Design and synthesis of EGFR dimerization inhibitors and evaluation of their potential in the treatment of psoriasis

Article abstract of DOI:10.1016/j.bmc.2012.07.048

Hit compounds from in silico screening for inhibitors of the EGFR dimerization process were evaluated for their anti-proliferative (CCD-1106 keratinocytes) and anti-oxidant (TBA assay) activity and their effect on EGFR dimerization (BS³ chemical crosslinking assay). 7-Benzyl-8-{N′- [1-(3-ethoxy-4-hydroxyphenyl)meth-(Z)-ylidene]hydrazino}-1,3-dimethylxanthine 2a (127 μM) leads to 37% inhibition of p-EGFR dimerization in the CCD-1106 cell line and also inhibits phosphorylation of proteins in the MAPK/ERK pathway, ERK 1/2 and p-38. Based on this initial data, 2a was selected for further study and was evaluated for its anti-proliferative activity in a range of keratinocyte (CCD-1106, HaCaT and NHEK) and monocyte (ThP1 and U937) cell lines. Xanthine 2a is pro-apoptotic in HaCaT keratinocytes, as shown by electron microscopy, caspase 3/7, and annexin V-FITC/PI flow cytometric assays. It is significantly less cytotoxic than the established antipsoriatic agent dithranol 14, as determined by MTT and LDH release assays, and thus has potential as a lead compound for the treatment of psoriasis.

Full text of DOI:10.1016/j.bmc.2012.07.048

Bioorganic & Medicinal Chemistry 20 (2012) 5901–5914
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design and synthesis of EGFR dimerization inhibitors and evaluation
of their potential in the treatment of psoriasis
Donna Petch a, Rosaleen J. Anderson a, Anne Cunningham a, Suja E. George a, David E. Hibbs c, Ran Liu c,
Simon P. Mackay b, Andrew Paul b, David A. P. Small a, Paul W. Groundwater a,
⇑
a Sunderland Pharmacy School, University of Sunderland, Wharncliffe Street, Sunderland, SR1 3SD, UK
b Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, 161 Cathedral Street, Glasgow, G4 0RE, UK
c Faculty of Pharmacy, Pharmacy and Bank Building A15, University of Sydney, Sydney, NSW 2006, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
Hit compounds from in silico screening for inhibitors of the EGFR dimerization process were evaluated for
their anti-proliferative (CCD-1106 keratinocytes) and anti-oxidant (TBA assay) activity and their effect on
EGFR dimerization (BS3 chemical crosslinking assay). 7-Benzyl-8-{N0-[1-(3-ethoxy-4-hydroxy-
Received 31 May 2012
Revised 16 July 2012
Accepted 24 July 2012
Available online 7 August 2012
phenyl)meth-(Z)-ylidene]hydrazino}-1,3-dimethylxanthine 2a (127 lM) leads to 37% inhibition of p-
EGFR dimerization in the CCD-1106 cell line and also inhibits phosphorylation of proteins in the
MAPK/ERK pathway, ERK 1/2 and p-38. Based on this initial data, 2a was selected for further study
and was evaluated for its anti-proliferative activity in a range of keratinocyte (CCD-1106, HaCaT and
NHEK) and monocyte (ThP1 and U937) cell lines. Xanthine 2a is pro-apoptotic in HaCaT keratinocytes,
as shown by electron microscopy, caspase 3/7, and annexin V-FITC/PI flow cytometric assays. It is signif-
icantly less cytotoxic than the established antipsoriatic agent dithranol 14, as determined by MTT and
LDH release assays, and thus has potential as a lead compound for the treatment of psoriasis.
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
EGFR dimerization
Xanthines
Psoriasis
Anti-proliferative
Apoptosis
1. Introduction
traditional systemic agents or phototherapy is associated with a
high likelihood of treatment failure—in any year 20% of patients
Psoriasis is a chronic inflammatory disease, which is character-
ized by the loss of normal cellular homeostasis, resulting in epider-
mal hyperproliferation, altered differentiation with parakeratosis,
and inflammation.1 It appears in the form of well-demarcated ery-
thematous plaques with a characteristic silvery scaling.2 Although
the pathogenesis of psoriasis is incompletely understood, it has
been acknowledged that it originates from T-cell activation and
is an autoimmune disease; however the identity of the antigen
remains unknown.3,4 The increased proliferation and abnormal
differentiation of keratinocytes is a consequence of the over-
expression of growth factors (and their receptors), cytokines, and
angiogenic peptides.4 The treatment of psoriasis relies on well
established agents, such as dithranol, acitretin (both of which in-
duce apoptosis but their mechanisms of action are still incom-
pletely understood), cyclosporine (an immunosuppressant which
inhibits calcineurin though complexation with cyclophilin) and
corticosteroids (which inhibit cytokine, e.g. interleukin-1, produc-
tion), which have a number of drawbacks in terms of efficacy, tox-
icity and side-effects, all of which contribute to poor patient
compliance. The treatment of moderate to severe psoriasis with
will experience treatment failure/inadequate response to treat-
ment, with the mean time to treatment failure following a change
of therapy being 3-6 months.5 A series of newly developed biolo-
gics, for example Ustekinumab (StelaraÒ), are now used for the
treatment of moderate-to-severe plaque psoriasis but the long
term effects of these agents has not been established.6 For exam-
ple, Efalizumab (RaptivaÒ), a once-weekly injection for the treat-
ment of severe to moderate plaque psoriasis, was withdrawn due
to cases of progressive multifocal leukoencephalopathy (PML)
which led to patient deaths.7
In comparison with normal skin, increased numbers of epider-
mal growth factor (EGF) receptors are found in the uppermost lay-
ers of skin in active psoriatic lesions,8 with the EGFR levels (and
distribution) returning to normal in regressing lesions. In addition,
when skin from non-psoriatic individuals was exposed to EGF in
organ culture, histological features which mimic those of psoriatic
lesions developed,9 while inhibition of the EGFR tyrosine kinase
ameliorated the psoriatic phenotype in organ-cultured skin, but
had no significant effect on the histological features of non-psori-
atic skin.10
High epidermal levels of EGFR activation are associated with
enhanced expression of the pro-inflammatory cytokine, granulo-
cyte/macrophage-colony stimulating factor (GM-CSF), in lesional
skin of psoriatic patients.11 Inhibition of EGFR activity reduced
⇑
Corresponding author. Present address: Faculty of Pharmacy, Pharmacy and
Bank Building A15, University of Sydney, Sydney, NSW 2006, Australia.
E-mail address: paul.groundwater@sydney.edu.au (P.W. Groundwater).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.07.048

5902
D. Petch et al. / Bioorg. Med. Chem. 20 (2012) 5901–5914
keratinocyte-derived GM-CSF expression both in vitro and in vivo.
GM-CSF is key activator of innate immunity and, as such, is in-
volved in chronic stages of inflammatory and autoimmune dis-
eases, such as psoriasis, where macrophages, granulocytes,
neutrophils, eosinophils and dendritic cells can contribute to tissue
damage and disease.
A number of agents which target the EGFR are currently in clin-
ical use in cancer chemotherapy and these include the small mol-
ecule tyrosine kinase inhibitors (gefitinib, erlotinib, and lapatinib)
and monoclonal antibodies (e.g. cetuximab and pertuzumab)
which target the extracellular domain. The clinical benefits of these
agents has been found to be highly variable, as a result of tumor
resistance due to variations in the levels of EGFR homo- and het-
ero-dimerization12 and mutations in the ATP-binding site of the
intracellular tyrosine kinase.13 However, a number of these EGFR
kinase-blocking drugs have been shown to exhibit anti-prolifera-
tive and pro-apoptotic activity in keratinocyte lesions14,15 and
improvements in chronic psoriasis for cancer patients receiving
EGFR inhibitors have been reported, for example erlotinib,16 lapat-
inib,17 and the anti-EGFR monoclonal antibody cetuximab.18
reduce the number of leads to a more manageable size. Com-
pounds with no chiral centres were initially chosen for ease of syn-
thesis and only compounds with LUDI scores P400 were selected,
as these were expected to have greater activity, giving 181 com-
pounds with Mr 6500 and belonging to twenty-one generic struc-
ture classes. The data for representatives of the top 5 scoring
structure classes 1–5 is given in Table 1, Figure 2.
The LUDI scores for each compound are given in Table 1, along
with the Ligand Scoring energy when the best conformer of each
compound was docked into the EGFR cleft. Each compound was
evaluated as an inhibitor of proliferation of the CCD-1106 rapidly
proliferating, immortalized keratinocyte cell line (which mimics
the hyper-proliferation of the epidermis) using the MTT assay
and as an inhibitor of EGFR dimerization (also in this cell line)
using an assay involving the chemical crosslinking of the EGFR
with BS3, Figure 3.21 In this assay, the cells are treated for only
30 min with the test compounds so, in order to observe their effect
on dimerization after such a short treatment time, concentrations
in the range (118–155 lM) were used, in line with those from a
previous study.21 The effects of these compounds on the phosphor-
ylation levels of some of the proteins (p-ERK1, p-ERK2 and p-p38)
involved in the EGFR downstream signaling cascade were also
investigated.
2. Results
All compounds were also evaluated as inhibitors of iron/ascor-
bate dependent bovine brain lipid peroxidation using the thiobar-
bituric acid (TBA) test. This assay has also been used in a separate
study to test anti-psoriatic compounds for their anti-oxidant activ-
ity.22 The assay was standardized using propyl gallate 6 Figure 4, a
commercially available anti-oxidant which gives 70% inhibition at
a concentration of 100 lM, as a positive control.
On the basis of its favorable activity profile, Table 1, xanthine 2a
was selected as a lead for the synthesis of further analogues. When
2.1. In silico screening and evaluation of EGFR dimerization
inhibition
The first step in EGF-stimulated cell proliferation is the binding
of EGF to the extracellular portion of its receptor (EGFR), resulting
in dimerization, to give an EGF(2):EGFR(2) complex, and the acti-
vation of the intracellular protein tyrosine kinase, ultimately lead-
ing to signal transduction, DNA synthesis, and cell proliferation.19
Our modeling of EGFR dimerization concentrated on key amino
acid residues in the dimerization arm (purple) of one monomeric
EGFR unit and its binding site (white) for the other EGFR unit,
Figure 1, from the crystal structure of the complex of human epi-
dermal growth factor and receptor extracellular domains.20 Using
in silico screening, we have identified potential inhibitors of this
interaction, which were then synthesized and tested as potential
anti-proliferative agents.
The screening area was refined to the ten main interaction res-
idues of the cleft to which the tip of the ‘dimerization arm’ binds,
Figure 1a. This screen had six queries each made up of six features
and, in order for a compound to satisfy a query, it must contain the
features stated for that query. This approach was successful, pro-
ducing 4900 hit compounds with positive LUDI scores from a
screen of the Aldrich Rare Chemicals Database (more than
102,000 commercially available drug-like molecules, 75% of which
obey all of Lipinski’s rules). A number of filters were applied to
CCD-1106 cells were treated with this xanthine 2a (126
30 min), levels of the phospho-EGFR dimer decreased by 37%, Fig-
ure 3. This xanthine was also a potent anti-oxidant (IC50 8 M) and
lM for
l
significantly reduced the levels of phospho-ERK 1 and 2. Figure 1b
shows the best conformer of xanthine 2a bound to the cleft of the
EGFR into which the dimerization arm binds.
2.2. Synthesis and anti-proliferative activity of xanthine
analogues 2a–m
In the three step synthesis of the xanthine analogues 2,
substituted benzyl bromides 8 were initially reacted with 8-chlo-
rotheophylline 7 using a literature method23 to form the 7-ben-
zyl-8-chloroxanthines 9a–g (Scheme 1), which were then reacted
with hydrazine hydrate to afford the 7-benzyl-8-hydrazinylxanth-
ines 10a–g in good yield (Scheme 2). In the final step, the
Figure 1. (a) Monomeric EGFR unit, showing dimerization arm (solid purple) and the binding site for the dimerization arm of another EGFR monomer (solid white); (b) A
docked stick conformer of 2a in the cleft of the EGFR dimerization arm binding site.

