEGF signalling (Chen
et al. 2014).
In recent years, our laboratory has focused on the dis-
covery of new drug candidates for potential application in
the TNBC setting (Chen et al. 2014; Haynes et al. 2016). As
part of these efforts, we were attracted to the possibilities
associated with purine-based compounds, given their long
and distinguished history in cancer drug development,
including clinically approved anti-leukaemia drugs such as
fludarabine (Rodriguez 1994) and clofarabine (Ghanem
et al. 2010). Purines represent a privileged heterocycle in
drug design, given the ability of purine analogues to bind
tightly within hydrophobic folds and inhibit a range of key
enzyme targets, such as members of the diverse protein
kinase, sulfatase and phosphorylase families. For example,
roscovitine (Selciclib) is a cyclin-dependent kinase inhibi-
tory purine analogue in clinical development in a range of
cancer, infectious disease and anti-inflammatory
settings (De Azevedo et al. 1997). The specific sub-class
of thiopurine analogues, such as 6-mercaptopurine and
thioguanine, are also used as standard cancer chemother-
apeutic agents, principally as anti-leukaemic drugs (Sahas-
ranaman et al. 2008). Figure 1 shows the chemical
structures of selected purine analogues in clinical use or
advanced development.
Fig. 1 Anticancer purine analogues in clinical use or advanced
development

Medicinal Chemistry Research
During our previous work on the identification of 3-
methylglutamic acid in disrupting CRYAB/VEGF165 inter-
action (Chen et al. 2014), we noted that the thioguanine core
structure was also able to potentially bind within the pro-
tein–protein interface binding pocket. In this paper, we
extend our studies on the identification of novel chemical
scaffolds active in models of TNBC through the synthesis
and in vitro antitumour evaluation of a series of substituted
thiopurines, and test their potential as CRYAB/VEGF
inhibitors.
added dropwise, then the reaction stirred for 3–5 h. On
completion of the reaction, the product was precipitated
with acetic acid, filtered, washed with water and dried.
Alkylation method 3
Substituted purine compound (1.0 eq) and 1,8-diazabicyclo
[5.4.0]undec-7-ene (1.1 eq) were dissolved in DMF at r.t.
for 5 min. The alkyl halide (1.2 eq) was added dropwise,
then the reaction stirred for 3–5 h. The reaction was then
quenched with cold water resulting in precipitation. The
precipitate was filtered, washed with water and dried prior
to purification by column chromatography.
Materials and methods
All solvents and reagents were used as obtained from
commercial sources unless otherwise indicated. Final
purification of target compounds was carried out by silica
gel column chromatography using CH2Cl2/MeOH as
2,6-bis(ethylthio)-7H-purin-8(9H)-one (4a)
According to general method 3 with 2,6-dimercapto-7H-
purin-8(9H)-one (200 mg, 1.0 mmol), DMF (2 mL), DBU
(0.15 ml, 1.0 mmol) and bromoethane (0.14 ml, 2.0 mmol)
The product was purified by column chromatography with
CH2Cl2/MeOH 90:10 as an eluent to afford a white solid of
yield 90 mg, 35%. 1H-NMR (500 MHz, DMSO-d6): δ
11.70 (s, br, 1H, NH), 11.16 (s, br, 1H, NH), 3.24 (q, 2H,
J = 7.5 Hz, SCH2), 3.09 (q, 2H, J = 7.5 Hz, SCH2), 1.32
(td, 6H, J1 = 7.5 Hz, J2 = 3.0 Hz, CH3). 13C-NMR (500
MHz, DMSO-d6): δ 161.45 (C2), 153.78 (C6), 149.40
(C8), 144.08 (C4), 116.81 (C5), 25.09 (SCH2), 23.39
(SCH2), 15.64 (CH3), 15.28 (CH3). m/z (FTMS + ESI) =
Found [M]+ (C9H12N4OS2) 256.9068 requires 256.3420.
1
eluent. Compound purity was established using H and
13C NMR, alongside accurate mass spectrometry. 1H
NMR and 13C NMR spectra were recorded in the appro-
priate deuterated solvents with
a Bruker Avance
DPX500 spectrometer operating at 500 MHz. Chemical
shifts (δ) are reported in parts per million (ppm) using the
following abbreviations: s, singlet; d, doublet; t, triplet, q,
quartet, quin, quintet; sex, sextet; sep, septet; m, multiplet,
br, broad, app.t, apparent triplet. All coupling constants
are reported in Hz. Mass spectra were recorded with a
Bruker microTOF mass spectrometer using electrospray
ionisation (ESI) conditions, or provided as a service at the
National Mass Spectrometry Facility at Swansea Uni-
versity, UK. Synthetic procedures and analytical/spec-
troscopic data for intermediate compounds can be found
in Supplementary Information.
2,6-bis(methylthio)-7H-purin-8(9H)-one (4b)
According to the general method 3 with 2,6-dimercapto-7H-
purin-8(9H)-one (200 mg, 1.0 mmol), DMF (2 ml), DBU
(0.15 ml, 1.0 mmol) and iodomethane (0.12 ml, 2.0 mmol).
The product was purified by column chromatography with
CH2Cl2/MeOH 90:10 as an eluent to afford a white solid of
yield 110 mg, 48%. 1H-NMR (500 MHz, DMSO-d6): δ
11.75 (s, br, 1H, NH), 11.23 (s, br, 1H, NH), 2.60 (s, 3H,
SCH3), 2.51 (s, 3H, SCH3). 13C-NMR (500 MHz, DMSO-
d6): δ 161.98 (C2), 153.78 (C6), 149.13 (C8), 144.58 (C4),
116.59 (C5), 14.24 (SCH3), 11.82 (SCH3). m/z (FTMS +
ESI) = Found [M+H]+ (C7H8N4OS2) 229.0212 requires
228.2880.
General methods for the alkylation of purines
Alkylation method 1
Substituted purine compound (1.0 eq) and potassium car-
bonate (1.1 eq) were dissolved in DMF (5 mL) at r.t. for 5
min. Bromoethane (1.2 eq) was added dropwise, then the
reaction stirred for 3–18 h, after which the reaction was
quenched with cold water resulting in precipitation. The
precipitate was filtered and washed with water. If no pre-
cipitate formed, the product was extracted with chloroform
or ethyl acetate, dried over sodium sulphate and con-
centrated under reduced pressure.
2,6-bis(propylthio)-7H-purin-8(9H)-one (4c)
According to the general method 3 with 2,6-dimercapto-7H-
purin-8(9H)-one (100 mg, 0.5 mmol), DMF (2 ml), DBU
(0.08 ml, 0.5 mmol) and 1-bromopropane (0.09 ml, 1.0
mmol). The product was purified by column chromato-
graphy with CH2Cl2/MeOH 90:10 as an eluent to afford a
Alkylation method 2
Substituted purine compound (1.0 eq) were dissolved in 1
M potassium hydroxide (1.1 eq). Bromoethane (1.2 eq) was
1
light yellow solid of yield 100 mg, 70%. H-NMR (500

