Synthesis and in vitro evaluation of pteridine analogues as monoamine oxidase B and nitric oxide synthase inhibitors

Article abstract of DOI:10.1016/j.bmc.2009.09.019

Monoamine oxidase B (MAO-B) and nitric oxide synthase (NOS) have both been implicated in the pathology of neurodegenerative diseases. In an attempt to design dual-target-directed drugs that inhibit both these enzymes, a series of pteridine-2,4-dione analo

Full text of DOI:10.1016/j.bmc.2009.09.019

Bioorganic & Medicinal Chemistry 17 (2009) 7523–7530
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and in vitro evaluation of pteridine analogues as monoamine oxidase
B and nitric oxide synthase inhibitors
*
Louis H. A. Prins, Jacobus P. Petzer, Sarel F. Malan
Pharmaceutical Chemistry, School of Pharmacy, North-West University, Private Bag X6001, Potchefstroom 2520, South Africa
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 15 July 2009
Revised 9 September 2009
Accepted 10 September 2009
Available online 15 September 2009
Monoamine oxidase B (MAO-B) and nitric oxide synthase (NOS) have both been implicated in the pathol-
ogy of neurodegenerative diseases. In an attempt to design dual-target-directed drugs that inhibit both
these enzymes, a series of pteridine-2,4-dione analogues were synthesised. The compounds were found
to be relatively weak NOS inhibitors but showed promising MAO-B activity with 6-amino-5-[(E)-3-(3-
chloro-phenyl)-prop-2-en-(E)-ylideneamino]-1,3-dimethyl-1H-pyrimidine-2,4-dione and 6-[(E)-2-(3-
chloro-phenyl)-vinyl]-1,3-dimethyl-1H-pteridine-2,4-dione inhibiting MAO-B with IC50 values of 0.602
and 0.314 lM, respectively. The pteridine-2,4-dione analogues thus show promise as scaffolds for the
development of potent reversible MAO-B inhibitors and as observed in earlier studies, the most potent
inhibitors were obtained with 3-chlorostyryl substitution.
Keywords:
Monoamine oxidase B
Nitric oxide synthase
Pteridine-2,4-dione
Dual-target-directed ligands
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
of a disease are combined, has been used as a means of multiple
targeting in the clinical setting. However, this strategy of combin-
Neurodegenerative disorders such as Parkinson’s and Alzhei-
mer’s diseases, are affecting the lives of a growing number of peo-
ple worldwide. These debilitating conditions lead to a general
decrease in quality of life and the need for therapeutics to effec-
tively prevent and treat these conditions are of increasing
importance.1
It has become evident that neurodegenerative diseases are not
the result of a single cause but that the processes involved are of
a multifactorial nature. It is hypothesised that genetic, environ-
mental and endogenous factors may be involved in neurodegener-
ative diseases such as Alzheimer’s disease, Parkinson’s disease,
Huntington’s disease and amyotrophic lateral sclerosis. A series
of general pathways has been identified that may be involved in
different pathogenic cascades and includes protein misfolding
and aggregation, oxidative stress and free radical formation, metal
dyshomeostasis, mitochondrial dysfunction and phosphorylation
impairment. All of the above seem to occur simultaneously, leading
to the demise of key neuronal cells.2
ing several drug molecules results in several challenges, such as a
combined or even multiplied toxicity and side-effect profile and
the occurrence of unforeseen drug–drug interactions.3 For these
reasons the design of single drug molecules that act on two or
more specific etiological targets of a particular disease may be of
value. The advantages associated with this strategy includes a low-
er likelihood of encountering unwanted side-effects and the possi-
bility to ‘design out’ any side-effects when only using one drug, as
opposed to using two or more drugs.1
One target for the treatment of neurodegenerative diseases is
the enzyme monoamine oxidase B (MAO-B). It is the predominant
isoform of MAO found in human brain4 where it is implicated in
age-related neurodegenerative diseases such as Parkinson’s dis-
ease and Alzheimer’s disease. It has been demonstrated that brain
MAO-B activity, but not MAO-A activity, increases with aging,5 and
since MAO-B appears to be located in glial cells this may be due to
gliosis associated with aging. MAO-B plays a dual role in the path-
ophysiology of Parkinson’s disease since it is a major dopamine
metabolising enzyme and is also implicated in the formation of
reactive oxygen species and other neurotoxic species.6 MAO-B
has also been implicated in the pathophysiology of Alzheimer’s
disease since increased MAO-B levels have been observed in
plaque-associated astrocytes in the brains of Alzheimer’s disease
patients.7 This increase in MAO-B activity may result in an eleva-
tion in hydroxyl radicals (ÅOH), which has been correlated with
amyloid-b (Ab) plaque formation. Hence, the therapeutic potential
of selective reversible MAO-B inhibitors does not rely solely on
Keeping this in mind, the paradigm of targeting a single disease
factor may not be an effective treatment strategy for neurodegen-
erative diseases. The polypharmaceutical approach, wherein sev-
eral drugs that act independently on different etiological targets
* Corresponding author at present address: School of Pharmacy, University of the
Western Cape, Private Bag X17, Bellville 7535, South Africa. Tel.: +27 21 9593190;
fax: +27 21 9591588.
E-mail address: sfmalan@uwc.ac.za (S.F. Malan).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.09.019