D. Petch et al. / Bioorg. Med. Chem. 20 (2012) 5901–5914
5903
Table 1
LUDI scores, Ligand scoring docking energies and initial biological testing data of hits 1–5
Compound LUDI score Ligand Scoring
CCD-1106
Anti-oxid. % Inhibition of p-EGFR % Inhibition of EGFR downstream signaling in CCD-1106 cells
(concn./ M)
docking energy Anti-prolif. IC50 (mM) dimer formation
l
(kcal molꢀ1
)
GI50
(lM)
(concn./lM)
Concn. (
lM)
p-ERK 1
p-ERK 2
p-p38
1
2a
3
4
5
536
478
477
449
426
ꢀ37.59
ꢀ54.45
ꢀ46.97
ꢀ46.58
ꢀ66.87
123.35
2.69
4.09
14.87
9.33
46.74
0.008
0.40
3.41
1.25
52 (155.4)
37 (126.7)
47 (134.5)
16 (123.6)
42 (118.1)
153.0
116.8
161.1
120.7
111.8
28
39
12
42
26
20
33
—
33
19
-
11
-
-
12
O
O
O2N
Me
O
N
N
N
H
OH
O
N
N
NH
NO2
Me
N
OEt
1
2a
Br
O
Me
O
CO2Et
OH
O
H
O
Ph
H
N
N
H
N
N
CHMe2
EtO2C
Me
O
H
O
O
N
O
N
H
Me
Me
3
4
5
Figure 2. Chemical structures of representatives of the top 5 generic classes of EGFR dimerization hit compounds.
group giving a singlet in the 1H NMR spectrum between d 7.74
and 7.92, and the CH giving a peak in the 13C NMR spectrum be-
tween d 143.4 and 144.6–as well as by elemental analysis.
3,4,5-Triethoxybenzaldehyde 11a was prepared from the corre-
sponding benzoic acid 12 in a two-step, reduction then oxidation,
process via the alcohol 13, Scheme 2, in 59% overall yield.
a
340 kDa
170 kDa
The xanthine analogues 2a–m were initially evaluated for their
in vitro anti-proliferative activity in the keratinocyte HPV-16
transformed cell line, CCD-1106, Table 2, using the MTT viability
assay. We have previously shown that there were no significant
differences between the MTT, NRU (neutral red uptake), SRB (sul-
forhodamine B), CVS (crystal violet staining), and radioactive
[3H]-thymidine incorporation assays for the determination of the
dithranol GI50 in keratinocytes.24 The established anti-psoriatic
agent dithranol 14, which had no effect on EGFR dimerization in
b
Lane
1
-
2
-
3
4
-
5
-
6
7
8
+
+
c
9
+
+
c
10 11 12 13 14
EGF 50 ng/mL
BS3 2mM
Compound
-
-
-
+
+
3
+
+
1
+
+
+
+
+
4
+
c
+
c
+
3
+
1
+
5
+
+
4
+
2a
5
2a
Figure 3. Immunoblots showing; (a) phospho-EGFR levels in CCD-1106 cells, (b)
total EGFR. Cells were exposed to compounds for 30 min, then EGF 50 ng/mL for
10 min and BS3 (extracellular crosslinking agent) 2 mM for 30 min (c = control [no
added inhibitor]). Immunoblots were quantified by densitometry.
the crosslinking assay even at a concentration of 348.8
lM, has a
GI50 of 0.51 0.03 M in this assay. Dithranol has previously been
l
shown to inhibit keratinocyte proliferation by acting on the EGFR
pathway and reducing the binding of EGF to EGFR.25
HO
Derivative 2a, with a GI50 of 2.69 lM, was the most active in the
OH
O
OH
O
O
assay, but the majority of the other derivatives also showed mod-
erate to high anti-proliferative activity, Table 3. Irrespective of the
R3 and R4 substituents, a carboethoxy or carboxyl group at R1
(2f,g,l,m) leads to a significant decrease in activity, while both
electron-donating and withdrawing R1 and R2 substituents can
give potent inhibitors.
HO
HO
CH3
14
6
Figure 4. Standards for the anti-proliferative and anti-oxidant assays.
2.3. Evaluation of the anti-proliferative activity and cytotoxicity
of xanthine 2a
hydrazines 10a–g were condensed with either 3,4,5-triethoxy-
benzaldehyde 11a or 3-ethoxy-4-hydroxybenzaldehyde 11b to
form the 7-benzyl-8-arylidenehydrazinylxanthines 2a–m in vari-
able yields (Scheme 2). These hydrazinylxanthines 2a–m were
characterized by spectroscopic analysis–the proton of the N@CH
On the basis of its activity in the initial screens, derivative 2a
was selected for further biological evaluation and we next investi-
gated its anti-proliferative effect on three different keratinocyte

5904
D. Petch et al. / Bioorg. Med. Chem. 20 (2012) 5901–5914
R1
R1
O
O
R2
Me
O
Me
O
a
N
NH
Cl
N
N
Br
+
R2
N
Me
N
N
Me
N
Cl
7
8a-g
9a - g
b
R1
R1
R2
R3
O
O
R2
R4
Me
O
Me
O
N
N
R3
c
N
N
+
R4
OEt
O
OEt
N
N
NH
N
N
Me
N
NH
NH2
H
Me
10a - g
2a-m
11a
11b
R
R
3 = R4 = OEt
3 = H, R4 = OH
d
2g
2m
2f
2l
Scheme 1. Reagents and conditions: (a) DMF, K2CO3, rt, overnight; (b) EtOH, hydrazine hydrate, reflux, overnight; (c) DCM, DMF, MgSO4, rt, overnight; (d) MeOH, LiOH, reflux,
3 h.
O
O
EtO
EtO
EtO
EtO
EtO
EtO
OH
OH
H
a
b
OEt
OEt
OEt
12
13
11a
Scheme 2. Reagents and conditions: (a) LiAlH4, Et2O, reflux, 4.5 h, 88%; (b) PCC, DCM, rt, 4 h, 67%.
Table 2
Table 4
GI50 values in the HPV-16 (E6/E7) transformed cell line, CCD-1106, using the MTT
assay for xanthine derivatives 2a–m
Mean GI50
(lM) values of 2a compared to dithranol 14 in monocyte cell lines, ThP1
and U937 determined by the Cell Titer 96 AQueous one assaya
Compound
R1
R2
R3
R4
Mean GI50
(
l
M) S.E.M.a
Cell line/compound
Mean GI50
(
l
M) S.E.M., n = 6
14
54 2.04
0.29
2a
2b
2c
2d
2e
2f
2g
2h
2i
H
H
H
H
NO2
COOEt
COOH
H
H
H
H
H
H
H
H
H
H
H
OEt
OEt
OEt
OEt
OEt
OEt
OH
OH
OH
OH
OH
OH
OH
OEt
OEt
OEt
OEt
OEt
OEt
2.69 0.79
5.47 1.53
9.75 0.12
5.21 0.14
8.50 3.06
105.94 2.43
178.28 6.48
4.65 1.60
18.91 1.63
9.83 0.55
5.16 0.27
48.26 1.38
117.01 2.44
2a
NO2
Me
Cl
H
H
H
H
NO2
Me
H
ThP1
U937
36 2.04
46 2.21
5
a
Data given as means S.E.M., n = 6. Incubation period was 72 h.
normal human epidermal keratinocytes (NHEK; available from
pooled donors and representative of 90% of the cells in the
epidermis).
As can be seen from the data presented in Table 3, xanthine 2a
was less active in all 3 cell lines than the established anti-psoriatic
agent dithranol 14. In the HaCaT cell line, the effects of xanthine 2a
were found to be reversible at the lower concentrations tested.
Xanthine 2a was also found to inhibit the growth of ThP1 and
U937 monocytes in a dose dependent manner, with GI50 values
2j
2k
2l
OMe
COOEt
COOH
H
H
2m
a
Standard error of the mean (six replicates were performed of each of 8 drug
concentrations)
of 36 and 46 lM, respectively, Table 4, and had a higher activity
Table 3
than that of dithranol in the ThP1 cell line.
Mean GI50 values of xanthine 2a and dithranol 14 in different keratinocyte cell lines
using the NRU assaya
Having thus established the promise of derivative 2a as an anti-
proliferative agent, we next evaluated its cytotoxicity, in the CCD-
1106 and HaCaT cell lines, Table 5. The use of the CCD-1106 and
HaCaT cell lines as in vitro model systems has the advantages of
having no donor specific variations, as found in primary human
keratinocytes, as well as providing an almost unlimited supply of
identical cells, assuring good reproducibility.
Cell line/compound
2a GI50
(lM)
14 GI50 (lM)
CCD-1106
HaCaT
NHEK
9
0.70
0.40 0.01
0.70 0.02
0.50 0.27
22 1.10
54 0.19
a
Data given as means S.E.M., n = 6. Incubation period was 72 h.
Dithranol 14 was found to be significantly more cytotoxic than
xanthine 2a, particularly in the HaCaT cell line, at both the initial
(6 h) and midpoint (24 h) cytotoxicity timepoints, as determined
cell lines; CCD-1106, HaCaT (a rapidly growing, spontaneously
transformed human epithelial cell line from adult skin), and