Medicinal Chemistry Research
MHz, DMSO-d6): δ 11.69 (s, br, 1H, NH), 11.15 (s, br, 1H,
NH), 3.21 (t, 2H, J = 7.0 Hz, SCH2), 3.06 (t, 2H, J = 7.0
Hz, SCH2), 1.65–1.73 (m, 4H, CH2), 0.97–1.00 (m, 6H,
CH3). 13C-NMR (500 MHz, DMSO-d6): δ 161.45 (C2),
153.78 (C6), 149.37 (C8), 144.07 (C4), 116.82 (C5), 32.68
(SCH2), 30.64 (SCH2), 23.41 (CH2), 23.02 (CH2), 13.79
(CH3), δ 13.63 (CH3). m/z (FTMS + ESI) = Found [M]+
(C11H16N4OS2) 284.9649 requires 284.3960.
7.19–7.31 (m, 10H, HBz), 3.53 (t, 2H, J = 7.5 Hz, SCH2),
3.36 (t, 2H, J = 7.5 Hz, SCH2), 2.95 (t, 4H, J = 7.5 Hz,
CH2). 13C-NMR (500 MHz, DMSO-d6): δ 161.35 (C2),
153.80 (C6), 149.45 (C8), 143.86 (C4), 140.72 (CBz),
140.32 (CBz), 128.98 (CHBz), 128.84 (CHBz), 128.82
(CHBz), 126.82 (CHBz), 126.72 (CHBz), 116.95 (C5), 35.82
(CH2), 35.60 (CH2), 32.04 (SCH2), 30.05 (SCH2). m/z
(FTMS + ESI) = Found [M+H]+ (C21H20N4OS2) 409.1150
requires 408.5380.
2,6-bis(isobutylthio)-7H-purin-8(9H)-one (4d)
2,6-bis((3-phenylpropyl)thio)-7H-purin-8(9H)-one (4g)
According to the general method 3 with 2,6-dimercapto-7H-
purin-8(9H)-one (200 mg, 1.0 mmol), DMF (2 ml), DBU
(0.15 ml, 1.0 mmol) and 1-bromo-2-methylpropane (0.22 ml,
2.0 mmol). The product was purified by column chromato-
graphy with CH2Cl2/MeOH 90:10 as eluent to afford a light
yellow solid of yield 90 mg, 29%. 1H-NMR (500 MHz,
DMSO-d6): δ 11.67 (s, br, 1H, NH), 11.17 (s, br, 1H, NH),
3.18 (d, 2H, J = 7.0 Hz, SCH2), 3.02 (d, 2H, J = 7.0 Hz,
SCH2), 1.92 (sep, 2 × 1H, J = 7.0 Hz, CH), 1.00 (d, 12H, J
= 7.0 Hz, CH3). 13C-NMR (500 MHz, DMSO-d6): δ 161.45
(C2), 153.81 (C6), 149.36 (C8), 144.00 (C4), 116.81 (C5),
39.20 (SCH2), 36.86 (SCH2), 28.80 (CH), 28.38 (CH), 22.21
(CH3), 22.01 (CH3). m/z (FTMS + ESI) = Found [M+H]+
(C13H20N4OS2) 313.0257 requires 312.4500.
According to the general method 3 with 2,6-dimercapto-7H-
purin-8(9H)-one (100 mg, 0.5 mmol), DMF (2 ml), DBU
(0.08 ml, 0.5 mmol) and 1-bromo-3-phenylpropane (0.15
ml, 1.0 mmol). The product was purified by column chro-
matography with CH2Cl2/MeOH 90:10 as an eluent to
1
afford an off-white solid of yield 110 mg, 50%. H-NMR
(500 MHz, DMSO-d6): δ 11.71 (s, br, 1H, NH), 11.17 (s, br,
1H, NH), 7.25–7.29 (m, 4H, HPh), 7.16–7.19 (m, 6H, HPh),
3.21 (t, 2H, J = 7.5 Hz, SCH2), 3.06 (t, 2H, J = 7.5 Hz,
SCH2), 2.69 (t, 4H, J = 7.0 Hz, CH2), 1.92–1.98 (m, 4H,
CH2). 13C-NMR (500 MHz, DMSO-d6): δ 161.33 (C2),
153.78 (C6), 149.44 (C8), 143.88 (C4), 141.67 (CPh),
141.47 (CPh), 128.84 (CHPh), 128.82 (CHPh), 128.76
(CHPh), 128.74 (CHPh), 126.38 (CHPh), 126.33 (CHPh),
116.97 (C5), 34.70 (CH2), 34.57 (CH2), 31.51 (CH2), 31.17
(CH2), 30.33 (SCH2), 28.40 (SCH2). m/z (FTMS + ESI) =
Found [M+H]+ (C23H24N4OS2) 437.2875 requires
436.5920.
2,6-bis(benzylthio)-7H-purin-8(9H)-one (4e)
According to the general method 3 with 2,6-dimercapto-7H-
purin-8(9H)-one (100 mg, 0.5 mmol), DMF (2 ml), DBU
(0.08 ml, 0.5 mmol) and benzyl bromide (0.11 ml, 1.0 mmol).
The product was purified by column chromatography with
CH2Cl2/MeOH 90:10 as an eluent to afford an off white solid
9-ethyl-2,6-bis(ethylthio)-8,9-dihydro-7H-purine-8-thiol (6a)
1
of yield 140 mg, 74%. H-NMR (500 MHz, DMSO-d6): δ
According to the general method 3 with 2,6-dimercapto-
7,9-dihydro-8H-purine-8-thione (100 mg, 0.46 mmol),
DMF (2 ml), DBU (0.07 ml, 0.46 mmol) and bromoethane
(0.07 ml, 0.92 mmol). The product was purified by col-
umn chromatography with CH2Cl2/MeOH 95:5 as an
eluent to afford a light yellow solid of yield 40 mg, 29%.
1H-NMR (500 MHz, DMSO-d6): δ 13.21 (s, br, 1H, NH),
3.27–3.31 (m, 4H, CH2), 3.10–3.16 (m, 2H, CH2), 1.32–
1.39 (m, 9H, CH3). 13C-NMR (500 MHz, DMSO-d6): δ
169.99 (C8), 163.58 (C2), 150.73 (C6), 147.44 (C4),
119.40 (C5), 25.82 (NCH2), 25.25 (SCH2), 23.49 (SCH2),
15.43 (CH3), 15.29 (CH3), 15.19 (CH3). m/z (FTMS +
ESI) = Found [M+H]+ (C11H16N4S3) 301.0609 requires
300.4570.
11.81 (s, br, 1H, NH), 11.76 (s, br, 1H, NH), 7.41 (d, 2H, J =
7.2 Hz, CHBz), 7.37 (d, 2H, J = 7.2 Hz, CHBz), 7.29–7.32 (m,
4H, CHBz), 7.23–7.27 (m, 2H, CHBz), 4.51 (s, 2H, SCH2),
4.40 (s, 2H, SCH2). 13C-NMR (500 MHz, DMSO-d6): δ
161.12 (C2), 153.77 (C6), 149.55 (C8), 143.52 (C4), 138.24
(CBz), 138.02 (CBz), 129.34 (CHBz), 129.23 (CHBz), 128.97
(CHBz), 128.89 (CHBz), 127.70 (CHBz), 127.50 (CHBz),
116.76 (C5), 34.95 (CH2), 32.73 (CH2). m/z (FTMS + ESI) =
Found [M+H]+ (C19H16N4OS2) 381.0796 requires 380.4840.
2,6-bis(phenethylthio)-7H-purin-8(9H)-one (4f)
According to the general method 3 with 2,6-dimercapto-7H-
purin-8(9H)-one (100 mg, 0.5 mmol), DMF (2 ml), DBU
(0.08 ml, 0.5 mmol) and 2-bromoethyl benzene (0.14 ml,
1.0 mmol). The product was purified by column chroma-
tography with CH2Cl2/MeOH 90:10 as an eluent to afford a
white solid of yield 100 mg, 49%. 1H-NMR (500 MHz,
DMSO-d6): δ 11.78 (s, br, 1H, NH), 11.20 (s, br, 1H, NH),
9-methyl-2,6-bis(methylthio)-7,9-dihydro-8H-purine-8-
thione (6b)
According to the general method 3 with 2,6-dimercapto-
7,9-dihydro-8H-purine-8-thione (100 mg, 0.46 mmol) DMF

Medicinal Chemistry Research
(2 ml), DBU (0.07 ml, 0.46 mmol) and iodomethane (0.06
ml, 0.92 mmol). The product was purified by column
chromatography with CH2Cl2/MeOH 95:5 as an eluent to
afford the tri-substituted product as an off-white solid of
yield 40 mg, 17%. 1H-NMR (500 MHz, DMSO-d6, tri-
substituted product): δ 2.54 (s, 3H, CH3), 2.52 (s, 3H, CH3),
2.47 (s, 3H, CH3). 13C-NMR (500 MHz, DMSO-d6): δ
163.84 (C8), 163.19 (C2), 156.40 (C6), 148.74 (C4),
133.58 (C5), 14.31 (CH3), 14.12 (CH3), 11.56 (CH3). m/z
(FTMS + ESI) = Found [M+H]+ (C8H10N4S3) 259.0133
requires 258.3760.
The tetra-substituted product (7) was an off-white solid
of yield 50 mg, 30%. 1H-NMR (500 MHz, DMSO-d6, tetra-
substituted product): δ 3.85 (s, 3H, NCH3), 2.75 (s, 3H,
NCH3), 2.64 (s, 3H, SCH3), 2.54 (s, 3H, SCH3). 13C-NMR
(500 MHz, DMSO-d6): δ 162.84 (C8), 156.54 (C2), 153.80
(C6), 151.99 (C4), 128.85 (C5), 33.45 (CH3), 29.08 (CH3),
14.09 (CH3), 11.68 (CH3). m/z (FTMS + ESI) = Found
[M+H]+ (C9H12N4S3) 273.0334 requires 272.4030.
(CH), 28.43 (CH), 22.23 (CH3), 22.08 (CH3), 21.95 (CH3).
m/z (FTMS + ESI) = Found [M+H]+ (C17H28N4S3)
385.1744 requires 384.6190.
9-benzyl-2,6-bis(benzylthio)-8,9-dihydro-7H-purine-8-thiol
(6e)
According to the general method 3 with 2,6-dimercapto-7,9-
dihydro-8H-purine-8-thione (100 mg, 0.46 mmol), DMF (2
ml), DBU (0.07 ml, 0.46 mmol) and benzyl bromide (0.11 ml,
0.92 mmol). The precipitate was filtered, washed with water
and dried. The product was purified by column chromato-
graphy with CH2Cl2/MeOH 95:5 as an eluent to afford a light
1
yellow solid of yield 120 mg, 54%. H-NMR (500 MHz,
DMSO-d6): δ 13.55 (s, br, 1H, NH), 7.42–7.47 (m, 6H,
CHBz), 7.30–7.33 (m, 6H, CHBz), 7.24–7.28 (m, 3H, CHBz),
4.59 (s, 2H, SCH2), 4.56 (s, br, 2H, NCH2), 4.46 (s, 2H,
SCH2). 13C-NMR (500 MHz, DMSO-d6): δ 162.29 (C8),
155.76 (C2), 152.73 (C6), 151.56 (C4), 138.21 (CBz), 137.53
(CBz), 129.47 (CHBz), 129.40 (CHBz), 129.28 (CHBz), 128.99
(CHBz), 128.95 (CHBz), 128.91 (CHBz), 127.97 (CHBz),
129.41 (C5), 127.66 (CHBz), 127.53 (CHBz), 35.24 (SCH2),
35.13 (SCH2), 32.38 (NCH2). m/z (FTMS + ESI) = Found
[M+H]+ (C26H22N4S3) 487.3201 requires 486.6700.
9-propyl-2,6-bis(propylthio)-8,9-dihydro-7H-purine-8-thiol
(6c)
According to general the method 3 with 2,6-dimercapto-7,9-
dihydro-8H-purine-8-thione (100 mg, 0.46 mmol), DMF (2
ml), DBU (0.07 ml, 0.46 mmol) and 1-bromopropane (0.08
ml, 0.92 mmol). The product was purified by column chro-
matography with CH2Cl2/MeOH 95:5 as an eluent to afford a
light yellow solid of yield 74 mg, 47%. 1H-NMR (500 MHz,
DMSO-d6): δ 3.24–3.29 (m, 4H, SCH2), 3.08–3.13 (m, 2H,
NCH2), 1.68–1.76 (m, 6H, CH2), 0.97–1.03 (m, 9H, CH3).
13C-NMR (500 MHz, DMSO-d6): δ 170.03 (C8), 163.56
(C2), 150.72 (C6), 147.41 (C4), 119.47 (C5), 32.88 (SCH2),
32.82 (SCH2), 30.69 (NCH2), 23.20 (CH2), 23.03 (CH2),
22.95 (CH2), 13.78 (CH3), 13.69 (CH3), 13.66 (CH3). m/z
(FTMS + ESI) = Found [M+H]+ (C14H22N4S3) 343.1079
requires 342.5380.
9-phenethyl-2,6-bis(phenethylthio)-8,9-dihydro-7H-purine-
8-thiol (6f)
According to the general method 3 with 2,6-dimercapto-7H-
purine-8(9H)-thione (100 mg, 0.46 mmol) DMF (2 ml),
DBU (0.07 ml, 0.46 mmol) and 2-bromoethyl benzene
(0.13 ml, 0.92 mmol). The precipitate was filtered, washed
with water and dried. The product was purified by column
chromatography with CH2Cl2/MeOH 95:5 as an eluent to
1
afford a light yellow solid of yield 102 mg, 42%. H-NMR
(500 MHz, DMSO-d6): δ 13.43 (s, br, 1H, NH), 7.29–7.34
(m, 12H, CHBz), 7.21–7.25 (m, 3H, CHBz), 3.52–3.59 (m,
4H, SCH2), 3.41–3.44 (m, 2H, NCH2), 2.99–3.05 (m, 6H,
CH2). 13C-NMR (500 MHz, DMSO-d6): δ 162.37 (C8),
156.00 (C2), 152.71 (C6), 151.60 (C4), 140.76 (CBz),
140.49 (CBz), 140.21 (CBz), 129.55 (C5), 129.09 (CHBz),
129.00 (CHBz), 128.87 (CHBz), 128.82 (CHBz), 126.91
(CHBz), 126.84 (CHBz), 126.73 (CHBz), 35.71 (SCH2),
35.57 (SCH2), 35.33 (NCH2), 32.62 (SCH2), 32.25 (SCH2),
29.79 (NCH2). m/z (FTMS + ESI) = Found [M+H]+
(C29H28N4S3) 529.15 requires 528.75.
9-isobutyl-2,6-bis(isobutylthio)-8,9-dihydro-7H-purine-8-
thiol (6d)
According to the general method 3 with 2,6-dimercapto-
7,9-dihydro-8H-purine-8-thione (100 mg, 0.46 mmol),
DMF (2 ml), DBU (0.07 ml, 0.46 mmol) and 1-bromo-2-
methylpropane (0.10 ml, 0.92 mmol). The product was
purified by column chromatography with CH2Cl2/MeOH
95:5 as an eluent to afford a light yellow solid of yield 80
1
mg, 45%. H-NMR (500 MHz, DMSO-d6): δ 13.36 (s, br,
9-(3-phenylpropyl)-2,6-bis((3-phenylpropyl)thio)-8,9-
1H, NH), 3.22 (dd, 4H, J = 6.8 Hz, SCH2), 3.08 (d, 2H, J =
6.8 Hz, NCH2), 1.90–2.02 (m, 3H, CH), 1.00–1.03 (m, 18H,
CH3). 13C-NMR (500 MHz, DMSO-d6): δ 162.43 (C8),
156.04 (C2), 152.58 (C6), 151.88 (C4), 129.41 (C5), 39.51
(S-CH2), 39.43 (SCH2), 36.51 (NCH2), 28.73 (CH), 28.52
dihydro-7H-purine-8-thiol (6g)
According to the general method 3 with 2,6-dimercapto-7,9-
dihydro-8H-purine-8-thione (100 mg, 0.46 mmol) DMF (2
ml), DBU (0.07 ml, 0.46 mmol) and 1-bromo-3-phenyl