7524
L. H. A. Prins et al. / Bioorg. Med. Chem. 17 (2009) 7523–7530
their ability to increase the biological half-life of dopamine but also
on their ability to reduce the levels of MAO-B generated reactive
oxygen species in the brain and their potential ability to inhibit
Ab plaque formation.
example, the caffeine analogue (E)-8-(3-chlorostyryl)-caffeine
(CSC) (1, Fig. 1), is an exceptionally potent MAO-B inhibitor, with
enzyme-inhibitor dissociation constants (Ki values) of 100 nano
molar (nM) for mouse brain mitochondrial MAO-B20 and 128 nM
for baboon liver mitochondrial MAO-B.21 Pteridine-2,4-dione (2)
bear structural resemblance to caffeine (3) and was therefore of
interest in the design of novel reversible MAO-B inhibitors, possi-
bly with comparable activity to the caffeinyl derivatives. The syn-
thesis of pteridine-2,4-diones conjugated to styryl and other
moieties were thus pursued, and the resulting compounds were
evaluated as MAO-B inhibitors.
Pteridine-2,4-dione is also structurally similar to the essential
NOS cofactor, tetrahydrobiopterin (BH4) (4) and certain pterin ana-
logues (5) have been developed to target this binding site.22,23 Be-
cause of this structural relationship with BH4, pteridine derivatives
have been described as NOS inhibitors. Since BH4 exhibits lower
affinity and selectivity for other pterin dependent enzymes, this
approach appears to be promising.24 In a series of BH4 analogues
Another enzyme implicated in the neurodegenerative process is
nitric oxide synthase (NOS). Nitric oxide (NO) is known to be an
important cell signalling agent that regulates an array of physio-
logical functions. These include blood pressure by regulation of
smooth muscle relaxation,8 platelet aggregation by acting as an
antithrombotic agent,9 antitumor, antibacterial, and antiviral ac-
tion of macrophages,10 brain development, learning and memory,11
and it is also the neuronal mediator of penile erection.12 However,
since NO is a free radical, overproduction thereof may result in
deleterious effects ranging from septic shock and pain13 to ischae-
mia14 and it may also be involved in neurodegenerative pro-
cesses15 associated with Parkinson’s and Alzheimer’s diseases.16
The harmful effects caused by an overproduction of NO are thought
to be mediated by peroxynitrate (ONOOꢀ), the product obtained
when NO and the superoxide radical (O2Åꢀ) react. ONOOꢀ causes
injury to the mitochondrial electron transport chain resulting in
damage and eventual death of neurons. Evidence that NO and
ONOOꢀ are neurotoxic is provided in the ability of superoxide dis-
mutase to protect cortical cultures from both glutamate and NO
donors. Removal of nNOS containing neurons from culture or elim-
ination of nNOS through transgenic technology, results in a culture
that is resistant to NMDA neurotoxicity indicating that nNOS neu-
rons are the source of neurotoxic NO.16,17
the 4-amino derivative, 5,6,7,8-tetrahydro-6-(D-threo-1,2-dihydr-
oxypropyl)pterin (6), was reported to be a potent inhibitor of recom-
binant rat brain NOS in vitro (Ki = 13 nM) as well as in vivo.25 Also of
interest is the 6-phenyl derivative (7) which inhibits NOS with an
IC50 value of 6 lM. Based on theNOS inhibitionactivityof pterinana-
logues, the structurally similar pteridine-2,4-dione analogues pre-
pared in the present study were also evaluated as NOS inhibitors.
Compounds that inhibit both MAO-B and NOS may find enhanced
application in the treatment of neurodegenerative disorders.
Several 8-substituted caffeinyl derivatives have been reported
to be moderate to potent reversible inhibitors of MAO-B.18,19 For
2. Results
2.1. Synthesis of pteridine-2,4-dione analogues
O
N
O
H
O
N
N
Cl
N
N
The target pteridine-2,4-dione derivatives were synthesised
according to the literature procedure (Scheme 1).26 The key
N
N
O
N
H
O
CSC (1)
Pteridine-2,4-dione (2)
NH2
O
N
i
O
O
H
N
OH
+
H
R
O
N
NH2
H
N
N
N
OH
8
N
O
N
H2N
N
N
H
O
O
N
N
R
N
N
R
Caffeine (3)
4
N
N
ii
O
N
NH2
O
O
NH2
N
H
N
OH
H
N
N
N
10a-b, d
9a-d
OH
H2N
N
N
H2N
N
H
Cl
6
5
R =
NH2
N
H
N
9a, 10a
9b, 10b
Cl
N
H2N
N
H
9d, 10d
9c
7
Scheme 1. Synthetic pathway to pteridine-2,4-dione (10) and pyrimidine (9)
analogues. Reagents and conditions: (i) EtOH, reflux, 4 h; (ii) CH(OCH2CH3)3, DMF,
reflux, 10 h.
Figure 1. The structures of CSC (1), pteridine-2,4-dione (2), caffeine (3) and
selected pterin analogues.