D. Petch et al. / Bioorg. Med. Chem. 20 (2012) 5901–5914
5905
Table 5
analysed by flow cytometry, after staining with Annexin V-FITC
and PI to measure PS exposure. Figure 6 is representative of the
flow cytometry plots obtained from control keratinocytes and drug
treated cells after only 9 h exposure, when PS exposure became
evident.
Mean LD50 values of xanthine 2a and dithranol 14 in the CCD-1106 and HaCaT
keratinocyte cell lines using a range of endpointsa
Compound/assay
Viability assay mean
LDH release assay mean
LD50 M) S.E.M., n = 3
LD50
(lM) S.E.M., n = 6
(l
MTT
CYTOX-96
CCD-1106
2a (6 h)
2a (24 h)
14 (6 h)
14 (24 h)
HaCaT
2a (6 h)
2a (24 h)
14 (6 h)
14 (24 h)
3. Discussion
234 4.01
112 3.94
53 1.17
>446
>446
310 4.81
442 10.42
Agents which interfere with the EGFR signaling pathway have
potential in the treatment of psoriasis and a number of these EGFR
kinase-blocking agents have been shown to exhibit anti-prolifera-
tive and pro-apoptotic activity in keratinocyte lesions.14–16 Half of
the amended crystal structure of the EGFR:EGF dimer, 1IVO, was
removed to leave just EGFR and its bound EGF ligand. In order to
perform an in silico screen for novel inhibitors of the EGFR, the
key dimerization interaction residues needed to be highlighted in
order to construct a target site for the screen. The eight key
residues (Tyr246 to Met253) in the tip of the ‘dimerization arm’ in-
volved in binding to the cleft of another EGFR during dimerization
have previously been identified. The ten key residues in the cleft of
the EGFR which bind the tip of the ‘dimerization arm’ have also
been identified.20,27 These ten residues are Asn86, Phe230,
Phe263, Gly264, Ala265, Tyr275, Thr278, Cys283, Arg285 and
Ala286. Details of the type of interactions between residues in
the tip of the dimerization arm and the cleft of the EGFR have pre-
viously been described.20,28
2
0.15
440 6.27
446 14.09
446 5.99
446 9.92
10 0.34
3
3
4.78
0.07
3
0.03
a
Data given as means S.E.M., n = 6.
by the viability (MTT) and lactate dehydrogenase (LDH) release as-
says. These assays have been used extensively to study the toxic ef-
fects of agents on a variety of different cell types grown in
monolayer cultures.26,27
2.4. Evaluation of the pro-apoptotic effects of xanthine 2a in
human keratinocytes
Established anti-psoriatic therapies such as dithranol 14, vita-
min-D3 analogues, low-dose methotrexate and PUVA (psoralen
plus UVA) induce clinical improvement by the initiation of apopto-
sis. The pro-apoptotic effects of hydrazinylxanthine 2a were thus
assessed with respect to morphological changes, caspase activation
and the binding of annexin V. HaCaT cells exposed to 2a showed
typical apoptotic morphology, with no signs of necrosis by electron
microscopy. Xanthine 2a was found to induce apoptosis in HaCaT
keratinocytes after 12 h incubation, through dose-dependent cas-
pase 3/7 activation, Figure 5, resulting in significant caspase 3/7
activation at all the concentrations tested, with remarkably high
caspase 3/7 activation even at the lowest concentration tested
LUDI was used to generate an interaction map for the active
site, showing three types of interaction sites; hydrogen bond
acceptors, donors and hydrophobic (lipophilic) sites. Each of these
interaction features were then clustered into groups of hydrogen
bond donors (HBD), acceptors (HBA) and hydrophobic (lipophilic)
groups and edited to give a manageable number of features. Next,
the number of queries and the number of features included in each
query were chosen and excluded volumes were added. Excluded
volumes are regions into which the screened compounds cannot
enter; they include heavy atoms in the protein structure, for exam-
ple C, N, O, S, but not hydrogen. The chosen queries were then gen-
erated and used to screen the Catalyst 3D database of compounds.
The Aldrich ‘Rare Chemical Database’ was chosen for the screen,
which involved flexible docking of each of the compounds in the
database into the defined rigid active site. Only one conformer of
each of the compounds was screened to achieve a manageable
screening time period. The binding affinity of each of the com-
pounds in the active site was assessed by LUDI, with the greater
the LUDI score, the greater the predicted binding affinity of the
compound. Each of the ligands was also scored using the Ligand
Scoring docking energy for the best conformer (generated by Li-
gand Fit).
(25 lg/ml = 55.8 lM). The MTT assay was performed in conjunc-
tion with the caspase assay in order to determine if there is any
change in the mitochondrial state of the cells during the caspase
activation, Figure 5, and the increase in caspase 3/7 activation
was always accompanied by a decrease in mitochondrial activity
for the cells exposed to 2a.
In order to quantify the apoptosis induced by 2a in keratino-
cytes, HaCaT cells incubated with different concentrations were
700
600
500
400
300
200
100
0
0.4
0.35
0.3
0.25
0.2
0.15
0.1
Representatives of the generic structures were then purchased
and evaluated for their activity in an EGFR dimerization assay. This
assay was recently employed in the discovery of NSC56452 15, the
first example of a small molecule which targets EGFR dimeriza-
tion.21 In common with xanthine 2a, NSC56452 is a purine deriva-
tive and significantly inhibits EGFR dimerization (in this case in
CHO cells). NSC56452 inhibits the proliferation of HeLa cells at a
Caspase activity
Cell viability
0.05
0
concentration of 50
lM but has little effect at lower doses.
Control
56
112
223
446
Concentration (µM)
Figure 5. Dose response of caspase 3/7 activity in HaCaT keratinocytes treated with
increasing concentrations of 3a as determined by Apo-ONE homogeneous caspase-
3/7 assay. Cell viability was evaluated by the MTT assay in parallel with the caspase
3/7 assays. Keratinocytes were seeded in 96 well microplates at 10,000 cells/0.2 ml/
well and allowed to attach overnight. Test compound was added to the cells and
incubated for 12 h. MTT and caspase 3/7 assays were performed as described in the
experimental section. Data shown represent means SEM, n P3 of two different
experiments. Statistical significance between the control and the treated samples
was determined by independent t-test, ⁄p <0.05.
Me
S
N
N
N
Me
Me
H2N
N
15

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D. Petch et al. / Bioorg. Med. Chem. 20 (2012) 5901–5914
Figure 6. Flow cytometric analysis of apoptosis induced by 2a in HaCaT keratinocytes. Keratinocytes were seeded at a density of 106 cells/ml and allowed to attach overnight.
Cells were exposed to different concentrations of the drug; (a) control (vehicle alone), (b) 111.6 lM (c) 223.2 lM and (d) 446 lM. After 9 h incubation, cells were trypsinized,
washed and stained with Annexin V-FITC/PI and two-color analysis was performed by flow cytometry. The lower left quadrant (AnnxVꢀ/PIꢀ) represents viable cells, whereas
the lower right quadrant (AnnxV+/PIꢀ) represents early-phase apoptotic and the upper right quadrant (AnnxV+/PI+) represents late-phase apoptotic/necrotic cells. The x-axis
shows log FL1-fluorescence intensity; the y axis indicates the log FL2-fluorescence intensity. Results are representative of three independent experiments.
Derivative 2a, with a GI50 of 2.69
l
M in the anti-proliferative
dimerization, downstream signaling (p-ERK 1, p-ERK2 and p-
p38), and CCD-1106 growth inhibition assays (Table 1), was used
to select xanthine 2a for further biological evaluation.
assay, was the most active, but the majority of the other deriva-
tives also showed moderate to high anti-proliferative activity, Ta-
ble 3. A 3D-QSAR Comparative Molecular Fields Analysis (CoMFA)
of the xanthines 2 was performed and the interactions of an active
(2a) and inactive (2g) compound with the CoMFA steric and elec-
trostatic fields are shown in Figures 7a and 7b. As can be clearly
seen from Figure 7a, the oxygen of the 3-ethoxy group of the aryl-
hydrazone group of xanthine 2a lies in a region of negative poten-
tial (red) and the interactions of this compound with the steric
field are mostly favorable. For the less active xanthine 2g, Figure 7b,
there are no favorable interactions with the electronic field and
more unfavorable interactions with the steric field than 2a. An ini-
tial PLS analysis was used to correlate the experimental inhibitory
activity of the xanthine growth inhibitors with the CoMFA values
containing the magnitude of steric and electrostatic potentials
and gave an excellent correlation (R2 = 0.96).
Monocyte cell lines are commonly used as in vitro models for
inflammatory disorders in the study of the effects of pro-inflamma-
tory cytokines. Although monocytes or macrophages can be ob-
tained from humans, they present many difficulties, such as
sample acquisition, donor variability and the difficulty associated
with obtaining a homogeneous population of human monocytes
in the amounts required for pre-clinical studies. In order to over-
come the problems associated with sample acquisition and donor
variability, monocyte cell lines such as ThP1 and U937 have been
used extensively as models for primary monocytes32 and the
appropriateness of using ThP1 and U937 cell lines as models for
primary peripheral blood monocytes (PBMs) for studying inflam-
matory disorders has previously been demonstrated.33,34 Several
studies have shown that monocyte cell lines are a sensitive target
for successful anti-psoriatic agents, such as dithranol,35,36 metho-
trexate,37,38 cyclosporin A,39 and corticosteroids.40 The novel xan-
thine 2a was found to be effective in inhibiting the growth of
both the monocyte cell lines, with a higher activity than that of
dithranol 14 in the ThP1 cell line.
Chronic inflammation is a pathological feature of psoriasis and
reactive oxygen species (ROS) and increased oxidative stress are
believed to be linked to inflammation.29–31 An anti-psoriatic drug
with anti-oxidant properties thus has great potential to aid in the
treatment of skin inflammation. With this in mind, all of the hit
compounds were also tested for their anti-oxidant activity.
Analysis of the cytotoxicity of xanthine 2a was performed at 6 h
and 24 h in order to determine the concentrations of test agents
causing initial cytotoxicity (6 h) and those causing midpoint
Xanthine 2a is a potent anti-oxidant (IC50
and this data, along with that for its activity in the EGFR
8 lM in the TBA assay)

D. Petch et al. / Bioorg. Med. Chem. 20 (2012) 5901–5914
5907
Figure 7. (a) Interaction of the CoMFA steric and electrostatic fields SD ꢁ coeff contour maps with ‘active’ xanthine 2a; (b) Interaction of the CoMFA steric and electrostatic
fields SD ꢁ coeff contour maps with ‘inactive’ xanthine 2g. Bulky groups in the green region favor the inhibitory activity, but bulky groups in the yellow region are not
favorable. Negative potentials in the red region favor the inhibitory activity, but negative potentials in the blue region are not desirable.
cytotoxicity (24 h). Such comparative data is very informative of
the kinetics of the cytotoxicity curves. The MTT assay is a measure
of mitochondrial reductase activity, and the CYTOX-96 assay deter-
mines lactate dehydrogenase (LDH) release and so can be used to
determine cell membrane damage (one of the final stages of cell
death). Korting et al. have confirmed that there is a close correla-
tion between the in vitro cytotoxicity values predicted by viability
assays in cultured human foreskin keratinocytes and the in vivo
irritancy data.41
A severe disadvantage of permeability assays is that the initial
damage sites of many cytotoxic agents are intracellular.42 Cells
may, therefore, be irreversibly damaged and committed to die
but the plasma membrane may still be intact. These assays are thus
likely to underestimate cellular damage caused by the cytotoxic
agents in comparison to other methods. The LDH release assay,
which is based on the measurement of cytoplasmic enzymes re-
leased by injured cells, is very informative, not only of cell growth
inhibition, but also of the extent of cell lysis caused by test
agents.43 It was observed that the cytotoxicity of 2a was detected
earliest by measuring the metabolic activities of the cells, whereas
the LDH leakage assay seems to be the most definitive marker of
cell death by membrane rupture. Dithranol 14 has greater growth
inhibitory activity in all 3 of the keratinocyte cell lines tested but
compound 2a proved to be much less cytotoxic than dithranol 14
in all cases, while there was no difference between the initial
and midpoint toxicities for 2a in the MTT viability assay in the Ha-
CaT cell line, or the LDH release assay in either cell line.
addition, been shown to be dysregulated at the cytokine level; it
has recently been shown that IL-15 does not display any relevant
effects in cell proliferation, but inhibits both the anti-Fas and the
methylcellulose-induced apoptosis of keratinocytes in vitro.48
Interestingly, increased expression of IL-15 was observed in the
epidermis of lesional psoriatic skin.48 It has been shown that sev-
eral genes which were reported to be related to apoptosis or
anti-proliferation show high expression levels only in uninvolved
psoriatic lesions.49 All psoriatic biopsies revealed absent apoptotic
keratinocytes and lymphocytes and these results are in agreement
with previous studies, where no apoptotic cells were detected in
any of the psoriatic lesions.47,49
In order to identify the cellular changes, the morphological dif-
ferences between the normal control and the treated cells were
compared by electron microscopy. Compared to the untreated con-
trol, cells treated with 2a exhibited typical morphological charac-
teristics of early phase apoptosis and late phase apoptosis/
necrosis, including; chromatin rearrangements and condensation,
nuclear fragmentation, an intact membrane and cytoplasm, the
formation of apoptotic bodies and cytoplasmic vacuolization, sur-
face blebbing and formation of apoptotic bodies. Cells undergoing
necrosis displayed nuclei with a nuclear pattern resembling that of
a normal cell but a degenerated cytoplasm and membranes as well
as clumped chromatin, swelling and the appearance of lesions on
the cell surface.
Apoptotic cell death is characterized by numerous morphologi-
cal and biological changes. One of the features of apoptotic cells is
the loss of cell membrane asymmetry during the early phases of
programed cell death. Phosphatidylserine (PS), a phospholipid that
can only be found in the inner layer of the intact cell membrane,
becomes exposed to the outer leaflet, where it can be detected
by the selective binding of Annexin V to PS under defined salt
and calcium concentrations and it is, therefore, considered to be
an early marker of apoptosis. Cells in the early phase of apoptosis
bind Annexin V (AnnxV) but exclude propidium iodide (PI). On the
other hand, double positive cells (stained by both Annexin V-FITC
and PI) may represent a late apoptotic/necrotic population, since
necrotic cells lose membrane integrity and cannot exclude PI. Flow
cytometric analysis of cells stained with Annexin V-FITC and PI al-
lows the identification of intact (unstained), apoptotic (AnnxV+/
PIꢀ) and necrotic (AnnxV+/PI+) populations.
Several studies have been carried out which have investigated
the role played by defective apoptosis in the pathogenesis of pso-
riasis. Experimental studies have revealed that keratinocytes de-
rived from psoriatic epidermis display resistance to apoptotic
stimuli, unlike the healthy keratinocytes of normal skin.44 Cathep-
sin D and zinc-a2-glycoprotein, catalytic enzymes linked with
apoptosis and flaking of the skin, are present within the stratum
corneum of normal skin, but are missing in psoriatic plaques.45
An inverse reaction of psoriatic keratinocytes in response to
IFN-
c
has also been reported. In normal keratinocytes, IFN-
c up-
regulates the expression of Cathepsin D and zinc-a2-glycoprotein,
while they are down regulated in keratinocytes from psoriatic epi-
dermis.45 Furthermore, expression of Bcl-2 is found to be increased
in psoriatic epidermis.46 Other studies have revealed an apoptotic
index of 0.12% in healthy skin, of 0.035% in psoriatic epidermis
and of 0.31% in regressing psoriasis after PUVA treatment and this
underlines the fact that loss of apoptosis within the epidermis
could be of some pathologic relevance for the development of pso-
riatic hyperplasia.47 Apoptosis in psoriatic keratinocytes has, in
Treatment with 2a resulted in a slow but steady increase in the
number of both early apoptotic and late apoptotic/necrotic cells,
accompanied by a decrease in the number of viable cells, Figures
6
and 8.
A significant increase in the number of early
apoptotic and late apoptotic/necrotic cells was observed for the