Medicinal Chemistry Research
propane (0.14 ml, 0.92 mmol). The reaction was quenched
with water resulting in precipitation. The precipitate was fil-
tered, washed with water and dried. The product was purified
by column chromatography with CH2Cl2/MeOH 95:5 as an
eluent to afford a light yellow solid of yield 116 mg, 44%. 1H-
NMR (500 MHz, DMSO-d6): δ 13.40 (s, br, 1H, NH), 7.26–
7.31 (m, 6H, CHPh), 7.16–7.24 (m, 9H, CHPh), 3.26–3.29 (m,
4H, SCH2), 3.13 (dd, J1 = 7.5 Hz, J2 = 2.5 Hz, 2H, NCH2),
1.95–2.07 (m, 6H, CH2). 13C-NMR (500 MHz, DMSO-d6): δ
162.33 (C8), 155.96 (C2), 152.66 (C6), 151.66 (C4), 141.68
(CPh), 141.53 (CPh), 141.44 (CPh), 129.54 (C5), 128.84
(CHPh), 128.83 (CHPh), 128.79 (CHPh), 128.77 (CHPh),
126.39 (CHPh), 126.37 (CHPh), 126.34 (CHPh), 34.70 (SCH2),
34.62 (SCH2), 34.39 (NCH2), 31.35 (SCH2), 31.15 (SCH2),
30.97 (NCH2), 30.87 (SCH2), 30.54 (SCH2), 38.00 (NCH2).
m/z (FTMS + ESI) = Found [M+H]+ (C34H34N4S3)
571.2016 requires 570.8320.
(CH3). m/z (FTMS + ESI) = Found [M+H]+ (C6H7N5O2)
196.0828 requires 195.1820.
2-amino-6-hydroxy-9-propyl-7,9-dihydro-8H-purin-8-one
(13c)
According to the general method with 2-amino-8-bromo-9-
propyl-9H-purin-6-ol (100 mg, 0.37 mmol) and sodium
acetate (160 mg, 1.95 mg to afford a tan solid of yield 20
1
mg, 26%. H-NMR (500 MHz, DMSO-d6): δ 10.64 (s, br,
1H, NH), 10.53 (s, 1H, OH), 6.49 (s, br, 2H, NH2), 3.54 (t,
2H, J = 7.5 Hz, NCH2), 1.60 (sext, 2H, J = 7.5 Hz, CH2),
0.83 (t, 3H, J = 7.5 Hz, CH3). 13C-NMR (500 MHz,
DMSO-d6): δ 153.97 (C2), 152.79 (C6), 151.45 (C4),
148.51 (C8), 98.55 (C5), 41.38 (NCH2), 21.99 (CH2), 11.48
(CH3). m/z (FTMS + ESI) = Found [M+H]+ (C8H11N5O2)
210.0986 requires 209.2090.
General method for the hydrolysis of brominated
alkyl-guanines: synthesis of 2-amino-6 hydroxy-9-
alkyl-7,9-dihydro-8H-purin-8-ones (13a-g)
2-amino-6-hydroxy-9-isobutyl-7,9-dihydro-8H-purin-8-one
(13d)
According to the general method with 2-amino-8-bromo-9-
isobutyl-9H-purin-6-ol (100 mg, 0.35 mmol) and sodium
acetate (152 mg, 1.85 mg to afford a tan solid of yield 45
A mixture of brominated alkyl-guanine (12a-g, 1 eq),
sodium acetate (5.3 eq), acetic acid (3 ml) and acetic
anhydride (0.5 ml) were heated under reflux and N2 for 15
h. The reaction was cooled and evaporated to dryness. The
resulting residue was suspended in water (5 ml) and the pH
adjusted to 13 with 10 M NaOH. The mixture was refluxed
for 20 min, cooled and the solid formed collected, triturated
with potassium phosphate buffer pH 7.5, filtered and dried.
1
mg, 58%. H-NMR (500 MHz, DMSO-d6): δ 10.73 (s, br,
1H, NH), 10.49 (s, 1H, OH), 6.48 (s, br, 2H, NH2), 3.39 (td,
2H, J = 7.5 Hz, NCH2), 2.10 (sept, 1H, J1 = 7.5 Hz, J2 =
6.5 Hz, CH), 0.82 (d, 6H, J = 6.5 Hz, CH3). 13C-NMR (500
MHz, DMSO-d6): δ 154.03 (C2), 153.02 (C6), 151.66 (C4),
148.74 (C8), 98.55 (C5), 46.68 (NCH2), 27.63 (CH), 20.22
(2xCH3).
m/z
(FTMS + ESI) = Found
[M+H]+
2-amino-6-hydroxy-9-methyl-7,9-dihydro-8H-purin-8-one (13a)
(C9H13N5O2) 224.1142 requires 223.2360.
According to the general method with 2-amino-8-bromo-9-
methyl-9H-purin-6-ol (100 mg, 0.41 mmol) and sodium
acetate (178 mg, 2.17 mg) to afford a tan solid of yield 62 mg,
2-amino-9-benzyl-6-hydroxy-7,9-dihydro-8H-purin-8-one
(13e)
1
63%. H-NMR (500 MHz, DMSO-d6): δ 10.41 (s, 1H, OH),
According to the general method with 2-amino-9-benzyl-8-
bromo-9H-purin-6-ol (100 mg, 0.31 mmol) and sodium
acetate (135 mg, 1.65 mg) to afford a tan solid of yield 40
6.90 (s, br, 2H, NH2), 3.08 (s, 3H, CH3). 13C-NMR (500
MHz, DMSO-d6): δ 154.69 (C2), 153.02 (C6), 152.08 (C4),
148.78 (C8), 98.55 (C5), 25.90 (CH3). m/z (FTMS + ESI) =
Found [M+H]+ (C6H7N5O2) 182.0672 requires 181.1550.
1
mg, 50%. H-NMR (500 MHz, DMSO-d6): δ 10.80 (s, br
1H, NH), 10.67 (s, 1H, OH), 7.24–7.33 (m, 5H, HBz), 6.52
(s, br, 2H, NH2), 4.80 (s, 2H, NCH2). 13C-NMR (500 MHz,
DMSO-d6): δ 154.17 (C2), 152.73 (C4), 148.38 (C6),
137.82 (C8), 128.94 (CBz), 127.67 (CHBz), 127.42 (CHBz),
98.74 (C5), 42.60 (NCH2). m/z (FTMS + ESI) = Found [M
+H]+ (C12H11N5O2) 258.0988 requires 257.2530.
2-amino-9-ethyl-6-hydroxy-7,9-dihydro-8H-purin-8-one
(13b)
According to the general method with 2-amino-8-bromo-9-
ethyl-9H-purin-6-ol (100 mg, 0.39 mmol) and sodium
acetate (170 mg, 2.05 mg) to afford a tan solid of yield 40
2-amino-6-hydroxy-9-phenethyl-7,9-dihydro-8H-purin-8-
one (13f)
1
mg, 40%. H-NMR (500 MHz, DMSO-d6): δ 10.65 (s, br,
1H, NH), 10.49 (s, 1H, OH), 6.48 (s, br, 2H, NH2), 3.62 (q,
2H, J = 7.0 Hz, NCH2), 1.16 (t, 3H, J = 7.0 Hz, CH3). 13C-
NMR (500 MHz, DMSO-d6): δ 153.97 (C2), 152.79 (C6),
151.45 (C4), 148.51 (C8), 98.55 (C5), 41.38 (NCH2), 11.48
According to the general method with 2-amino-8-bromo-9-
phenethyl-9H-purin-6-ol (100 mg, 0.30 mmol) and sodium
acetate (129 mg, 1.57 mg) to afford a tan solid of yield