L. H. A. Prins et al. / Bioorg. Med. Chem. 17 (2009) 7523–7530
7525
Table 1
starting material, 1,3-dimethyl-5,6-diaminouracil (8), was reacted
with the appropriate aldehyde to yield the pyrimidines 9a–d.
The pyrimidines were cyclised by the addition of triethyl ortho-
formate under reflux, to obtain the final pteridine-2,4-diones
10a–b, d.26 Cyclisation of 9c using this procedure was not suc-
cessful and the corresponding pteridine-2,4-dione could not be
obtained. The structures of the newly synthesised compounds
were confirmed by 1H NMR, 13C NMR, IR, MS and elemental
analyses.
MAO-B and NOS inhibition by pteridine-2,4-dione (10) and pyrimidine (9) analogues
Compound
MAO-B inhibition
M)c
NOS
inhibition
IC50 (lM)
IC50
(
lM)
Ki (l
O
N
N
Not
Not
466.9
determineda determineda
O
N
NH2
2.2. MAO-B inhibition
9a
It has previously been shown that baboon liver tissue is devoid
of MAO-A activity while exhibiting a high degree of MAO-B cata-
lytic activity.27 The interaction of reversible inhibitors with baboon
liver MAO-B also appears to be similar to human liver MAO-B.19
Recent results from our laboratory (unpublished data) have also
indicated that a variety of structurally unrelated compounds inhi-
bit membrane bound baboon liver and recombinant human MAO-B
with similar potencies. Mitochondrial fractions obtained from
baboon liver were thus used to determine the extent by which
the pteridine-2,4-dione analogues (10a–b, d) and the pyrimidines
(9a–d) inhibit this enzyme.
Cl
O
N
N
7.033
4.060
6968
O
N
NH2
9b
O
IC50 values (concentration of the inhibitor that inhibits 50% of
the enzyme activity) for the test compounds were determined by
measuring the extent by which different concentrations thereof
N
N
12.052
6.958
Not
determinedd
slowed the rate of
a-carbon oxidation of 1-methyl-4-(1-methyl-
O
N
NH2
pyrrol-2-yl)-1,2,3,6-tetrahydropyridine (MMTP) to the correspond-
ing dihydropyridinium metabolite, MMDP+ (Fig. 2). The production
of MMDP+ was measured spectrophotometrically at a wavelength
of 420 nm, at which neither the substrate nor the test compounds
absorb UV light.
9c
Cl
O
The results of the MAO-B activity measurements indicate that
10d is the most potent MAO-B inhibitor of the analogues examined
N
N
0.602
0.348
577.2
in this study, with an IC50 value of 0.314
most potent inhibitor was pyrimidine 9d with with an IC50 value of
0.602 M. It is noteworthy that 10d and 9d are structurally closely
lM (Table 1). The second
O
N
NH2
l
related to CSC (1), a known MAO-B inhibitor. Using the Cheng–
Prusoff equation28 the Ki values (enzyme–inhibitor dissociation
constants) for the inhibition of MAO-B by 10d and 9d were calcu-
lated to be 181 and 348 nM, respectively, indicating that 10d is
only slightly less potent than CSC (Ki = 128 nM)21 as a MAO-B
inhibitor.
9d
O
N
N
N
Not
Not
443.3
determinedb determinedb
O
N
10a
2.5
2.0
1.5
1.0
0.5
Cl
O
N
N
N
4.889
2.823
Not
determinedd
O
N
10b
Cl
0
1
2
3
4
5
6
O
N
Log [10d]
N
N
N
0.314
0.181
1057
Figure 2. The sigmoidal dose–response curve of the initial rates of oxidation of
MMTP versus the logarithm of concentration of inhibitor 10d (expressed in nM).
The concentration of the baboon liver mitochondrial preparation was 0.15 mg
protein/mL. The rates are expressed as nmol MPDP+ formed/mg protein/min and
O
10d
the concentration of MMTP used was 50
in duplicate and the values are expressed as mean SEM.
lM. The determinations were carried out
(continued on next page)