5908
D. Petch et al. / Bioorg. Med. Chem. 20 (2012) 5901–5914
100
80
60
40
20
0
125 MHz, respectively. Column chromatography was performed
viable
using Fluka silica gel 60. Thin layer chromatography was per-
formed using Merck aluminum sheets, silica gel 60 F264. Mass spec-
trometry was performed on an Esquire 3000plus or Bruker ApexII,
for low and high resolution spectra, respectively. Elemental analy-
ses were performed by Medac Ltd, Brunel Science Centre, Surrey,
United Kingdom or on an Exeter Analytical CE-440 elemental ana-
lyser at Chemispec, University of Sunderland, United Kingdom.
Dimethylsulfoxide (DMSO), trypan blue, and MTT were pur-
chased from Sigma–Aldrich, Dorset, UK. Alamar blue was obtained
from Serotec (Oxford, UK). Phosphate buffered saline (PBS) was
purchased from Gibco, Renfrewshire, UK. An HPV-16 immortalised
human keratinocyte cell line obtained from the American Tissue
Culture Collection (ATCC), CRL-2309 and reference CCD-1106
KERTr was used. Keratinocyte serum free medium (SFM) pur-
chased from Gibco, was used successfully in previous studies to
cultivate a HPV-16 cell line50,51 and was used in this study. The
keratinocyte growth supplements bovine pituitary extract (BPE)
and epidermal growth factor (rEGF) were purchased from Gibco.
Cells were grown under conditions of 5% CO2 at 37 °C. Manipula-
tion of the cells was performed in a Class II Safety Cabinet under
aseptic conditions.
early apoptotic
lateapoptotic/necrotic
#
*
Control
112
223
Concentration (µM)
446
Figure 8. Effect of xanthine 2a on the induction of apoptosis and necrosis in human
keratinocytes. Cells were seeded at a density of 106 cells/ml and allowed to attach
overnight. Cells were exposed to different concentrations of the drug for 9 h. after
the incubation period, cells were then trypsinized, washed and stained with
Annexin V-FITC/PI and two-color analysis was performed by flow cytometry. Data
shown represent means SEM,
n P3 of two different experiments. Statistical
significance between the control and the treated samples were determined by
independent t-test, ⁄p <0.05 and #p <0.01.
concentration range tested, with the increase being more pro-
nounced at the highest concentration, Figure 8. As the incubation
period for these studies was only 12 h (compared to the 72 h incu-
bation times for the growth inhibition assays), the concentrations
Cell Titer 96 Aqueous One solution cell proliferation assay kit,
and Cyto Tox 96 assay kits were purchased from Promega (Madi-
son, USA). Annexin V-FITC (K101-100) was obtained from Biovision
(Mountain View, CA) and the caspase-3/7 activity assay kit from
Promega (Madison, WI). p-EGFR (Tyr1173): sc-12351, p-ERK (E-
4): sc-7383 and p-p38 [D-8] antibodies were purchased from Santa
Cruz Biotechnology, Santa Cruz, CA, USA.
chosen for these apoptosis studies were greater than the GI50
,
but well below the LD50, as it would be difficult to detect any ef-
fects with lower concentrations after such a short incubation time.
The Annexin V-FITC/PI flow cytometric assays provided quanti-
tative information about the apoptotic process and, in agreement
with the electron microscopy observations, 2a was found to induce
apoptosis in keratinocytes. The percentage of cells in the late apop-
totic/necrotic phase was observed to be low (15%), even at the
highest concentration (446 lM) tested, indicating that the com-
pound has very low cytotoxicity. This was also supported by the
viability assay (MTT), Figure 5, which showed 62% viability after
12 h incubation with a high concentration (446 lM) of 2a.
5.2. In silico screening
The crystal structure of the EGFR:EGF 2:2 dimer, determined by
X-ray diffraction, was obtained from the RCSB protein databank
(1IVO).20 Insight II 2005, Cerius2 4.10L, Catalyst 4.10 and DS Viewer-
Pro 6.0 programs from Accelrys were used with a Silicon or Linux
Graphics workstation. The force field used in Insight II was CFF91
and for Cerius2 was CFF. The compound database screened was
the Aldrich ‘Rare Chemicals Database’ (SALOR). All modeling images
were taken from screen shots using Insight II or DS ViewerPro 6.0.
The crystal structure of the EGFR:EGF dimer, 1IVO, was
amended for use by adding hydrogens and removing all water mol-
ecules and NAG ligands. An assembly was created including three
subsets; all atoms (heavy), backbone, and backbone and side
chains, then the structure were minimised using Insight II Discover
module.
Half of the amended crystal structure dimer was removed to
leave just EGFR and its bound EGF. The binding site to be screened
was defined by entering the coordinates of the centre of the site
and radius by highlighting the ten key binding residues of the
‘cleft’. Next, LUDI was used to generate an interaction map for
the active site, showing three types of interaction sites; hydrogen
bond acceptors, donors and hydrophobic (lipophilic) sites. Each
of these interaction features were then clustered into groups of
hydrogen bond donors (HBD), acceptors (HBA) and hydrophobic
(lipophilic) groups and edited to give a manageable number of fea-
tures. The number of queries and the number of features included
in each query were chosen and excluded volumes were added. The
chosen queries were then generated and used to screen the Catalyst
3D database of the Aldrich ‘Rare Chemical Database’, containing
102,000 compounds. This screen involved flexible docking of each
of the compounds in the database into the defined rigid active site.
Only one conformer of each of the compounds was screened to
achieve a manageable screening time period. The binding affinity
of each of the compounds in the active site was assessed by LUDI.
Each compound was given a LUDI score to represent the predicted
4. Conclusion
In conclusion, based on the results from growth inhibition as-
says in human keratinocytes and monocytes and an anti-oxidant
assay, xanthine 2a was identified as a potential agent for the treat-
ment of psoriasis. This xanthine inhibits the growth of keratino-
cytes and monocytes and is significantly less cytotoxic than the
established antipsoriatic agent dithranol 14. These findings were
further confirmed by electron microscopy, caspase 3/7 assays,
and flow cytometric analysis which showed that this novel com-
pound induces apoptosis in highly proliferating human keratino-
cytes and is, therefore, therapeutically relevant for the treatment
of psoriasis.
5. Experimental
5.1. General
Melting points were determined on a Stuart Scientific SMP10
apparatus and are uncorrected. IR spectra were recorded on an
ATl Mattson Generis series FTIR or Unicam Research series 1 FTIR
using KBr disks or on a Perkin Elmer Inc Spectrum BX FT-IR system.
1H NMR spectra were recorded in DMSO or CDCl3 on a Bruker
AVANCE 300 or 500, at 300 MHz or 500 MHz. Coupling constants
are given in Hz and all chemical shifts, which are relative to the
peaks for residual protonated solvent, are given in ppm. 13C NMR
spectra were obtained on the AVANCE 300 or 500 at 75 or