Medicinal Chemistry Research
1
69 mg, 85%. H-NMR (500 MHz, DMSO-d6): δ 10.50 (s,
147.82 (C8), 22.87 (SCH2), 15.42 (CH3). m/z (FTMS +
ESI) = Found [M+H]+ (C7H8N4S) 181.0541 requires
180.2290.
1H, OH), 7.26–7.30 (m, 2H, HBz), 7.19–7.22 (m, 3H, HBz),
6.93 (s, br, 4H, NH2), 3.81–3.88 (m, 2H, CH2), 2.94–3.00
(m, 2H, CH2). 13C-NMR (500 MHz, DMSO-d6): δ 154.66
(C2), 152.66 (C4), 148.69 (C6), 139.01 (C8), 138.84 (CBz),
129.03 (CHBz), 128.86 (CHBz), 126.80 (CHBz), 98.47 (C5),
34.38 (NCH2), 30.80 (CH2). m/z (FTMS + ESI) = Found
[M+H]+ (C13H13N5O2) 272.1146 requires 271.2800.
6-(propylthio)-7H-purine (15c)
According to the general alkylation method 3 with 6-
mercaptopurine monohydrate (200 mg, 1.18 mmol), DMF
(3 ml), DBU (0.18 ml, 1.18 mmol) and 1-bromopropane
(0.21 ml, 2.35 mmol) for 3 h. The product extracted with
chloroform, and purified by column chromatography with
CH2Cl2/MeOH 95:5 as an eluent to afford a white solid of
yield 140 mg, 61%. 1H-NMR (500 MHz, DMSO-d6): δ
13.50 (s, br, 1H, NH), 8.69 (s, 1H, H2), 8.43 (s, 1H, H8),
3.33 (t, 2H, J = 7.5 Hz, SCH2), 1.73 (sext, 2H, J = 7.5 Hz,
CH2), 1.01 (t, 3H, J = 7.5 Hz, CH3). 13C-NMR (500 MHz,
DMSO-d6): δ 151.93 (C2), 143.27 (C8), 30.15 (SCH2),
23.10 (CH2), 13.70 (CH3). m/z (FTMS + ESI) = Found [M
+H]+ (C8H10N4S) 195.0696 requires 194.2560.
2-amino-6-hydroxy-9-(3-phenylpropyl)-7,9-dihydro-8H-
purin-8-one (13g)
According to the general method with 2-amino-8-bromo-9-
(3-phenylpropyl)-9H-purin-6-ol (100 mg, 0.29 mmol) and
sodium acetate (125 mg, 1.52 mg) to afford a tan solid of
1
yield 5 mg, 6%. H-NMR (500 MHz, DMSO-d6): δ 10.65
(s, br 1H, NH), 10.55 (s, 1H, OH), 7.26–7.32 (m, 2H, HBz),
7.16–7.22 (m, 3H, HBz), 6.49 (s, br, 2H, NH2), 3.64 (t, 2H,
J = 7.5 Hz, CH2), 2.57 (t, 2H, J = 7.5 Hz, CH2), 1.91 (quin,
2H, J = 7.5 Hz, CH2). 13C-NMR (500 MHz, DMSO-d6): δ
154.48 (C2), 152.86 (C4), 146.62 (C6), 141.71 (C8),
140.81 (CBz), 131.10 (CHBz), 131.06 (CHBz), 128.78
(CHBz), 128.70 (CHBz), 126.30 (CHBz), 98.42 (C5), 33.21
(NCH2), 30.37 (CH2), 29.06 (CH2). m/z (FTMS + ESI) =
Found [M+H]+ (C14H15N5O2) 286.1301 requires 285.3070.
6-(isobutylthio)-7H-purine (15d)
According to the general alkylation method 3 with 6-
mercaptopurine monohydrate (200 mg, 1.18 mmol), DMF
(3 ml), DBU (0.18 ml, 1.18 mmol) and 1-bromo-2-
methylpropane (0.26 ml, 2.35 mmol) for 3 h. The product
extracted with chloroform, and purified by column chro-
matography with CH2Cl2/MeOH 95:5 as an eluent to afford
6-(methylthio)-9H-purine (15a)
1
According to the general alkylation method 3 with 6-
mercaptopurine monohydrate (200 mg, 1.18 mmol), DMF
(3 ml), DBU (0.18 ml, 1.18 mmol) and iodomethane (0.15
ml, 2.35 mmol) for 3 h. The product extracted with
chloroform, and purified by column chromatography with
CH2Cl2/MeOH 95:5 as an eluent to afford a white solid of
a white solid of yield 90 mg, 37%. H-NMR (500 MHz,
DMSO-d6): δ 13.50 (s, br, 1H, NH), 8.68 (s, 1H, H2), 8.44
(s, 1H, H8), 3.29 (d, 2H, J = 6.5 Hz, SCH2), 1.97 (sep, 1H,
J1 = 6.5 Hz, J2 = 7.0 Hz, CH), 1.02 (d, 6H, J = 6.5 Hz,
2xCH3). 13C-NMR (500 MHz, DMSO-d6): δ 151.86 (C2),
143.68 (C8), 36.38 (SCH2), 28.68 (CH2), 22.09 (2xCH3).
1
yield 110 mg, 56%. H-NMR (500 MHz, DMSO-d6): δ
m/z
(FTMS + ESI) = Found
[M+H]+
(C9H12N4S)
13.51 (s, br, 1H, NH), 8.71 (s, 1H, H2), 8.44 (s, 1H, H8),
2.67 (s, 3H, CH3). 13C-NMR (500 MHz, DMSO-d6): δ
151.99 (C2), 143.63 (C8), 11.66 (CH3). m/z (FTMS +
ESI) = Found [M+H]+ (C6H6N4S) 167.0383 requires
166.2020.
209.1011 requires 208.2830.
6-(benzylthio)-9H-purine (15e)
According to the general alkylation method 3 with 6-
mercaptopurine monohydrate (200 mg, 1.18 mmol), DMF
(3 ml), DBU (0.18 ml, 1.18 mmol) and benzyl bromide
(0.28 ml, 2.35 mmol) for 3 h. The product extracted with
chloroform, and purified by column chromatography with
CH2Cl2/MeOH 95:5 as an eluent to afford a white solid of
yield 140 mg, 49%. 1H-NMR (500 MHz, DMSO-d6): δ
13.55 (s, br, 1H, NH), 8.75 (s, 1H, H2), 8.46 (s, 1H, H8),
7.46 (d, 2H, J = 7.5 Hz, HBz), 7.32 (t, 2H, J = 7.0 Hz, HBz),
7.25 (t, 1H, J1 = 7.0 Hz, J2 = 7.5 Hz, HBz), 4.66 (s, 2H,
SCH2). 13C-NMR (500 MHz, DMSO-d6): δ 151.92 (C2),
143.97 (C8), 138.35 (CBz), 129.46 (CHBz), 128.96 (CHBz),
127.64 (CHBz), 32.10 (SCH2). m/z (FTMS + ESI) = Found
[M+H]+ (C12H10N4S) 243.0716 requires 242.3000.
6-(ethylthio)-7H-purine (15b)
According to the general alkylation method 3 with 6-
mercaptopurine monohydrate (200 mg, 1.18 mmol), DMF
(3 ml), DBU (0.18 ml, 1.18 mmol) and bromoethane (0.18
ml, 2.35 mmol) for 3 h. The product extracted with
chloroform, and purified by column chromatography with
CH2Cl2/MeOH 95:5 as an eluent to afford a white solid of
yield 170 mg, 80%. 1H-NMR (500 MHz, DMSO-d6): δ
13.51 (s, br, 1H, NH), 8.70 (s, 1H, H2), 8.44 (s, 1H, H8),
3.34 (q, 2H, J = 7.0 Hz, SCH2), 1.37 (t, 3H, J = 7.0 Hz,
CH3). 13C-NMR (500 MHz, DMSO-d6): δ 151.96 (C2),

Medicinal Chemistry Research
6-(phenethylthio)-9H-purine (15f)
potassium carbonate (150 mg, 1.1 mmol) and bro-
moethane (0.09 ml, 1.2 mmol) for 3 h. The product was
purified by column chromatography with CH2Cl2/MeOH
95:5 as an eluent to afford an off-white solid of yield 99
According to the general alkylation method 3 with 6-
mercaptopurine monohydrate (200 mg, 1.18 mmol), DMF
(3 ml), DBU (0.18 ml, 1.18 mmol) and 2-bromoethylbenzene
(0.32 ml, 2.35 mmol) for 3 h. The product extracted with
chloroform, and purified by column chromatography with
CH2Cl2/MeOH 95:5 as an eluent to afford a white solid of
yield 160 mg, 53%. 1H-NMR (500 MHz, DMSO-d6): δ 13.52
(s, br, 1H, NH), 8.73 (s, 1H, H2), 8.45 (s, 1H, H8), 7.31–7.32
(m, 4H, HBz), 7.21–7.25 (m, 1H, HBz), 3.61 (t, 2H, J = 7.0
Hz, CH2), 3.03 (t, 2H, J = 7.0 Hz, CH2). 13C-NMR (500
MHz, DMSO-d6): δ 152.01 (C2), 143.33 (C8), 140.58 (CBz),
129.07 (CHBz), 128.85 (CHBz), 126.82 (CHBz), 35.61 (SCH2),
29.72 (CH2). m/z (FTMS + ESI) = Found [M+H]+
(C13H12N4S) 257.0850 requires 256.3270.
1
mg, 76%. H-NMR (500 MHz, MeOD): δ 3.20 (q, 2H, J
= 7.0 Hz, SCH2), 3.08 (9, 2H, J = 7.0 Hz, SCH2), 1.37 (t,
3H, J = 7.0 Hz, CH3), 1.36 (t, 3H, J = 7.5 Hz, CH3). 13C-
NMR (500 MHz, MeOD): δ 158.58 (C2), 152.99 (C6),
148.97 (C4), 118.60 (C5), 24.52 (SCH2), 23.86 (SCH2),
14.49 (CH3), 14.18 (CH3). m/z (FTMS + ESI) = Found
[M+H]+ (C8H14N4S2) 230.9 requires 230.3.
2,6-bis(propylthio)pyrimidine-4,5-diamine (16c)
According to the general alkylation method 1 with 4,5-dia-
mino-2,6-dimercaptopyrimidine (200 mg, 1.0 mmol), potas-
sium carbonate (150 mg, 1.1 mmol) and 1-bromopropane
(0.06 ml, 1.2 mmol) for 3 h. The product was purified by
column chromatography with CH2Cl2/MeOH 95:5 as an
eluent to afford an off-white solid of yield 180 mg, 63%. 1H-
NMR (500 MHz, MeOD): δ 3.18 (t, 2H, J = 7.5 Hz, SCH2),
3.06 (t, 2H, J = 7.5 Hz, SCH2), 1.73 (sext, 4H, J = 7.5 Hz,
CH2), 1.05 (td, 6H, J1 = 7.5 Hz, J2 = 1.0 Hz, CH3). 13C-
NMR (500 MHz, MeOD): δ 158.59 (C2), 152.92 (C6),
149.11 (C4), 118.49 (C5), 32.31 (SCH2), 31.40 (SCH2),
23.33 (2xCH2), 23.04 (2xCH2), 12.44 (2xCH3), 12.31
(CH3). m/z (FTMS + ESI) = Found [M+H]+ (C10H18N4S2)
259.1045 requires 258.4020.
6-((3-phenylpropyl)thio)-9H-purine (15g)
According to the general alkylation method 3 with 6-
mercaptopurine monohydrate (200 mg, 1.18 mmol), DMF
(3 ml), DBU (0.18 ml, 1.18 mmol) and 1-bromo-3-
phenylpropane (0.37 ml, 2.35 mmol) for 3 h. The product
extracted with chloroform, and purified by column chro-
matography with CH2Cl2/MeOH 95:5 as an eluent to afford
1
a white solid of yield 200 mg, 63%. H-NMR (500 MHz,
DMSO-d6): δ 13.52 (s, br, 1H, NH), 8.68 (s, 1H, H2), 8.45
(s, 1H, H8), 7.28–7.31 (m, 2H, HPh), 7.17–7.24 (m, 3H,
HPh), 3.35 (t, 2H, J = 7.3 Hz, CH2), 2.76 (t, 2H, J = 7.5 Hz,
CH2), 2.03 (quin, 2H, J1 = 7.3, J2 = 7.5 Hz, CH2). 13C-
NMR (500 MHz, DMSO-d6): δ 151.94 (C2), 141.61 (CPh),
128.82 (CHPh), 126.36 (CHPh), 39.48 (SCH2), 31.28 (CH2),
27.87 (CH2). m/z (FTMS + ESI) = Found [M+H]+
(C14H14N4S) 271.1011 requires 270.3540.
2,6-bis(isobutylthio)pyrimidine-4,5-diamine (16d)
According to the general alkylation method 1 with 4,5-
diamino-2,6-dimercaptopyrimidine (100 mg, 0.57 mmol),
potassium carbonate (150 mg, 1.1 mmol) and 1-bromo-2-
methylpropane (0.13 ml, 1.2 mmol) for 3 h. The product
was purified by column chromatography with CH2Cl2/
MeOH 95:5 as an eluent to afford a light brown solid of
2,6-bis(methylthio)pyrimidine-4,5-diamine (16a)
1
According to the general alkylation method 1 with 4,5-
diamino-2,6-dimercaptopyrimidine (200 mg, 1.0 mmol),
potassium carbonate (150 mg, 1.1 mmol) and iodomethane
(0.07 ml, 1.2 mmol) for 3 h. The product was purified by
column chromatography with CH2Cl2/MeOH 95:5 as an
eluent to afford an off-white solid of yield 112 mg, 49%.
1H-NMR (500 MHz, MeOD): δ 2.57 (s, 3H, SCH3), 2.50 (s,
3H, SCH3). 13C-NMR (500 MHz, MeOD): δ 159.25 (C2),
152.82 (C6), 149.96 (C4), 118.09 (C5), 12.75 (2xSCH3),
11.55 (SCH3). m/z (FTMS + ESI) = Found [M+H]+
(C6H10N4S2) 203.0419 requires 202.2940.
yield 90 mg, 50%. H-NMR (500 MHz, MeOD): δ 3.14 (d,
2H, J = 6.5 Hz, SCH2), 3.01 (d, 2H, J = 7.0 Hz, SCH2),
1.89–2.00 (m, 2H, CH), 1.06 (d, 3H, J = 2.0 Hz, CH3), 1.05
(d, 3H, J = 2.5, CH3). 13C-NMR (500 MHz, MeOD): δ
158.57 (C2), 152.87 (C6), 149.11 (C4), 118.43 (C5), 38.94
(SCH2), 37.91 (SCH2), 28.81 (CH), 28.47 (CH), 20.93
(4xCH3).
m/z
(FTMS + ESI) = Found
[M+H]+
(C12H22N4S2) 287.0228 requires 286.4560.
2,6-bis(benzylthio)pyrimidine-4,5-diamine (16e)
According to the general alkylation method 1 with 4,5-
diamino-2,6-dimercaptopyrimidine (200 mg, 1.0 mmol),
potassium carbonate (150 mg, 1.1 mmol) and benzyl bro-
mide (0.14 ml, 1.2 mmol) for 3 h. The product was purified
by column chromatography with CH2Cl2/MeOH 95:5 as an
2,6-bis(ethylthio)pyrimidine-4,5-diamine (16b)
According to the general alkylation method 1 with 4,5-
diamino-2,6-dimercaptopyrimidine (100 mg, 0.57 mmol),

Medicinal Chemistry Research
eluent to afford an off-white solid of yield 190 mg, 50%.
1H-NMR (500 MHz, MeOD): δ 7.37–7.39 (m, 2H, HBz),
7.19–7.31 (m, 10H, HBz), 4.38 (s, 2H, SCH2), 4.34 (s, 2H,
SCH2). 13C-NMR (500 MHz, MeOD): δ 158.08 (C2),
153.16 (C6), 147.77 (C4), 138.50 (CBz), 138.25 (CBz),
128.02 (CHBz), 128.36 (CHBz), 128.02 (CHBz), 127.97
(CHBz), 126.66 (CHBz), 126.48 (CHBz), 119.16 (C5), 34.66
(SCH2), 33.89 (SCH2). m/z (FTMS + ESI) = Found [M
+H]+ (C18H18N4S2) 355.1015 requires 354.4900.
Modified Eagle’s Media (DMEM) supplemented with
10% FCS. Human microvascular endothelial (HMVECs)
were maintained in microvascular endothelial cell basal
medium 131 with growth supplement (Gibco). MCF10A
cells were maintained in mammary epithelial cell growth
medium (Lonza) optimized for the growth of mammary
epithelial cells in a serum-free environment. All cells were
incubated at 37 °C in 5% CO2. Cells were seeded in a 96-
well plate at a density of 5 × 103 cells/ml and incubated for
24 h at 37 °C and 5% CO2. The cells were then treated
with different concentrations of the test compounds and
controls and incubated for 72 h. After this period, 20 µL of
sterile MTT solution in PBS (5 mg/mL) was added to each
well. The plates were then incubated for 5 h at 37 °C and
5% CO2. Following this, 100 μL of acidified isopropanol
was added (0.02% 2M HCl) and the mixture was allowed
to stand until the formed formazan crystals had dissolved.
The absorbance was measured at 570 nm (BioTeK), and
the cell viability was expressed as a percentage of the
absorbance of non-treated cells. The experiments were
performed in three independent repeats, and the results
were expressed as the mean the standard error of the
mean (SEM).
2,6-bis(phenethylthio)pyrimidine-4,5-diamine (16f)
According to the general alkylation method 1 with 4,5-
diamino-2,6-dimercaptopyrimidine (200 mg, 1.0 mmol),
potassium carbonate (150 mg, 1.1 mmol) and 2-bromoethyl
benzene (0.16 ml, 1.2 mmol) for 5 h. The product was
purified by column chromatography with CH2Cl2/MeOH
95:5 as an eluent to afford an off-white solid of yield 260
1
mg, 64%. H-NMR (500 MHz, MeOD): δ 7.15–7.26 (m,
10H, HBz), 3.45 (t, 2H, J = 7.5 Hz, SCH2), 3.34 (t, 2H, J =
7.5 Hz, SCH2), 2.96 (dt, 4H, J1 = 7.5 Hz, J2 = 3.5 Hz,
2xCH2). 13C-NMR (500 MHz, MeOD): δ 158.39 (C2),
153.07 (C6), 149.11 (C4), 140.64 (CBz), 140.23 (CBz),
128.24 (CHBz), 128.21 (CHBz), 128.03 (CHBz), 125.95
(CHBz), 125.83 (CHBz), 118.95 (C5), 36.18 (CH2), 35.98
(CH2), 31.73 (SCH2), 30.93 (SCH2). m/z (FTMS + ESI) =
Found [M+H]+ (C20H22N4S2) 383.1298 requires 382.5440.
Cytotoxicity assay
(Essen Bioscience 2017) MDA-MB-231, MDA-MB-436,
HMVECs and MCF10A cells were seeded (100 μL per
well) at a cell density of 5000 cells/well into a 96-well plate
and incubated at 37 °C in 5% CO2 within the IncuCyte
ZOOM® system for 24 h. Contrast images were captured
every 2 h and analysed using an integrated confluence
algorithm. The test compounds and controls were prepared
in the media containing the IncuCyte™ Cytotox Reagent at
3x the final concentration, then 50 μL of this solution was
added to each well to obtain a dilution of 1:3 with a final
assay volume of 150 μL (final assay concentration of
Cytotox Reagent 250 nM). Images of the plate were cap-
tured at 10x objective, two images per well with phase and
fluorescence scanning every 2 h until completion of the
assay (72 h). The data were analysed using integrated
software.
2,6-bis((3-phenylpropyl)thio)pyrimidine-4,5-diamine (16g)
According to the general alkylation method 1 with 4,5-dia-
mino-2,6-dimercaptopyrimidine (169 mg, 1.0 mmol), potas-
sium carbonate (128 mg, 1.1 mmol) and 1-bromo-3-
phenylpropane (0.15 ml, 1.2 mmol) for 5 h. The product was
purified by column chromatography with CH2Cl2/MeOH 95:5
as an eluent to afford an off-white solid of yield 260 mg, 60%.
1H-NMR (500 MHz, MeOD): δ 7.25 (dd, 4H, J1 = 15.5 Hz, J2
= 8.0 Hz, HPh), 7.15 (m, 6H, HPh), 3.14 (dd, 2H, J1 = 13 Hz,
J2 = 7.0 Hz, SCH2), 3.03 (t, 2H, J = 7.0 Hz, SCH2), 2.72 (t,
4H, J = 7.0 Hz, CH2), 1.92–2.00 (m, 4H, 2xCH2). 13C-NMR
(500 MHz, MeOD): δ 158.43 (C2), 152.96 (C6), 148.84 (C4),
141.53 (CPh), 141.33 (CPh), 128.12 (CHPh), 128.11 (CHPh),
128.03 (CHPh), 128.01 (CHPh), 125.54 (CHPh), 125.50 (CHPh),
118.72 (C5), 34.49 (CPh), 34.32 (CH2), 31.59 (CH2), 31.37
(SCH2), 29.78 (SCH2), 28.95 (SCH2). m/z (FTMS + ESI) =
Found [M+H]+ (C22H26N4S2) 411.2652 requires 410.5980.
PathHunterTM cell-based CRYAB/VEGF165 interaction assay
The cells were maintained in a PathHunter Assaycom-
pleteTM culture media and incubated at 37 °C and 5% CO2
until confluent. The U2OS cells were detached with
AssaycompleteTM cell detachment reagent, suspended in
AssaycompleteTM cell plating reagent and counted. In
total, 20 µL of the cell suspension was transferred to each
well of a 384-well white-walled, clear bottom plate at the
indicated cell densities (5000, 10000, 20000 cells/ml).
Biological evaluation
MTT assay
(Mosmann 1983) Breast cancer cell lines MDA-MB-231
and MDA-MB-436 were maintained in Delbecco’s