D. Petch et al. / Bioorg. Med. Chem. 20 (2012) 5901–5914
5909
binding affinity and this score was used to prioritise the lead com-
pounds. This LUDI score uses the same algorithms as the LUDI 1
score of the program Ligand Scoring. The greater the LUDI score,
the greater the predicted binding affinity of the compound.
ethanol, recrystallized from methanol and acetonitrile and dried
in a vacuum oven to give the hydrazine.
5.3.4. General procedure for the synthesis of 7-benzyl-8-{N0-
[1-(3-ethoxy-4-hydroxyphenyl)meth-(Z)-ylidene]hydrazino}-
1,3-dimethylxanthine (2a) and analogues (2b–m)
5.3. Synthetic chemistry
To a stirred suspension of aldehyde 11a or 11b (0.50 mmol) in
dichloromethane (5 mL) and hydrazine 10a–g (0.50 mmol), N,N-
dimethylformamide was added dropwise until a clear solution
was formed. Magnesium sulfate (0.1 g) was added and the reaction
mixture was stirred overnight at room temperature. The magne-
sium sulfate was filtered from the reaction solution, the solvents
were removed under reduced pressure, and the residue recrystal-
lized from methanol and acetonitrile to afford the product.
5.3.1. 3,4,5-Triethoxybenzaldehyde (11a)
5.3.1.1. 3,4,5-Triethoxyphenylmethanol (13).
3,4,5-Trieth-
oxybenzoic acid 12 (5.03 g, 19.77 mmol) was added slowly to an
ice-cooled grey suspension of LiAlH4 (1.42 g, 37.41 mmol) in dry
diethyl ether (50 mL). The mixture was then refluxed for 4.5 h.
The suspension was cooled, quenched with water/ethyl acetate
(100 mL 1:1) and filtered through celite. The organic layer was sep-
arated, dried over MgSO4, filtered and the solvent evaporated un-
der reduced pressure to give (3,4,5-triethoxyphenyl)methanol, 13
as an off-white solid (4.20 g, 88%); mp 38–39 °C (lit. mp 29–
30 °C);52 Rf = 0.38 (pet.ether/ethyl acetate 1:1); mmax (KBr)/cmꢀ1
3283 (OH), 1585 (Ar C@C), 1436 (Ar C@C); 1H NMR (300 MHz,
DMSO): d 1.13 (3H, t, J = 6.9 Hz, CH3), 1.23 (6H, t, J = 6.9 Hz,
2 ꢁ CH3), 3.81 (2H, q, J = 6.9 Hz, CH2), 3.91 (4H, q, J = 6.9 Hz,
2 ꢁ CH2), 4.31 (2H, d, J = 5.7 Hz, CH2), 4.99 (1H, s, OH), 6.49 (2H,
s, H-2,6); 13C NMR (75 MHz, DMSO): d 15.3 (2 ꢁ CH3), 15.9 (CH3),
63.5 (CH2), 64.4 (2 ꢁ CH2), 68.2 (CH2), 105.2 (2 ꢁ CH), 136.2 (quat.),
138.4 (quat.), 152.7 (2 ꢁ quat.); Anal. Calcd for C13H20O4: C, 65.0;
H, 8.4. Found: C, 64.9; H, 8.4.
5.3.4.1. 7-Benzyl-8-{N0-[1-(3-ethoxy-4-hydroxyphenyl)meth-
(Z)-ylidene]hydrazino}-1,3-dimethylxanthine (2a).
Prepared
from 3-ethoxy-4-hydroxybenzaldehyde 11b (0.61 g, 3.60 mmol)
and 7-benzyl-8-hydrazino-1,3-dimethyl-3,7-1H-dihydropurine-2,6-
dione 10a (0.99 g, 3.29 mmol), 7-benzyl-8-{N0-[1-(3-ethoxy-4-
hydroxylphenyl)meth-(Z)-ylidene]hydrazine}-1,3-dimethyl-3,7-1H-
dihydropurine-2,6-dione 2a was obtained as pale brown crystals
(1.04 g, 70%); Rf = 0.50 (ethyl acetate); mp 243–244 °C; mmax (KBr)/
cmꢀ1 3512 (NH), 3403 (OH), 1693 (C@O), 1653 (C@O), 1621 (Ar
C@C), 1477 (Ar C@C), 1286 (C–O); 1H NMR (300 MHz, DMSO): d
1.29 (3H, t, J = 6.9 Hz, CH3), 3.17 (3H, s, N1-CH3), 3.40 (3H, s, N3-
CH3), 3.81 (2H, q, J = 6.9, CH2), 5.80 (2H, s, CH2), 6.77 (1H, d,
J = 8.2 Hz, H-5), 6.89 (1H, dd, J = 8.2, 1.5 Hz, H-6), 6.99 (1H, d,
J = 1.5 Hz, H-2), 7.18–7.41 (5H, m), 7.90 (1H, s, CH), 9.32 (1H, s,
NH), 11.49 (1H, s, OH); 13C NMR (75 MHz, DMSO): d 15.4 (CH3),
28.2 (CH3), 30.2 (CH3), 48.8 (NCH2), 64.6 (CH2), 103.4 (quat.), 110.8
(CH), 116.3 (CH), 121.9 (CH), 126.5 (quat.), 127.3 (2 ꢁ CH), 128.0
(CH), 129.3 (2 ꢁ CH), 138.8 (quat.), 144.5 (CH), 147.9 (quat.), 149.2
(quat.), 149.5 (quat.), 151.1 (quat., C@O), 151.9 (quat.), 153.9 (quat.,
C@O); Anal. Calcd for C23H24N6O4: C, 61.6; H, 5.4; N, 18.7. Found: C,
61.4; H, 5.4; N, 18.6.
5.3.1.2. 3,4,5-Triethoxybenzaldehyde (11a).
To a mixture of
PCC (2.15 g, 9.9 mmol) and powdered molecular sieves (3 Å,
1.30 g) in dry CH2Cl2 (20 mL) was added (3,4,5-triethoxyphe-
nyl)methanol 9 (1.60 g, 6.7 mmol) at 0 °C. The reaction mixture
was stirred at room temperature for 4 h. The solvent was evapo-
rated and diethyl ether (10 mL) was added. The slurry was stirred
and filtered through celite and the solvent was evaporated under
reduced pressure to produce 3,4,5-triethoxybenzaldehyde 10 as
pale brown crystals (1.06 g, 67%); mp 67–69 °C (lit. mp 68 °C);53
Rf = 0.71 (pet.ether/ethyl acetate 1:1); mmax (KBr)/cmꢀ1; 1685
(C@O), 1583 (Ar C@C), 1439 (Ar C@C), 1120 (C–O), 1100 (C–O);
1H NMR (300 MHz, DMSO): d 1.16 (3H, t, J = 6.9 Hz, CH3), 1.27
(6H, t, J = 6.9 Hz, 2 ꢁ CH3), 3.62 (2H, q, J = 6.9 Hz, CH2), 4.02 (4H,
q, J = 6.9 Hz, 2 ꢁ CH2), 7.11 (2H, s, H-2,6), 9.76 (1H, s, CHO); 13C
5.3.4.2. 8-{N0-[1-(3-Ethoxy-4-hydroxyphenyl)meth-(Z)-ylidene]
hydrazino}-1,3-dimethyl-7-(4-nitrobenzyl)xanthine (2b).
Prepared from 3-ethoxy-4-hydroxybenzaldehyde 11b (0.55 g,
3.25 mmol) and 8-hydrazino-1,3-dimethyl-7-(4-nitrobenzyl)-3,7-
1H-dihydropurine-2,6-dione 10b (0.99 g, 2.90 mmol), 8-{N0-[1-(3-
ethoxy-4-hydroxyphenyl)meth-(Z)-ylidene]hydrazino}-1,3-di-
methyl-7-(4-nitrobenzyl)-3,7-1H-dihydropurine-2,6-dione 2b was
obtained as yellow crystals (0.64 g, 49%); Rf = 0.41 (ethyl acetate);
mp 267–268 °C; mmax (KBr)/cmꢀ1 3436 (NH), 3069 (OH), 1685
(C@O), 1619 (C@O), 1596 (Ar C@C), 1525 (NO2), 1508 (Ar C@C),
1332 (NO2), 1268 (C–O); 1H NMR (300 MHz, DMSO): d 1.28 (3H,
t, J = 6.9 Hz, CH3), 3.18 (3H, s, N1-CH3), 3.42 (3H, s, N3-CH3), 3.74
(2H, q, J = 6.9 Hz, CH2), 5.90 (2H, s, CH2), 6.69–6.77 (2H, m), 6.82
(1H, d, J = 1.5 Hz, H-2), 7.45 (2H, d, J = 8.7 Hz, H-20,60), 7.86 (1H, s,
CH), 8.13 (2H, d, J = 8.7 Hz, H-30,50), 8.98 (1H, bs, NH), 11.43 (1H,
s, OH); 13C NMR (75 MHz, DMSO): d 14.9 (CH3), 27.6 (CH3), 29.7
(CH3), 48.4 (NCH2), 64.2 (CH2), 103.1 (quat.), 110.2 (CH), 115.7
(CH), 121.6 (CH), 123.8 (2 ꢁ CH), 125.9 (quat.,), 127.9 (2 ꢁ CH),
144.6 (CH), 146.1 (quat.), 147.1 (quat.), 147.4 (quat.), 148.8 (quat.),
149.1 (quat.), 150.8 (quat., C@O), 151.5 (quat., C-8), 153.5 (quat.,
C@O, C-6); Anal. Calcd for C23H23N7O6: C, 55.9; H, 4.7; N, 19.9.
Found: C, 55.9; H, 4.7; N, 19.9.
NMR (75 MHz, DMSO):
d
15.1 (2 ꢁ CH3), 15.9 (CH3), 64.7
(2 ꢁ CH2), 68.7 (CH2), 108.1 (2 ꢁ CH), 131.9 (quat.), 142.9 (quat.),
153.3 (2 ꢁ quat.), 192.3 (CHO); Anal. Calcd for C13H18O4: C, 65.5;
H, 7.6. Found: C, 65.6; H, 7.6.
5.3.2. General procedure for the synthesis of 7-benzyl-8-chloro-
1,3-dimethylxanthine (9a) and analogues (9b–g)
7-Benzyl-8-chloroxanthines 9a–g were prepared by the method
of Vollmann and Müller.23 8-Chlorotheophylline 7 (10 mmol) was
added to dry dimethylformamide (30 mL) then K2CO3 (20 mmol)
and the appropriate benzyl bromide 8 (20 mmol) were added
and the reaction mixture was stirred overnight at room tempera-
ture. Water (60 mL) was added and the reaction mixture was
cooled in ice for 2 h. The precipitated product was collected by vac-
uum filtration, washed with water (10 mL) and dried in a vacuum
oven to give the 8-chloroxanthine 9a–g.
5.3.3. General procedure for the synthesis of 7-benzyl-8-
hydrazino-1,3-dimethylxanthine (10a) and analogues (10b–g)
To a warm solution of xanthine 10a–g (1.65 mmol) in ethanol
(20 mL), hydrazine hydrate (0.10 mL, 3.30 mmol) was added. The
reaction mixture was then refluxed overnight. After cooling, the
precipitate formed was filtered under suction, washed with cold
5.3.4.3. 8-{N0-[1-(3-Ethoxy-4-hydroxyphenyl)meth-(Z)-ylidene]
hydrazino}-1,3-dimethyl-7-(4-methylbenzyl)xanthine (2c).
Prepared from 3-ethoxy-4-hydroxybenzaldehyde 11b (0.59 g,
3.51 mmol) and 8-hydrazino-1,3-dimethyl-7-(4-methylbenzyl)-3,

5910
D. Petch et al. / Bioorg. Med. Chem. 20 (2012) 5901–5914
7-1H-dihydropurine-2,6-dione 10c (0.99 g, 3.17 mmol), 8-{N0-
[1-(3-ethoxy-4-hydroxyphenyl)meth-(Z)-ylidene]hydrazino}-1,3-di-
methyl-7-(4-methylbenzyl)-3,7-1H-dihydropurine-2,6-dione 2c was
obtained as pale yellow solid (0.59 g, 40%); Rf = 0.56 (pet. ether/ethyl
acetate, 1:9); mp 195–198 °C; mmax (KBr)/cmꢀ1 3654 (NH), 3220
(OH), 1689 (C@O), 1624 (C@O), 1578 (Ar C@C), 1480 (Ar C@C),
1273 (C–O); 1H NMR (300 MHz, DMSO): d 1.20 (3H, t, J = 7.2 Hz,
CH3), 2.14 (3H, s, CH3), 3.08 (3H, s, N1-CH3), 3.30 (3H, s, N3-CH3),
3.71 (2H, q, J = 7.2 Hz, CH2), 5.65 (2H, s, CH2), 6.67 (1H, d,
J = 8.4 Hz, H-5), 6.81 (1H, dd, J = 8.4, 1.8 Hz, H-6), 6.91 (1H, d,
J = 1.8 Hz, H-2), 6.96–7.04 (4H, m), 7.81 (1H, s, CH), 9.20 (1H, s,
NH), 11.36 (1H, s, OH); 13C NMR (75 MHz, DMSO): d 15.1 (CH3),
21.1 (CH3), 27.8 (CH3), 29.8 (CH3), 48.2 (NCH2), 64.1 (CH2), 103.0
(quat.), 110.3 (CH), 116.0 (CH), 121.6 (CH), 126.2 (quat.), 127.0
(2 ꢁ CH), 129.4 (2 ꢁ CH), 135.4 (quat.), 136.8 (quat.), 144.0 (CH),
147.6 (quat.), 148.8 (quat.), 149.1 (quat.), 150.7 (quat., C@O), 151.5
(quat.), 153.5 (quat., C@O); Anal. Calcd for C24H26O4N6: C, 62.3; H,
5.7; N, 18.2. Found: C, 62.0; H, 5.7; N, 18.1.
5.3.4.6.
hydroxyphenyl)meth-(Z)-ylidene]hydrazino}-1,3-dimethylxan-
thine (2f). Prepared from 3-ethoxy-4-hydroxybenzaldehyde
7-(2-Ethoxycarbonylbenzyl)-8-{N0-[1-(3-ethoxy-4-
11b (0.25 g, 1.50 mmol) and 8-hydrazino-1,3-dimethyl-7-(1-eth-
oxycarbonyl benzyl)-3,7-1H-dihydropurine-2,6-dione 10g (0.51 g,
1.37 mmol),
8-{N0-[1-(3-ethoxy-4-hydroxyphenyl)meth-(Z)-yli-
dene] hydrazino}-1,3-dimethyl-7-(1-ethoxycarbonylbenzyl)-3,7-
1H-di hydropurine-2,6-dione 2f was obtained as a fine white
powder (0.55 g, 78%); mp 277–278 °C; mmax (KBr)/cmꢀ1 3679
(NH). 3458 (OH), 1698 (C@O), 1654 (C@O), 1627 (C@O), 1580 (Ar
C@C), 1478 (Ar C@C), 1261 (C–O); 1H NMR (300 MHz, DMSO): d
1.28 (3H, t, J = 6.9 Hz, CH3), 1.34 (3H, t, J = 6.9 Hz, CH3), 3.12 (3H,
s, N1-CH3), 3.44 (3H, s, N3-CH3), 3.71 (2H, q, J = 6.9 Hz, CH2), 4.34
(2H, q, J = 6.9 Hz, CH2), 6.10 (2H, s, CH2), 6.67–6.77 (4H, m), 7.40
(1H, m, H-40), 7.53 (1H, m, H-50), 7.83 (1H, s, CH), 8.01 (1H, dd,
J = 7.8, 1.5 Hz, H-30), 9.29 (1H, bs, NH), 11.50 (1H, s, OH); 13C
NMR (75 MHz, DMSO: d 14.6 (CH3), 15.0 (CH3), 27.7 (CH3), 29.9
(CH3), 55.2 (NCH2), 61.3 (CH2), 64.2 (CH2), 103.1 (quat.), 108.9
(CH), 115.9 (CH), 121.4 (CH), 126.0 (CH), 127.6 (CH), 128.7 (quat.),
131.0 (CH), 133.5 (CH), 139.5 (quat.), 144.2 (CH), 147.5 (quat.),
148.7 (quat.), 148.8 (quat.), 149.0 (quat.), 151.0 (quat., C@O),
151.5 (quat.), 152.5 (quat., C@O), 166.7 (quat., C@O); Anal. Calcd
for C26H28O6N6: C, 60.0; H, 5.4; N, 16.1. Found: C, 60.0; H, 5.5; N,
16.3.
5.3.4.4. 7-(4-Chlorobenzyl)-8-{N0-[1-(3-ethoxy-4-hydroxyphenyl)
meth-(Z)-ylidene]hydrazino}-1,3-dimethylxanthine
(2d).
Prepared from 3-ethoxy-4-hydroxybenzaldehyde 11b (0.55 g,
3.26 mmol) and 8-hydrazino-1,3-dimethyl-7-(4-chlorobenzyl)-3,7-
1H-dihydropurine-2,6-dione 10d (0.66 g, 1.97 mmol), 8-{N0-
[1-(3-ethoxy-4-hydroxyphenyl)meth-(Z)-ylidene]hydrazino}-1,3-
dimethyl-7-(4-chlorobenzyl)-3,7-1H-dihydropurine-2,6-dione
2d
5.3.4.7. 7-Benzyl-8-{N0-[1-(3,4,5-triethoxyphenyl)meth-(Z)-yli-
was obtained as a fine cream solid (0.90, 95%); Rf = 0.54 (ethyl ace-
tate); mp 266–267 °C; mmax (KBr)/cmꢀ1 3492 (NH), 3231 (OH),
1690 (C@O), 1655 (C@O), 1621 (Ar C@C), 1513 (Ar C@C), 1276 (C–
O); 1H NMR (300 MHz, DMSO): d 1.31 (3H, t, J = 6.9 Hz, CH3), 3.18
(3H, s, N1-CH3), 3.41 (3H, s, N3-CH3), 3.77 (2H, q, J = 6.9 Hz, CH2),
5.77 (2H, s, CH2), 6.74 (1H, d, J = 8.1 Hz, H-5), 6.84 (1H, dd, J = 8.1,
1.8 Hz, H-6), 6.92 (1H, d, J = 1.8 Hz, H-2), 7.22 (2H, d, J = 8.7 Hz, H-
20,60), 7.31 (2H, d, J = 8.7 Hz, H-30,50), 7.90 (1H, s, CH), 9.17 (1H, bs,
NH), 11.43 (1H, s, OH); 13C NMR (75 MHz, DMSO): d 15.0 (CH3),
27.7 (CH3), 29.7 (CH3), 47.9 (NCH2), 64.1 (CH2), 103.0 (quat.), 110.1
(CH), 115.8 (CH), 121.6 (CH), 126.1 (quat.), 128.7 (2 ꢁ CH), 128.8
(2 ꢁ CH), 132.4 (quat.), 137.3 (quat.), 144.3 (CH), 147.5 (quat.),
148.8 (quat.), 149.1 (quat.), 150.7 (quat., C@O), 151.4 (quat.), 153.5
(quat., C@O); Anal. Calcd for C23H23ClN6O4: C, 57.2; H, 4.8; N, 17.4.
Found: C, 57.2; H, 4.8; N, 17.3.
dene]hydrazino}-1,3-dimethylxanthine (2h).
3,4,5-triethoxybenzaldehyde 11a (0.71 g, 2.97 mmol) and 7-ben-
zyl-8-hydrazino-1,3-dimethyl-3,7-1H-dihydro purine-2,6-dione
Prepared from
10a (0.81 g, 2.69 mmol), 7-benzyl-1,3-dimethyl -8-{N‘-[1-(3,4,5-
triethoxyphenyl)meth-(Z)-ylidene] hydrazino}-3, 7-1H-dihydropu-
rine-2,6-dione 2h was obtained as fine dark yellow crystals (0.63 g,
45%); Rf = 0.50 (ethyl acetate); mp 199–200 °C; mmax (KBr)/cmꢀ1
3223 (NH), 1690 (C@O), 1639 (C@O), 1618 (Ar C@C), 1498 (Ar
C@C), 1121 (C–O); 1H NMR (300 MHz, DMSO): d 1.21 (3H, t,
J = 6.9 Hz, CH3), 1.29 (6H, t, J = 6.9 Hz, 2 ꢁ CH3), 3.16 (3H, s, N1-
CH3), 3.40 (3H, s, N3-CH3), 3.81 (4H, q, J = 6.9 Hz, 2 ꢁ CH2), 3.94
(2H, q, J = 6.9 Hz, CH2), 5.81 (2H, s, CH2), 6.70 (2H, s, H-20,60),
7.18–7.34 (5H, m), 7.90 (1H, s, CH), 11.72 (1H, bs, NH); 13C NMR
(75 MHz, DMSO): d 15.1 (2 ꢁ CH3), 15.9 (CH3), 27.8 (CH3), 29.8
(CH3), 48.6 (NCH2), 64.4 (2 ꢁ CH2), 68.4 (CH2), 103.1 (quat.),
105.1 (2 ꢁ CH), 126.8 (2 ꢁ CH), 127.6 (CH), 128.9 (2 ꢁ CH), 129.9
(quat.), 138.4 (quat.), 138.7 (quat.), 143.4 (CH), 148.7 (quat.),
150.3 (quat.), 151.4 (quat., C@O), 153.1 (2 ꢁ quat.), 153.5 (quat.,
C@O); Anal. Calcd for C27H32N6O5: C, 62.3; H, 6.2; N, 16.1. Found:
C, 62.4; H, 6.2; N, 16.2.
5.3.4.5. 8-{N0-[1-(3-Ethoxy-4-hydroxyphenyl)meth-(Z)-ylidene]
hydrazino}-1,3-dimethyl-7-(2-nitrobenzyl)xanthine (2e).
Prepared from 3-ethoxy-4-hydroxybenzaldehyde 11b (0.28 g,
1.67 mmol) and 8-hydrazino-1,3-dimethyl-7-(2-nitrobenzyl)-3,
7-1H-dihydropurine-2,6-dione 10e (0.52 g, 1.51 mmol), 8-{N0-
[1-(3-ethoxy-4-hydroxyphenyl)meth-(Z)-ylidene]hydrazino}-1,3-
5.3.4.8.
8-{N0-[1-(3,4,5-Triethoxyphenyl)meth-(Z)-ylidene]
hydrazino}-1,3-dimethyl-7-(4-nitrobenzyl)xanthine (2i).
dimethyl-7-(2-nitrobenzyl)-3,7-1H-dihydropurine-2,6-dione
2e
Prepared from 3,4,5-triethoxybenzaldehyde 11a (0.78 g,
3.29 mmol) and 8-hydrazino-1,3-dimethyl-7-(4-nitrobenzyl)-
3,7-1H-dihydropurine-2,6-dione 10b (1.00 g, 2.90 mmol),
1,3-dimethyl-7-(4-nitrobenzyl)-8-{N0-[1-(3,4,5-triethoxyphe-
nyl)meth-(Z)-ylidene]hydrazino}-3,7-1H-dihydropurine-2,6-dione
2i was obtained as a pale yellow solid (0.12 g, 7%); Rf = 0.41 (ethyl
was obtained as dark orange crystals (0.29 g, 39%); Rf = 0.57 (ethyl
acetate); mp 265–267 °C; mmax (KBr)/cmꢀ1 3523 (NH), 3077 (OH),
1703 (C@O), 1627 (C@O), 1580 (Ar C@C), 1510 (NO2), 1478 (Ar
C@C), 1330 (NO2), 1283 (C–O); 1H NMR (300 MHz, DMSO): d 1.27
(3H, t, J = 6.9 Hz, CH3), 3.12 (3H, s, N1-CH3), 3.44 (3H, s, N3-CH3),
3.71 (2H, q, J = 6.9 Hz, CH2), 6.06 (2H, s, CH2), 6.70 (2H, s, H-5,6),
6.81 (1H, s, H-2), 7.00 (1H, dd, J = 8.4, 1.2 Hz, H-6), 7.55 (1H, m),
7.71 (1H, m), 7.85 (1H, s, CH), 8.20 (1H, dd, J = 8.1, 1.2 Hz, H-3),
9.80 (1H, bs, NH), 11.46 (1H, s, OH); 13C NMR (75 MHz, DMSO): d
14.5 (CH3), 27.2 (CH3), 29.4 (CH3), 47.1 (NCH2), 63.6 (CH2), 102.5
(quat.), 106.7 (CH), 109.8 (CH), 115.4 (CH), 125.4 (CH), 124.9
(quat.), 128.4 (CH), 133.8 (quat.), 127.4 (CH), 127.6 (CH), 144.5
(CH), 146.6 (quat.), 147.0 (quat.), 148.7 (quat.), 149.4 (quat.),
151.6 (quat., C@O), 152.3 (quat.), 152.9 (quat., C@O); Anal. Calcd
for C23H23N7O6: C, 56.0; H, 4.7; N, 19.9. Found: C, 55.7; H, 4.7; N,
19.8.
acetate); mp 187–190 °C; m
max (KBr)/cmꢀ1 3334 (NH), 1681 (C@O),
1619 (C@O), 1575 (Ar C@C), 1530 (NO2), 1499 (Ar C@C), 1338
(NO2), 1126 (C–O); 1H NMR (300 MHz, DMSO): d 1.13 (3H, t,
J = 6.9 Hz, CH3), 1.17 (6H, t, J = 6.9 Hz, 2 ꢁ CH3), 3.05 (3H, s, N1-
CH3), 3.32 (3H, s, N3-CH3), 3.71 (4H, q, J = 6.9 Hz, 2 ꢁ CH2), 3.82
(2H, q, J = 6.9 Hz, 2 ꢁ CH2), 5.81 (2H, s, CH2), 6.54 (2H, s, H-20,60),
7.38 (2H, d, J = 8.7 Hz, H-2,6), 7.92 (1H, s, CH), 8.12 (2H, d,
J = 8.7 Hz, H-3,5), 11.90 (1H, bs, NH); 13C NMR (75 MHz, DMSO):
d 15.1 (2 ꢁ CH3), 15.9 (CH3), 27.7 (CH3), 29.9 (CH3), 48.6 (NCH2),
64.2 (2 ꢁ CH2), 68.5 (CH2), 105.2 (CH), 107.1 (quat.), 124.1