Medicinal Chemistry Research
The seeded cells were incubated for 24 h at 37 °C and 5%
CO2, 5 µL of the test compounds (10 μM) dissolved in the
vehicle (2% DMSO) were added to the appropriate wells
and incubated at room temperature for 90 min. After
incubating, 25 µL per well of PathHunter Detection
Reagents were added to the plates, incubated for 60 min at
room temperature in the dark and the plate read using the
POLARstar Omega luminescence reader.
Results and discussion
Design of novel analogues and synthetic chemistry
We began our synthetic studies around a dithiopurine-8-one
heterocyclic core, proposing the introduction of substituents
to improve lipophilicity and enhance cellular penetration.
We decided to explore alkylation chemistry to derivatise the
core dithiopurine-8-one heterocyclic structure particularly at
the sulphur and/or the N7/N9 positions, as well as intro-
ducing functional flexibility at position C8. Figure 2 shows
the proposed structural changes and exploratory chemistry
leading to five small series of new molecules for in vitro
anticancer evaluation.
ELISA assay
VEGF165 secreted by MDA-MB-231 cells were assayed
using Quantio® ELISA assay kit (R&D Systems). The assay
was performed according to the manufacturers’ instructions.
MDA-MB-231 cells were grown in a T25 flask at 37 °C and
5% CO2 until 80% confluence. The cells were then treated
with the test compounds dissolved in DMSO (0.1%). The
cell supernatant was harvested by centrifugation at 2000 × g
for 15 min at 6, 12 and 24 h. VEGF165 concentrations were
measured in triplicate in each sample. The results are
expressed as VEGF165 pg/ml for at least three separate
experiments.
There is clear evidence in the literature that reaction
conditions play a crucial role in determining the selectivity
of alkylation of purines and pyrimidines. Therefore, various
conditions reported in the literature were examined to
ascertain the optimal reactions conditions required to obtain
selective alkylation at the appropriate positions of the pur-
inone core (Pathak et al. 2004; Cheng and Robins 1958;
Robins 1958; Biagi et al. 1996; Corder et al. 2013; Johnston
et al. 1958; Robins 1957). Scheme 1 outlines the synthetic
route employed in the synthesis of Series 1 analogues 4a–g,
characterised by derivatisation of the sulphur groups at
positions 2 and 6 with various alkyl groups. The purine core
structure 2 was prepared via a thermal cyclisation reaction
between the commercially available pyrimidine 1 and urea
(Levin et al. 1960). As suggested in the literature, it was
expected that under alkaline conditions, alkylation of
compound 2 would selectively produce the exclusively S-
alkylated product (Joule et al. 1995; Montgomery et al.
1966). In addition, it has been reported that low reaction
temperatures favour S- over N-alkylation. However, it was
noted that when using K2CO3 or KOH as a base, the tri-
substituted product (3) was produced regardless of the
stoichiometry of the reagents. The best conditions found to
facilitate selective S-alkylation to provide the double alky-
lation products (4a-g) involved the non-nucleophilic base,
1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) in DMF (Biagi
et al. 1996), in yields of 29–74% following purification by
column chromatography.
Molecular modelling
All molecular modelling studies were performed with
Molecular Operating Environment (MOE) 2015.10 model-
ling software. The crystal structure of the ACD domain of
CRYAB was obtained from the Protein Data Bank (PDB
code: 2KLR). Hydrogen atoms were added to the crystal
structure minimised with MOE until a gradient of 0.05 Kcal
mol−1 Å−1 was reached, using the MMFF94x forcefield.
The partial charges were automatically calculated. To sim-
plify calculations and analysis within this hydrophobic
active site, no water molecules were considered. The
docking site was defined by selecting the important amino
acid residues (Tyr122, Arg 123 and Arg 149) and extending
to 4.5 Å to include other β7 and β9 amino acid residues.
Ligand docking was performed using MOE default settings.
After docking, all docked molecules were visually
inspected.
SR
N
SR
N
OH
SR
SR
H
N
H
N
H
N
N
NH2
NH2
N
N
N
N
N
O
S
O
N
H
N
R
N
R
N
H
RS
RS
H2N
N
N
RS
N
series 1 (4a-g)
S-substituted
purinones
series 2 (6a-g)
S/N-substituted
thiopurinones
series 3 (13a-g)
N-substituted
purinones
series 4 (15a-g)
S-substituted
purines
series 5 (16a-g)
S-substituted
purine precursors
Fig. 2 Target molecule series derived from the dithiopurin-8-one core structure

Medicinal Chemistry Research
S
N
H
N
(b)
(c)
(d)
N
O
N
S
3
SH
N
SH
S
H
N
H
N
(a)
NH2
N
N
N
N
O
O
O
N
H
N
HS
HS
N
1
NH2
S
N
2
3
4a (R = CH2CH3)
4b (R = CH3)
SR
N
H
N
4c (R = CH2CH2CH3)
4d (R = CH2CH(CH3)2
4e (R = CH2Ph)
N
H
RS
4f (R = CH2CH2Ph)
4g (R = CH2CH2CH2Ph)
(29-74%)
Scheme 1 Synthesis of Series 1 analogues. Reagents and conditions: a urea, 180–195 °C, 20 min; b CH2CH3Br, K2CO3, DMF, r.t., 2.5 h; c
CH2CH3Br, 1 M KOH, r.t., 2.5 h; d R-Br/I, DBU, DMF, r.t., 2.5 h
S
H
(b)
N
N
S
N
S
N
6a
SH
SH
S
H
N
H
N
(a)
NH2
NH2
(c)
(d)
N
N
N
N
S
S
S
N
H
N
HS
N
HS
N
S
N
6a
5
1
6a (R = CH2CH3)
6b (R = CH3)
6c (R = CH2CH2CH3)
6d (R = CH2CH(CH3)2
6e (R = CH2Ph)
SR
H
N
N
R
RS
N
6f (R = CH2CH2Ph)
6g (R = CH2CH2CH2Ph)
(17-54%)
Scheme 2 Synthesis of Series 2 analogues. Reagents and conditions: a thiourea, 180–195 °C, 20 min; b CH2CH3Br, K2CO3, DMF, r.t., 2.5 h; c
CH2CH3Br, 1 M KOH, r.t., 2.5 h; d R-Br/I, DBU, DMF, r.t., 2.5 h
The synthetic route employed in the synthesis of Series
2 analogues 6a–g is outlined in Scheme 2. A thermal
cyclisation reaction between pyrimidine (1) and thiourea
resulted in the 8-thiopurine (5), which was followed by
alkylation reactions with various alkyl halides as described
in Scheme 1. Under all the investigated reaction conditions,
the products of the alkylation reactions were the tri-
substituted purines similar to the products obtained under
the previously described alkaline conditions (K2CO3 or
KOH), in low-to-moderate yields of 17–54% (Scheme 1).
The acidity of the thiopurine NH group adjacent to the
pyrimidine N3-atom provides a rationale for alkylation at
the N9-position under basic conditions. Although tri-
substituted purines have been reported in the literature,
they are usually constructed from alkylated pyrimidines
cyclised with alkoxides and not in a one-pot synthesis as
we achieved (Yang et al. 2005; Villatoro et al. 2015;
Ibrahim and Legraverend 2009).
Surprisingly in addition to the tri-substituted thiopurine
(6b), methylation of compound 5 also produced the tetra-
substituted product 7 (Scheme 3). We postulated that
methylation at position N7 is possible since the methyl

Medicinal Chemistry Research
group is not affected by steric hindrance compared with the
bulkier alkyl substituents.
moderate-to-high yield (37–80%) following the previously
described alkylation methods for further SAR studies
(Scheme 5).
Scheme 4 was employed to give access to selectively N-
alkylated purines 13a–g that were deemed necessary for
SAR studies. Selective N-alkylation was readily achieved
by a reaction between 2-amino-6-chloropurine 8 and var-
ious alkyl halides to afford compounds 9a–g/10a–g as a
mixture of regioisomers, with the N9-isomer (9a–g) being
the major product as previously reported in the literature
(Chhabra et al. 2013). Both isomers were successfully
separated by silica gel chromatography and fully char-
Biological evaluation—anti-proliferative activity
The analogues were tested for their in vitro inhibitory
effect on the viability of the invasive TNBC cell lines
MDA-MB-231 and MDA-MB-436, using the MTT end-
point assay (72 h). Additionally, endothelial cell lines,
MCF10A and human microvascular endothelial
(HMVEC-d) cell lines were used to assess the effects of
the analogues on endothelial cells. Table 1 details the
result of this in vitro screen expressed as IC50 values from
dose-response studies.
In general, several analogues from Series 1 (4a–g),
Series 2 (6a–g) and Series 5 (16a–g) showed the most
potent anti-proliferative effects, significantly reducing the
viability of one or both TNBC cells with IC50 values in the
low micromolar range. It should be noted that active
analogues were also generally more active as growth
inhibitors in the endothelial cell lines HMVEC and
MCF10A, perhaps not surprisingly for these early stage
lipophilic drug candidates. The selectivity profiles
between the different cell types certainly underscore the
importance of carrying out more detailed toxicity studies
in the future.
1
acterised by NMR. The H NMR spectrum showed the
alkyl peaks of the N7 product to be shifted downfield due to
the deshielding effect of the bulky chloro group. The N9
products were then taken forward in a multi-step synthesis
to give the desired N-alkylated purinones 13a–g by intro-
ducing a keto-group at position 8. Compounds 9a–g were
hydrolysed in aqueous HCl to afford the intermediates 11a–
g, which were then brominated to afford the 8-bromopurine
intermediates 12a–g (Michael et al. 1993). Finally, the
brominated purines were transformed to the desired 8-
oxopurines 13a–g through hydrolysis of the bromo group,
in low to good yields (6–85%) (Verones et al. 2010).
Lastly, substituted 6-thiopurines 15a–g and 2,6-dithio-
pyrimindines 16a–g, which represent Series 4 and 5 ana-
logues, respectively, were also synthesised from
commercially available precursor compounds 14 and 31, in
SH
S
S
Scheme 3 Further methylation
of thiopurine 6b
H
N
H
N
MeI, DBU
DMF, rt
N
N
N
N
N
+
S
S
S
N
H
N
HS
N
S
N
S
N
7 (30%)
6b (17%)
5
Cl
Cl
Cl
R
N
N
(a)
N
N
N
N
+
N
N
R
N
N
H2N
N
H2N
N
H2N
H
9a-g
8
10a-g
a: R = CH3
(b)
b: R = CH2CH3
c: R = CH2CH2CH3
d: R = CH2CH(CH3)2
e: R = CH2Ph
f: R = CH2CH2Ph
g: R = CH2CH2CH2Ph
OH
OH
OH
H
N
N
(c)
(d)
N
N
N
N
Br
O
N
R
N
R
N
R
H2N
N
H2N
N
H2N
N
11a-g
12a-g
13a-g (6-85%)
Scheme 4 Synthesis of Series 3 analogues. Reagents and conditions: a RBr/I, K2CO3, DMF, r.t., 2.5 h; b 1 M HCl, reflux, 3 h; c Br2, AcOH, 50–
60 °C, 18 h; d CH3COONa, AcOH/Ac2O, reflux, 18 h