D. Petch et al. / Bioorg. Med. Chem. 20 (2012) 5901–5914
5911
(2 ꢁ CH), 127.9 (2 ꢁ CH), 129.8 (quat.), 139.4 (quat.), 144.0 (CH),
147.1 (quat.), 148.7 (quat.), 148.8 (quat.), 150.6 (quat.), 151.5
(quat., C@O), 153.1 (2 ꢁ quat.), 153.5 (quat., C@O); Anal. Calcd
for C27H31N7O7: C, 57.3; H, 5.5; N, 17.3. Found: C, 57.0; H, 5.2; N,
17.0.
3.90 (2H, q, J = 6.9 Hz, CH2), 4.33 (2H, q, J = 7.2 Hz, CH2), 6.1 (2H,
s, CH2), 6.56 (2H, s, H-20,60), 6.79 (1H, d, J = 7.5 Hz, H-6), 7.39 (1H,
m, H-4), 7.54 (1H, m, H-5), 7.88 (1H, s, CH), 8.00 (1H, dd, J = 7.5,
1.2 Hz, H-3), 11.74 (1H, bs, NH); 13C NMR (75 MHz, DMSO): d
14.6 (CH3), 15.1 (2 ꢁ CH3), 15.9 (CH3), 27.7 (CH3), 29.9 (CH3),
48.4 (NCH2), 61.3 (CH2), 64.4 (2 ꢁ CH2), 68.4 (CH2), 103.2 (quat.),
105.1 (2 ꢁ CH), 126.1 (CH), 127.4 (CH), 127.7 (quat.), 129.8 (quat.),
130.9 (CH), 133.5 (CH), 138.7 (quat.), 140.4 (quat.), 143.7 (CH),
148.7 (quat.), 150.8 (quat.), 151.5 (quat., C@O), 153.1 (2 ꢁ quat.),
153.4 (quat., C@O), 166.7 (quat., C@O); Anal. Calcd for
5.3.4.9.
8-{N0-[1-(3,4,5-Triethoxyphenyl)meth-(Z)-ylidene]
hydrazino}-1,3-dimethyl-7-(4-methylbenzyl)xanthine (2j).
Prepared from 3,4,5-triethoxybenzaldehyde 11a (0.84 g,
3.52 mmol) and 8-hydrazino-1,3-dimethyl-7-(4-methylbenzyl)-
3,7-1H-dihydropurine-2,6-dione 10c (0.99 g, 3.19 mmol), 1,3-
dimethyl-7-(4-methylbenzyl)-8-{N‘-[1-(3,4,5-triethoxyphenyl)
meth-(Z)-ylidene]hydrazino}-3,7-1H-dihydropurine-2,6-dione 2j
was obtained as a cream solid (1.16 g, 68%); Rf = 0.52 (ethyl ace-
tate); mp 216–219 °C; mmax (KBr)/cmꢀ1 3244 (NH), 1692 (C@O),
1653 (C@O), 1622 (Ar C@C), 1501 (Ar C@C), 1126 (C–O); 1H NMR
(300 MHz, DMSO): d 1.21 (3H, t, J = 6.9 Hz, CH3), 1.29 (6H, t,
J = 6.9 Hz, 2 ꢁ CH3), 2.24 (3H, s, CH3), 3.16 (3H, s, N1-CH3), 3.40
(3H, s, N3-CH3), 3.84 (4H, q, J = 6.9 Hz, 2 ꢁ CH2), 3.94 (2H, q,
J = 6.9 Hz, 2 ꢁ CH2), 5.75 (2H, s, CH2), 6.73 (2H, s, H-20,60), 7.06-
7.13 (4H, m), 7.91 (1H, s, CH), 11.69 (1H, bs, NH); 13C NMR
(75 MHz, DMSO): d 15.1 (2 ꢁ CH3), 15.9 (CH3), 21.1 (CH3), 27.8
(CH3), 29.8 (CH3), 48.4 (NCH2), 64.4 (2 ꢁ CH2), 68.4 (CH2), 103.1
(quat.), 105.1 (2 ꢁ CH), 126.9 (2 ꢁ CH), 129.5 (2 ꢁ CH), 129.9
(quat.), 135.3 (quat.), 136.7 (quat.), 138.7 (quat.), 143.4 (CH),
148.7 (quat.), 150.3 (quat.), 151.5 (quat., C@O), 153.1 (2 ꢁ quat.),
153.5 (quat., C@O); Anal. Calcd for C28H34O5N6: C, 62.9; H, 6.4; N,
15.7. Found: C, 63.2; H, 6.5; N, 15.6.
C30H36O7N6: C, 60.8; H, 6.1; N, 14.2. Found: C, 60.6; H, 6.0; N, 13.9.
5.3.5. General procedure for the synthesis of 7-(1-
carboxybenzyl)-8-{N0-[1-(3-ethoxy-4-hydroxyphenyl)meth-(Z)-
ylidene]hydrazino}-1,3-dimethylxanthine (2g) and 7-(1-
carboxybenzyl)-8-{N0-[1-(3,4,5-triethoxyphenyl)meth-(Z)-
ylidene]hydrazino}-1,3-dimethylxanthine (2m)
To a suspension of 2f or 2l, (0.64 mmol) in methanol (20 mL), a
solution of lithium hydroxide (0.03 g, 1.34 mmol) in distilled water
(10 mL) was added and the mixture refluxed for 3 h. The solvent
was evaporated under reduced pressure. The resulting solid resi-
due was dissolved in distilled water (20 mL), then acidified with
1% aq. HCl. The precipitated solid was filtered, dried in a vacuum
oven, then recrystallized from methanol and acetonitrile to give
2g or 2m, respectively.
5.3.5.1.
phenyl)meth-(Z)-ylidene]hydrazino}-1,3-dimethylxanthine
(2g).
Prepared from 8-{N0-[1-(3-ethoxy-4-hydroxyphenyl)-
7-(2-Carboxybenzyl)-8-{N0-[1-(3-ethoxy-4-hydroxy-
5.3.4.10.
8-{N0-[1-(3,4,5-Triethoxyphenyl)meth-(Z)-ylidene]
meth-(Z)-ylidene]hydrazino}-1,3-dimethyl-7-(1-ethoxycarbonylb-
enzyl)-3,7-1H-dihydropurine-2,6-dione 2f (0.33 g, 0.64 mmol),
8-{N0-[1-(3-ethoxy-4-hydroxyphenyl)meth-(Z)-ylidene]hydrazino}-
1,3-dimethyl-7-(1-carboxybenzyl)-3,7-1H-dihydropurine-2,6-dione
2g was obtained as a fine pink solid (0.03 g, 10%); mp 285–286 °C;
mmax (KBr)/cmꢀ1 3234 (NH), 3105 (OH), 17.17 (C@O), 1679 (C@O),
1619 (C@O), 1579 (Ar C@C), 1477 (Ar C@C), 1200 (C–O); 1H NMR
(300 MHz, DMSO): d 1.20 (3H, t, J = 6.9 Hz, CH3), 1.98 (1H, s, OH),
3.03 (3H, s, N1-CH3), 3.37 (3H, s, N3-CH3), 3.59 (2H, q, J = 6.9 Hz,
CH2), 6.02 (2H, s, CH2), 6.58–6.66 (4H, m), 7.27 (1H, m, H-40),
7.42 (1H, m, H-50), 7.74 (1H, s, CH), 7.91 (1H, dd, J = 7.5, 1.5 Hz,
H-30), 9.18 (1H, bs, NH), 11.46 (1H, s, OH); 13C NMR (75 MHz,
DMSO): d 15.0 (CH3), 27.7 (CH3), 29.9 (CH3), 48.7 (NCH2), 64.1
(CH2), 103.1 (quat.), 110.2 (CH), 115.9 (CH), 121.5 (CH), 126.0
(CH), 127.2 (CH), 124.9 (quat.), 131.2 (CH), 133.1 (CH), 140.6
(quat.), 144.1 (CH), 147.4 (quat.), 147.5 (quat.), 148.8 (quat.),
149.0 (quat.), 151.0 (quat., C@O), 151.5 (quat.), 153.3 (quat.,
C@O,), 168.6 (quat., C@O); Anal. Calcd for C24H24N6O6: C, 58.5; H,
4.9; N, 17.1. Found: C, 58.4; H, 4.9; N, 16.9.
hydrazino}-1,3-dimethyl-7-(2-methoxybenzyl)xanthine (2k).
Prepared from 3,4,5-triethoxybenzaldehyde 11a (0.81 g,
3.40 mmol) and 8-hydrazino-1,3-dimethyl-7-(2-methoxybenzyl)-
3,7-1H-dihydropurine-2,6-dione 10f (1.06 g, 3.20 mmol), 1,3-
dimethyl-7-(2-methoxybenzyl)-8-{N‘-[1-(3,4,5-triethoxyphenyl)-
meth(Z)-ylidene]hydrazino}-3,7-1H-dihydropurine-2,6-dione 2k
was obtained as fine yellow crystals (0.87 g, 83%); Rf = 0.50 (ethyl
acetate); mp 224–228 °C; mmax (KBr)/cmꢀ1 3221 (NH), 1694
(C@O), 1639 (C@O), 1622 (Ar C@C), 1496 (Ar C@C), 1120 (C–O);
1H NMR (300 MHz, DMSO): d 1.20 (3H, t, J = 6.9 Hz, CH3), 1.30
(6H, t, J = 6.9 Hz, 2 ꢁ CH3), 3.13 (3H, s, N1-CH3), 3.43 (3H, s, N3-
CH3), 3.82 (3H, s, OCH3), 3.84–4.10 (6H, m, 3 ꢁ CH2), 5.71 (2H, s,
CH2), 6.57 (1H, d, J = 7.8 Hz, H-3), 6.64 (2H, s, H-20,60), 6.84 (1H,
m, H-5), 7.02 (1H, d, J = 7.8 Hz, H-6), 7.22 (1H, m, H-4), 7.87 (1H,
s, CH), 11.59 (1H, bs, NH); 13C NMR (75 MHz, DMSO): d 15.2
(2 ꢁ CH3), 15.9 (CH3), 27.7 (CH3), 29.9 (CH3), 44.8 (NCH2), 55.9
(OCH3), 64.5 (2 ꢁ CH2), 68.5 (CH2), 103.2 (quat.), 105.3 (2 ꢁ CH),
110.9 (CH), 120.9 (CH), 125.7 (CH), 126.3 (quat.), 128.4 (CH),
129.9 (quat.), 138.8 (quat.), 143.5 (CH), 148.7 (quat.), 150.9 (quat.),
151.5 (quat., C@O), 153.1 (2 ꢁ quat.), 153.4 (quat., C@O), 156.3
(quat.); Anal. Calcd for C28H34O6N6: C, 61.1; H, 6.2; N, 15.3. Found:
C, 60.8; H, 6.2; N, 15.0.
5.3.5.2.
7-(2-Carboxybenzyl)-8-{N0-[1-(3,4,5-triethoxyphenyl)
meth-(Z)-ylidene]hydrazino}-1,3-dimethylxanthine (2m).
Prepared from 1,3-dimethyl-7-(1-ethoxycarbonylbenzyl)-8-{N‘-
[1-(3,4,5-triethoxy phenyl)meth-(Z)-ylidene]hydrazino}-3,7-1H-
dihydropurine-2,6-dione 2l (0.34 g, 0.57 mmol), 1,3-dimethyl-7-
(1-carboxybenzyl)-8-{N‘-[1-(3,4,5-triethoxyphenyl)meth-(Z)-yli-
5.3.4.11. 7-(2-Ethoxycarbonylbenzyl)-8-{N0-[1-(3,4,5-triethoxy-
phenyl)meth-(Z)-ylidene]hydrazino}-1,3-dimethylxanthine
(2l).
Prepared from 3,4,5-triethoxybenzaldehyde 11a (0.36 g,
dene]hydrazino}-3,7-1H-dihydropurine-2,6-dione
2m
was
1.49 mmol) and 8-hydrazino-1,3-dimethyl-7-(1-ethoxycarbonyl
benzyl)-3,7-1H-dihydropurine-2,6-dione 10g (0.50 g, 1.35 mmol),
1,3-dimethyl-7-(1-ethoxycarbonylbenzyl)-8-{N‘-[1-(3,4,5-triethoxy-
phenyl)meth-(Z)-ylidene]hydrazino}-3,7-1H-dihydropurine-2, 6-
obtained as a yellow solid (0.12 g, 36%); mp 259–260 °C; mmax
(KBr)/cmꢀ1 3223 (NH), 3119 (OH), 1714 (C@O), 1679 (C@O),
1615 (C@O), 1574 (Ar C@C), 1472 (Ar C@C), 1121 (C–O); 1H NMR
(300 MHz, DMSO): d 1.19 (3H, t, J = 6.9 Hz, CH3), 1.28 (6H, t,
J = 6.9 Hz, 2 ꢁ CH3), 3.11 (3H, s, N1-CH3), 3.44 (3H, s, N3-CH3),
3.78 (4H, q, J = 6.9 Hz, 2 ꢁ CH2), 3.90 (2H, q, J = 6.9 Hz, CH2), 6.11
(2H, s, CH2), 6.55 (2H, s, H-20,60), 6.77 (1H, d, J = 7.8 Hz, H-6), 7.37
(1H, m, H-4), 7.50 (1H, m, H-5), 7.88 (1H, s, CH), 8.00 (1H, dd,
J = 7.8, 1.5 Hz, H-3), 11.81 (1H, bs, NH); 13C NMR (75 MHz, DMSO):
d 15.1 (2 ꢁ CH3), 15.9 (CH3), 27.7 (CH3), 29.9 (CH3), 48.6 (NCH2),
dione 2l was obtained as
a white solid (0.56 g, 70%); mp
227–228 °C; mmax (KBr)/cmꢀ1 3599 (NH), 1696 (C@O), 1648
(C@O), 1622 (C@O), 1577 (Ar C@C), 1467 (Ar C@C), 1122 (C–O);
1H NMR (300 MHz, DMSO): d 1.19 (3H, t, J = 6.9 Hz, CH3), 1.28
(6H, t, J = 6.9 Hz, 2 ꢁ CH3), 1.33 (2H, t, J = 6.9 Hz, CH3), 3.11 (3H,
s, N1-CH3), 3.44 (3H, s, N3-CH3), 3.77 (4H, q, J = 6.9 Hz, 2 ꢁ CH2),

5912
D. Petch et al. / Bioorg. Med. Chem. 20 (2012) 5901–5914
64.4 (2 ꢁ CH2), 68.4 (CH2), 103.1 (quat.), 105.0 (2 ꢁ CH), 125.9
(CH), 127.2 (CH), 129.8 (quat.), 131.2 (CH), 131.6 (quat.), 133.1
(CH), 138.7 (quat.), 140.4 (quat.), 143.7 (CH), 148.7 (quat.), 150.8
(quat.), 151.5 (quat., C@O), 153.1 (2 ꢁ quat.), 153.3 (quat., C@O),
168.6 (quat., C@O); Anal. Calcd for C28H32O7N6: C, 59.6; H, 5.7; N,
14.9. Found: C, 59.5; H, 5.7; N, 14.8.
the plates were incubated for 3 h at 37 °C. Formazan formation
was determined using a microplate reader at 492 nm.
5.4.1.3. Cyto Tox 96 cytotoxicity assays.
The Cyto Tox 96 as-
say was performed following the manufacturer’s (Promega)
protocol.
5.4.2. Effect of inhibitors on protein levels in CCD-110 cells
Cells were grown on 6-well plates and were exposed to vehicle,
EGF or novel compounds (added from stock solutions dissolved in
medium/DMSO 1:1 or DMSO, for 30 min) for the required period of
time, at 37 °C,and then placed on ice to stop the reaction. Cells
were then washed with PBS (2 ꢁ 1 mL). For cross-linking assays,
5.4. Biological testing
5.4.1. Cell culture
Cells were seeded in 96 well microtiter plates at an optimal
density so that the untreated cells were in the exponential growth
phase at the time of harvest (10,000 cells/well which was the opti-
mal cell density from preliminary studies). All the plates were
incubated overnight to allow cell attachment. The 96 well plates
were set up with six in-plate replicates of eight concentrations of
the drug. Control wells were treated with fresh media containing
no drug. The same solvent (DMSO) used to dissolve the test com-
pound was added to the control wells so that the concentration
of the solvent present in all the wells was <1%. Wells were also
set up with just the medium to serve as the negative control for
the determination of any background absorbance that may be
present. The absorbance (or fluorescence) levels from drug treated
cells were corrected against untreated control absorbance (or fluo-
rescence) values. All of the compounds tested were soluble at all
concentrations and did not interfere with the colorimetric assay
chemistry. Assays were based on comparative growth with the
control, set as 100% growth. The IC50 was defined as 50% reduction
of absorbance.
Normal human epidermal keratinocytes (Promocell; NHEK
pooled adult) were grown in keratinocyte growth medium (Promo-
cell), with the growth supplement (supplied with the medium)
consisting of insulin, hydrocortisone, transferrin, epinephrine, bo-
vine pituitary extract, and epidermal growth factor. Trypsinization
was performed using the detach kit C-14200, Promocell) recom-
mended by the supplier. The HPV-16 transformed human keratino-
cyte cell line, CCD-1106, was cultured in serum free medium
(GIBCO) with the growth supplement (supplied with the medium)
consisting of L-glutamine, bovine pituitary extract, and epidermal
growth factor. HaCaT cells were grown in Dulbecco’s modified Ea-
gle’s medium (GIBCO) supplemented with 10% fetal bovine serum
BS3 (500
l
l) was then added for 30 min at room temperature, the
L 1 M Tris 20 mM (pH 7.5) for
15 min at room temperature, then washed with PBS (2 ꢁ 1 mL) be-
fore 250
l of 4 ꢁ sample buffer [63 mM Tris–HCl, pH 6.8, 2 mM
reaction was quenched with 10
l
l
Na4P2O7, 5 mM EDTA, 10% (v/v) glycerol, 2% (w/v) SDS, 50 mM
DTT, 0.007% (w/v) bromophenol blue] was added. The cells were
then scraped and dispensed into Eppendorf tubes using a gauge
needle, a hole was impregnated in each of the sample lids and
the tubes were boiled for 5 min to denature the protein. The sam-
ples were then frozen and stored at ꢀ20 °C.
Resolving gels were prepared containing an appropriate amount
[5% (w/v), 7.5% (w/v) or 10% (w/v)] acrylamide: N0-methylenebis-
acrylamide (30:0.8), 0.375 M tris pH 8.8, 0.1% (w/v) SDS and
0.05% (w/v) ammonium persulfate (APS). Polymerization was initi-
ated by the addition of 0.05% (v/v) N,N,N0,N0-tetramethylethylene-
diamine (TEMED). The solution was poured between two glass
plates, and overlayed with 200 lL 0.1% (w/v) SDS. After polymeri-
zation, a stacking gel containing 10% (v/v) acrylamide: N0-methyl-
enebisacrylamide (30:0.8) in 125 mM tris, pH 6.8, 0.1% (w/v) SDS,
0.05% (w/v) ammonium persulfate and 0.05% (v/v) TEMED was
poured directly on top of the resolving gel, and a comb inserted.
After polymerization was complete the comb was removed and
the polyacrylamide gels were assembled in a Bio-Rad Mini-PRO-
TEAN II™ electrophoresis tank, with both reservoirs filled with
electrophoresis buffer (25 mM tris, 192 mM glycine, 0.1% (w/v)
SDS). Aliquots of samples (10–30 lL) from the cell lysates were
loaded into the wells using a microsyringe. A prestained SDS–PAGE
molecular weight marker of known molecular weights was run to
identify the polypeptide of interest. Samples were electrophoresed
at a constant voltage (90–150 V), for 90 min or until the bromo-
phenol blue dye reached the bottom of the gel.
and 2 mM L-glutamine.
The human monocytic cell lines, THP-1 and U937, were grown
in RPMI medium (GIBCO) supplemented with 10% fetal bovine ser-
The proteins separated by SDS–PAGE were transferred to nitro-
cellulose membranes by electrophoretic blotting. The gel was
pressed firmly against a nitrocellulose sheet and assembled in a
transfer cassette sandwiched between Whatmann 3MM paper
and two sponge pads. The cassette was immersed in blotting buffer
(25 mM Tris, 195 mM glycine, 20% (v/v) methanol) in a Bio-Rad
Mini Trans-Blot™ tank and a constant current of 0.3–0.4 Å was ap-
plied for 2–5 h whilst the tank was cooled by inclusion in an ice
reservoir.
Following transfer of the proteins to the nitrocellulose mem-
brane, the membrane was removed and any remaining protein
blocked by incubation in a solution of 3% (w/v) BSA in NaTT
(150 mM NaCl, 20 mM Tris, pH 7.4, 0.2% (v/v) Tween-20) for 2 h.
Blocking buffer was then replaced with a solution of 0.2% (w/v)
um and 2 mM L-glutamine.
Streptomycin (100
added to the media.
lg/mL) and penicillin (100 U/mL) were also
5.4.1.1. MTT assay.
The MTT assay was performed according
to the method developed by Mosmann.54 After the 72 h incubation
period, cells in 96 well plates were exposed to 200
(0.5 mg/ml in PBS) and incubated for 2 h at 37 °C/5% CO2. The cells
were washed with PBS after the removal of the dye and 200 l of
90% isopropanol/10% DMSO was added to the plates. The plates
were incubated in the dark for 10 min and the absorbance at
590 nm was recorded immediately using a microplate reader (Te-
can, Reading, UK).
ll of MTT
l
BSA in NaTT and an appropriate volume (1–5 lL) of the antibody
specific for the protein of interest was added. This mixture was agi-
5.4.1.2. Cell Titer 96 Aqueous One solution assay.
Cells were
seeded in round bottomed 96 well microtiter plates at 10,000 cells/
well and exposed to the test compounds for 72 h. At the end of the
incubation period, the Cell titer 96 Aqueous One solution cell pro-
liferation assay was used to determine the growth inhibitory ef-
fects of the compounds, with the assay performed according to
the manufacturer’s instructions. After the addition of the reagent,
tated overnight and then washed every 15 min for 1.5–2 h in NaTT.
A secondary antibody (1–2 lL) directed against the primary anti-
body, was then added in 0.2% (w/v) BSA solution and incubated
for a further 2 h prior to further washing every 15 min over a
1.5 h period. The membranes were then exposed to an enhanced
chemiluminescence (ECL) reagent for 1.5 min with agitation before

D. Petch et al. / Bioorg. Med. Chem. 20 (2012) 5901–5914
5913
being placed in an exposure cassette and covered with Saran-
5.4.5. Anti-oxidant assay
wrapTM. Blots were then exposed to Kodak X-OMAT LS film for
2–5 min under darkroom conditions and were then developed
using a Kodak M35-M X-OMAT processor.
Exact details of the anti-oxidant assay can be found in a sepa-
rate study.22
Densitometry measurements were recorded using Scion image
for Windows, 2000–2001, Scion corporation, Alpha 4.0.3.2.
Acknowledgements
We thank Stiefel International R&D (now Stiefel, a GSK com-
pany) for sponsoring this research.
5.4.2.1. Effect of inhibitors on EGFR dimerization.
Cells
were exposed to varying concentrations of the test compounds
for 30 min, then stimulated with EGF at 50 ng/mL for 10 min at
37 °C, and cross-linked with BS3 at 2 mM for 30 min at room tem-
perature. After solubilization and SDS–page gel electrophoresis, the
samples were transferred onto nitrocellulose paper and then
immunoblotted with anti-EGFR antibodies. In the absence of
cross-linker, the dimers separate into monomers upon SDS–page
gel electrophoresis.55 The protein species at 170 kDa is proposed
to be the EGFR monomer and at the 340 kDa, the covalently linked
dimer. It is assumed during the procedure that both the monomer
and dimer are equally transferred onto the nitrocellulose paper and
that the both species exhibit equal recognition by the anti-EGFR
antibodies. Immunoblots were quantified by densitometry as opti-
cal density (% control).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.07.048.
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