Medicinal Chemistry Research
SH
SR
N
was performed using the cyanine nucleic acid dyes devel-
oped by Essen BioScience with their IncuCyte™ live-cell
imaging system that enables real time detection of cell
death. The cells were co-cultured with the IncuCyte™
Cytotox reagent, and test compounds 4c, 4e–f, 6d–e, 15f
and 16d were added at different concentrations over a
period of 72 h. Images were captured every 2 h for the
duration of the assay and the cytotoxicity effect was
quantified in real-time with the integrated analysis software.
Generally, all the tested compounds showed a significant
increase in the cell death characteristics of a cytotoxicity
effect compared with the no-drug control, with a few
notable exceptions (Fig. 3). All the seven compounds
induced a cytotoxic effect at a concentration of 100 μM
against the TNBC cells, while cytotoxic effects were
detected at a concentration of 50 μM against endothelial
cells. These results appear to correlate with the observed
anti-proliferative activity and thus, provided confidence for
these analogues to undergo further evaluation, notably in
our custom cell-based CRYAB/VEGF165 interaction assay
to probe their ability to disrupt this interaction.
a: R = CH3
H
N
b: R = CH2CH3
(a)
N
N
N
c: R = CH2CH2CH3
d: R = CH2CH(CH3)2
e: R = CH2Ph
f: R = CH2CH2Ph
g: R = CH2CH2CH2Ph
N
H
N
N
15a-g
(37-80%)
14
SH
SR
NH2
NH2
(b)
NH2
NH2
N
N
HS
N
RS
N
16a-g
(49-76%)
1
Scheme 5 Synthesis of Series 4 and 5 analogues. Reagents and con-
ditions: a R-Br/I, DBU, DMF, r.t., 2.5 h; b RBr/I, K2CO3, DMF, r.t.,
2.5 h
Notably, benzyl/phenethyl substituted purinone/thiopur-
inone analogues 4e, 4f, and 6e, were the most potent in
TNBC cell lines, suggesting that this group may be impor-
tant for biological activity. Across all series, it was also
evident that the chain length of the alkyl substituents has an
influence on biological activity. For analogues with aliphatic
substituents, it was observed that isobutyl > propyl > ethyl >
methyl in terms of their anti-proliferative activity within cell
lines examined. For example, ethyl substituted analogue 4a
showed IC50 values of 97 and 100 μM against MDA-MB-
231 and MDA-MB-436 cells, respectively, compared with
the propyl substituted analogue 4c, which showed an IC50
value of 29 μM against both TNBC cell lines. However, for
analogues with alkyl benzyl substituents, an increase in the
chain length of the alkyl benzyl group appears to have a
detrimental effect on biological activity. For example,
compound 4f with a phenethyl group showed an IC50 value
of 3–4 μM in TNBC cell lines, while increasing the chain
length to a phenylpropyl group as seen for compound 4g
resulted in a complete loss of activity (IC50 > 100 μM).
Overall, analogues from Series 3 and 4 showed no
inhibitory effects against either TNBC cell line apart from
compound 15f (Series 4). This suggests that the substitution
at positions 2 and 6 and the keto group at position-8 may
contribute to the observed biological effect. As noted above,
the most potent analogues across all series also showed
potent growth inhibitory effects on the epithelial and
endothelial cell lines MCF10A and HMVEC-d, respec-
tively, without the desired selectivity for TNBC cell lines.
PathHunterTM cell-based CRYAB/VEGF165 interaction
assay
The PathHunterTM cell-based CRYAB/VEGF165 interaction
assay is a commercially available, custom cell-based assay
designed by DiscoverX. The assay utilizes the enzyme
fragment complementation (EFC) technology for studying
protein–protein interactions. For the purposes of this
research, the assay was designed to monitor the interaction
between CRYAB and a cytosolically localised VEGF165
.
The two candidate proteins were fused to complementation
fragments of the β-galactosidase (β-Gal) enzyme. CRYAB
was co-expressed with the larger sequence encoding the
majority of enzyme, termed enzyme acceptor (EA), while
VEGF165 was fused to a small peptide epitope called Pro-
link (PK). The two separate enzyme fragments are inactive;
however, after the constructs were transduced into U2OS
cells, the recombined active enzyme is generated as a result
of CRYAB/VEGF165 protein–protein interaction. The dis-
ruption of the CRYAB/VEGF165 interaction was quantified
by measuring the chemiluminescence signal produced as a
consequence of the conversion of a non-luminescent sub-
strate to a luminescent product by the active enzyme.
The U2OS cells were seeded at three cell densities
(20,000, 10,000 and 5000 cells/well), allowed to adhere to
the wells and challenged with different concentrations of the
test compounds. Tunicamycin was used as a positive con-
trol for this assay because of its ability to elicit the unfolded
protein response and thus inhibit angiogenesis (Banerjee
et al. 2011). After 90-min incubation period, the non-
luminescent substrates were introduced into the cells and
Cytotoxicity evaluation
Compounds that demonstrated the most potent inhibitory
effect in the anti-proliferative assay (IC50 ≤ 50 µM) were
evaluated for their ability to induce cytotoxicity against the
cell lines under investigation. The in vitro cytotoxicity assay

Medicinal Chemistry Research
Table 1 Anti-proliferative
activity of the synthesised
analogues expressed as IC50
SEM (three independent
experiments)
Compound
R group
IC50 SEM (μM)
MDA-MB-231
MDA-MB-436
HMVEC
MCF10A
Series 1
4a
4b
4c
4d
4e
4f
CH2CH3
97 0.22
>100
100
>100
>100
CH3
>100
>100
>100
CH2CH2CH3
CH2CH(CH3)2
CH2Ar
29 1.3
>100
29 0.5
>100
14 0.07
12 3.2
1.5 4.4
3
4
1
2
0.16
0.23
0.23
0.48
17 1.3
45 1.1
3 0.12
CH2CH2Ar
CH2CH2CH2Ar
3
0.35
4
1.05
9.6 0.12
0.6 0.08
4g
>100
>100
Series 2
6a
6b
6c
6d
6e
6f
CH2CH3
84 1.1
>100
57 1.5
19 0.72
31 0.14
CH3
80 0.06
56 0.89
35 0.11
>100
45 0.13
28 0.71
CH2CH2CH3
CH2CH(CH3)2
CH2Ar
99 0.44
36 0.20
37 0.08
>100
3
1
1.2
5
3
0.14
0.08
0.21
5
0.05
1.2 0.17
1.3 0.1
19 0.06
CH2CH2Ar
CH2CH2CH2Ar
91 0.15
>100
19 0.19
>100
6g
>100
Series 3
13a
13b
13c
13d
13e
13f
CH3
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
CH2CH3
>100
>100
CH2CH2CH3
CH2CH(CH3)2
CH2Ar
>100
>100
85 0.10
69 0.05
>100
>100
>100
CH2CH2Ar
CH2CH2CH2Ar
80 0.09
72 0.04
13g
>100
Series 4
15a
15b
15c
15d
15e
15f
CH3
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
CH2CH3
>100
>100
>100
CH2CH2CH3
CH2CH(CH3)2
CH2Ar
>100
>100
>100
>100
85 0.10
69 0.05
15 0.05
49 0.05
>100
>100
>100
CH2CH2Ar
CH2CH2CH2Ar
49 0.06
>100
83 0.12
59 0.37
15g

Medicinal Chemistry Research
Table 1 (continued)
Compound
R group
IC50 SEM (μM)
MDA-MB-231
MDA-MB-436
HMVEC
MCF10A
Series 5
16a
16b
16c
16d
16e
16f
CH3
>100
>100
30 0.07
17 0.08
34 0.25
33 0.29
14 0.07
2.1 0.08
1.4 0.11
1.9 0.16
0.2 0.18
0.7 0.24
0.6 0.34
CH2CH3
>100
>100
CH2CH2CH3
CH2CH(CH3)2
CH2Ar
61 0.30
21 0.11
78 0.07
>100
>100
43 0.11
>100
1
0.06
10 0.07
0.07
CH2CH2Ar
CH2CH2CH2Ar
>100
16g
>100
>100
8
MDA-MB-231 (100 µM)
MDA-MB-436 (100 µM)
8000
30000
20000
6000
4000
2000
0
10000
4000
3000
2000
1000
0
4c
4e
4f
6f
6d
15f
4c 4e
Ctrl
4f
6f
15f
16d
6d
Ctrl
16d
HMVEC-d (50 µM)
MCF10A (50 µM)
20000
10000
8000
6000
4000
2000
0
15000
10000
5000
0
6f
4f
6f
4f
4c
4e
4c
6d
4e
6d
15f
15f
16d
16d
Ctrl
Ctrl
Fig. 3 Cytotoxic effect of selected analogues against TNBC and control cell lines. Data are expressed as mean standard error of the mean (SEM),
n = 3
luminescent readings taken with a spectrophotometer after
60 min. All experiments were carried out in triplicate. The
seven analogues that demonstrated inhibitory and cyto-
toxicity effects in previous experiments were selected for
further evaluation in the cell-based CRYAB/VEGF165
interaction assay. Out of the seven compounds, analogues
4e and 4f were found to significantly attenuate the chemi-
luminescence signal of U2OS cells at a density of 20,000
cells/well. No significant inhibition effect was observed for
the remaining five analogues. The response (RLU) of the

Medicinal Chemistry Research
Fig. 4 Concentration-response curves of analogues 4e and 4f and
controls in the PathHunterTM cell-based CRYAB/VEGF165 interaction
assay, indicating chemiluminescence quantification relative to the
vehicle control; data are expressed as mean standard error of the
mean (SEM), n = 3, ***P < 0.001
Fig. 5 ELISA measurement of VEGF165 levels secreted by MDA-
MB231 cells treated with 100 µM of compounds 4e and 4f. Data are
expressed as mean standard error of the mean (SEM), n = 3; ***P <
0.05
analogues at 10 μM and the controls are shown in Fig. 4.
Both analogues and tunicamycin demonstrated a significant
decrease in chemiluminescence signal in comparison with
the vehicle control (DMSO). This inhibitory effect is indi-
cative of the disruption of the interaction between CRYAB
α-crystallin domain (ACD) domain made up of 84 amino
acids, and finally a C-terminal region composed of 26
amino acids. The ACD domain consists of eight β strands
(β2 to β9) and helical sequences with strong interactions to
regulatory proteins. The aforementioned sequence studies
demonstrated that the β strands (β3, β7, β8 and β9) play a
crucial role in the interactions between many growth factors
and CRYAB, including VEGF165. Additionally, an inhibi-
tory competition study with decoy peptides corresponding
to CRYAB and VEGF165 confirmed the significance of β7
and β9 in the protein–protein interaction between CRYAB
and VEGF165 (Chen et al. 2014). Fortunately from the
standpoint of computational drug design, a good crystal
structure of the α-crystallin domain (ACD) of human
CRYAB obtained from solid-state NMR, small angle X-ray
scattering, and computational modelling studies has been
published (PDB code: 2KLR) (Jehle et al. 2010).
On this basis, for docking studies a hydrophobic struc-
tural pocket was selected that was formed by discontinuous
segments of β7 and β9 and containing the amino acids
Try122/Arg123 (β7) and Arg149 (β9). In a series of dock-
ing simulations carried out with Molecular Operating
Environment (MOE 2017), all the analogues and one
additional monosubstituted purinone, 6-mercapto-2-
(methylthio)purin-8-one, were docked into the selected
structural pocket to probe their interaction with the target
site.
and VEGF165
.
VEGF165 ELISA measurement
As previously discussed, one of the proposed mechanisms
by which CRYAB promotes angiogenesis is by preventing
the proteolytic degradation of misfolded or unfolded VEGF
resulting in the upregulation of VEGF and maintenance of
VEGF signalling (Ruan et al. 2011). Hence, we postulated
that disrupting the interaction between CRYAB/VEGF
would affect the levels of VEGF produced by breast cancer
cells. The concentration of VEGF secreted by MDA-MB-
231 cells was measured by the ELISA assay. MDA-MB-
231 cells treated with 100 μM of compound 4e showed a
significant reduction in VEGF levels compared with the
control.
A
concentration of 160 pg/ml of soluble
VEGF was detected by the assay compared with the control
(260 pg/ml), representing a 40% decrease in VEGF con-
centration (Fig. 5). No significant reduction was observed in
cells treated with compound 4f. This result supports our
hypothesis that the disruption of the interaction between
CRYAB/VEGF can lead to inhibition of VEGF production
by breast tumour cells.
Figure 6 shows the predicted binding pose of 6-mer-
capto-2-(methylthio)purin-8-one. The methythio side group
spatially occupies the structural pocket, albeit partially.
Crucially, one of the nitrogen groups of the imidazolidine
ring forms an H-bond with Tyr122. Moreover, there were
other hydrophobic interactions between the purine ring and
surrounding amino acids, including Asn78 of the β3 strand.
Comparatively, 4e (Fig. 7) also retained the key interaction
Molecular modelling studies
Previous work has reported sequence studies for the inter-
action of important regulatory proteins with CRYAB
(Ghosh et al. 2007). Typical of most proteins of the Hsp
family, the structure of CRYAB consists of an N-terminal
region of ~65 amino acids, preceding a conserved

Medicinal Chemistry Research
Fig. 6 a Predicted binding mode
of 6-mercapto-2-(methylthio)
purin-8-one (orange) in selected
docking site of CRYAB (grey),
and b ligand interaction diagram
(the compound is shown in
orange while protein residues
are in green)
with Tyr122 and an additional interaction with β7 through a
H-bond between a thiol group and Ile124. Compound 4f
(Fig. 8) interacts with β7 through hydrophobic interaction
between the purine core and Arg123. Furthermore, one of
the nitrogen groups of the imidazolidine ring of both ana-
logues forms a H-bond with Lys121 also of β7. Finally, it
was noted that the benzyl/phenethyl rings penetrates further
and occupies more of the binding pocket compared with 6-
mercapto-2-(methylthio)purin-8-one. Interestingly, even the
aromatic ring of the benzyl/phenethyl groups does not fully
occupy this pocket hence the presence of bulky substituents
on the ring could promote better insertion, as well as new
interactions that could improve on the biological activities
of the analogues. Also noticeable is the fact that none of the
compounds interact with β9; this may be indicative of its
distance (26 amino acids away) from β7. Therefore, intro-
ducing bulky substituents to the aromatic ring could bring
this group closer to β9 and enhance the interaction between
the molecules and CRYAB.
in vitro activities of the synthesised analogues and will
inform future endeavours to design more efficacious
CRYAB inhibitors. These observations are summarised
below.
Effects of S-alkyl substituents
To improve on the lipophilicities of the purine core
structure, various alkyl substituents were introduced to the
thio- and/or nitrogen groups. It was observed that across
all series, analogues with aromatic phenalkyl substituents
demonstrated the most potent biological effects in the
various assays. However, the chain length of the phe-
nalkyl group was found to be a limiting factor, that is, an
increase in chain length led to a decrease in activity.
Furthermore, compounds 4e and 4f, with this benzyl/
phenethyl functionality were able to disrupt the interac-
tion between CRYAB/VEGF165 in the protein–protein
interaction assay. In addition, compound 4e also
decreased the levels of VEGF secreted by MDA-MB-231
cells. In our docking studies, the benzyl/phenethyl group
was also found to form key interactions with the selected
structural pocket on CRYAB confirming the significance
of this group.
Discussion
The biological and docking studies have highlighted
structural features that could be responsible for the observed

Medicinal Chemistry Research
Fig. 7 a Predicted binding mode
of compound 4e (orange) in
selected docking site of CRYAB
(grey), b ligand interaction of
compound 4e
Fig. 8 a Predicted binding mode
of compound 4f (orange) in
selected docking site of CRYAB
(grey), b ligand interaction of 4f
Effects of N-substitutions
Functional flexibility at position-8
No improvement in activity was observed for the series of
analogues with N-substitutions (Series 3). Additionally, the
docking studies showed no interactions between this sub-
stitution and CRYAB (data not shown).
As Series 4 analogues are effectively non-toxic against the
control cell lines, it can be deduced that this substitution
may contribute to the observed toxicity of the analogues.
However, the toxicity effect may not be limited to this
substituent but also the substituted thiol group at position-2,
since analogues without this substitution (Series 3 and 4)
showed little or no cytotoxic effect.
Effects of removing the thio group at position-2
Removal of the thio-group at position-2 as seen with Series 3
and 4 analogues could be responsible for the loss of activity in
both the various in vitro assays. Crucially, in the docking
studies, it appears that this substitution interacts with the
structural pocket of CRYAB indicating that this substitution
contributes to the biological activities of the analogues.
CRYAB confers protection against apoptosis induced
by chemotherapeutic agents by inhibiting caspase-3 acti-
vation, sequestration of the anti-apoptotic protein Bcl-2
and prevention of Bcl-2 translocation into the mitochon-
dria. In a study where breast epithelial carcinoma cells
were treated with the anticancer agent vinblastine, the

Medicinal Chemistry Research
phosphorylated form of CRYAB was found to inhibit
apoptosis by interacting with Bcl-2, while non-
phosphorylated CRYAB induced resistance against vin-
blastine (Launay et al. 2010). The silencing of CRYAB
with antisense or RNAi molecules reversed this effect and
sensitised tumour cells to chemotherapeutic and apoptotic
agents (Lee et al. 2012). Angiogenesis, a segment of the
metastatic cascade and the role of CRYAB as a facilitator
of this phenomenon is of particular interest to our group.
In the tumour microenvironment, CRYAB modulates
angiogenesis through its effect on both cancer and endo-
thelial cells. Studies have shown that CRYAB can prevent
proteolytic degradation of VEGF by binding to and cor-
recting unfolded/misfolded VEGF leading to preservation
of intracrine VEGF signalling. Inhibition of CRYAB
leads to a decrease in endogenous VEGF levels, providing
clear evidence that targeting CRYAB may represent a
novel approach to suppress angiogenesis. It should be
noted that CRYAB is also expressed at high levels in the
the heart and cardiomyocytes, conferring protection
against stress (such as an ischaemic event) by inhibiting
apoptosis (Pereira et al. 2014; Gonçalves et al. 2016;
Morrison et al. 2003). Therefore, direct inhibition of
CRYAB could lead to unwanted cardiotoxicity. Conse-
quently, we propose a novel strategy of targeting angio-
genesis by disrupting the interaction between CRYAB
and VEGF with small molecules that competitively bind
to the interacting surface. This approach could produce a
therapeutic effect while potentially avoiding the unwanted
side effects associated with direct inhibition of CRYAB,
and serve as a novel anti-angiogenic therapy in the
treatment of TNBC.
Following this trend, we propose a novel approach of
supressing angiogenesis in TNBC with small molecules that
target the PPI between CRYAB and VEGF. There is a cri-
tical need for targeted therapies in TNBC since the only
established treatment for this aggressive disease is che-
motherapy, which has been shown to be ineffective in terms
of medium- to long-term survival. Angiogenesis is one of
the main targets in TNBC due to its role in disease pro-
gression, however, most anti-angiogenic therapies have
shown mixed and sometimes disappointing results. In the
tumour microenvironment, CRYAB is a chaperone protein
that has been shown to bind to and correct unfolded/mis-
folded VEGF promoting angiogenesis. This protein is
overexpressed in TNBC and has been found to correlate
with poor prognosis in this disease setting, making it an
attractive target for the development of new anti-angiogenic
therapies. Several studies looking at the interaction between
CRYAB and regulator proteins identified a “hotspot” on the
surface of CRYAB where VEGF interacts. Aided by
molecular docking methods, small molecule hit compounds
were identified which bind to this “hotspot” and could
potentially inhibit this PPI.
In this paper, we report a proof-of-concept study devised
to validate this target. The work started with the synthesis of
novel purine-based compounds for structural activity rela-
tionship studies. The newly synthesised analogues were
then subjected to biological evaluation, firstly, in a cell
viability assay where several of the analogues were found to
significantly reduce the viability of MDA-MB-231 and
MDA-MB-436 TNBC cell lines. The most potent of these
analogues were then tested for their ability to disrupt the
CRYAB/VEGF interaction in a custom designed cell-based
CRYAB/VEGF165 PPI assay. The PPI assay showed that
compounds 4e and 4f successfully disrupted the interaction
between CRYAB and VEGF165. Additionally compound 4e
(100 μM) was found to decrease the amount of VEGF
secreted by MDA-MB231 TNBC cells by 40%. Molecular
docking studies also suggested that these two analogues
interfere with the target “hotspot” through key interactions
with specific amino acids. In conclusion, for this work to
progress to pre-clinical trials as a viable therapeutic option
in TNBC, further design, synthesis and in vitro studies are
required to validate this novel concept. Additional studies
on lead compound stability and other pharmacokinetic
properties will also be required prior to evaluation within
relevant in vivo models and identification of a new pre-
clinical drug candidate.
Conclusions
Even though protein–protein interactions (PPI) are
involved in many fundamental biological processes,
designing small molecules to target PPI’s has been
deemed challenging due to the frequent occurrence of
large and featureless protein interfaces. Over the last two
decades, as our understanding of the interfaces have
progressed, PPI’s have become attractive molecular tar-
gets in drug development. Although the interfaces of
PPI’s are extensive, studies have shown that not all resi-
dues of these interfaces contribute towards the stability of
the binding between target- and client proteins, but only
the so-called “hotspots” (a number of conserved amino
acid residues) are critical for interaction. Thus, most of
these “hotspots” have become targets in the drug devel-
opment process and the last few years have seen a number
of small molecule PPI inhibitors enter clinical trials
(White et al. 2008; Arkin et al. 2014).
Acknowledgements We thank Cancer Research Wales for a post-
doctoral research associate award (to NF-M), and acknowledge the
EPSRC UK National Mass Spectrometry Facility at Swansea Uni-
versity for provision of mass spectrometry analysis. We thank Essen
BioScience for providing IncuCyte™ live-cell imaging system and
reagents for cytotoxicity measurements.

Medicinal Chemistry Research
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