7526
L. H. A. Prins et al. / Bioorg. Med. Chem. 17 (2009) 7523–7530
2.4. Molecular modelling study
Table 1 (continued)
Compound
MAO-B inhibition
NOS
inhibition
IC50 (lM)
In an attempt to clarify the relationships between the observed
MAO-B inhibition potencies and the structures of the inhibitors
examined here, molecular docking studies with the active pyrimi-
dine (9b–d) and pteridine (10b, d) analogues were performed. CSC
(1) and safinamide were also included in the docking study. The
crystallographic structure of recombinant human MAO-B in com-
plex with the reversible inhibitor safinamide (PDB code: 2V5Z)32
was selected for the docking studies. Since safinamide spans both
the entrance and substrate cavities of the enzyme active site, the
side chain of the gating residue, Ile-199, is rotated out of its normal
conformation to allow for the fusion of the two cavities and the
accommodation of larger structures such as those examined in
the present study.33 To evaluate the accuracy of the docking proce-
dure, the co-crystallised ligand was redocked within the active site
using the LigandFit application within Discovery StudioÒ 1.7. This
procedure was repeated three times and the best ranked solution
of safinamide in each instance exhibited a RMSD of 1.73 Å from
the co-crystallised ligand (Fig. 4). In general, RMSD values smaller
than 2.0 Å indicate that the docking protocol is capable of accu-
rately predicting the binding orientation of the co-crystallised li-
gand.34 This protocol was thus deemed to be suitable for docking
of the inhibitors into the active site model of MAO-B.
IC50
(lM)
Ki (
l
M)c
H
H2N
N
19.41e
NH2
—
—
NH
AG
N
N
—
—
0.111e
H
NO2
7-NI
a
b
c
At the limit of solubility (30
At the limit of solubility (100
The experimentally determined IC50 values were used to calculate the Ki values
with
l
l
M) 17.8% of the MAO-B activity was inhibited.
M) 12.2% of the MAO-B activity was inhibited.
according to the equation by Cheng and Prusoff: Ki = IC50/(1 + [S]/Km
)
[S] = 50 M and Km (MMTP) = 68.3 1.60
l
l
M.28
d
Values could not be calculated as these compounds absorb UV light within the
assay detection range (390–430 nm).
Values obtained from Joubert et al. (2008).31
e
The best ranked docking solutions of the pyrimidine (9b–d) and
pteridine (10b, d) analogues examined here show that all inhibi-
tors span both the entrance and substrate cavities of the enzyme.
As shown for example with 9d (Fig. 5) and 10d (Fig. 6), the pyrim-
idine-2,4-dione rings of 9b–d and the pteridine-2,4-dione rings of
10b, d are located within the substrate cavity nearer to the FAD
cofactor. The phenyl (9b, 10b) and styryl (9c–d, 10d) side chains
extend towards the entrance cavity. These binding modes are ex-
pected since the polar regions in the substrate cavity would better
2.3. NOS inhibition
In order to determine the NOS inhibition potencies of the pter-
idine-2,4-dione (10a–b, d) and pyrimidine (9a–d) analogues, the
oxyhaemoglobin (Hb) assay29 was used. This assay is based on
the measurement of the conversion of oxyHb to metHb by NO
(Fig. 3). Rat brain homogenate was used as NOS enzyme source
since it has been shown to have high constitutive NOS activity.30
A disadvantage of brain homogenate as enzyme source is that iso-
form selectivity is unaccounted for since the crude rat brain extract
contains several NOS isoforms. For the purpose of screening poten-
tial NOS inhibitors for central NOS inhibition activity, this enzyme
source was deemed appropriate.
Comparing the obtained IC50 values for the pteridine-2,4-diones
(10a–b, d) and pyrimidines (9a–d) to those of two known NOS
inhibitors, 7-nitroindazole (7-NI; IC50 = 0.111
nidine (AG; IC50 = 19.41 M), reveals that none of the test com-
pounds are promising as potential NOS inhibitors (Table 1). The
most potent inhibitors were 10a (IC50 = 443.3 M) and 9a
(IC50 = 466.9 M), which are approximately 23-fold less potent
lM) and aminogua-
l
Figure 4. RMSD overlay of the best ranked solutions of safinamide on the original
co-crystallised ligand, displayed in bright green.
l
l
than AG. IC50 values could not be calculated for compounds 9c
and 10b since these compounds absorb UV light in the detection
range (390–430 nm) of the assay.
0.15
401 nm
0.10
0.05
411 nm
0.00
•
-0.05
-0.10
-0.15
390
400
410
420
430
Wavelength (nm)
Figure 5. Putative binding mode of 9d to the human MAO-B active site. Hydrogens
are hidden, except those involved in hydrogen bonds, and selected amino acid
residues are displayed in dark green. The FAD cofactor is indicated in red,
interacting water molecules in blue and hydrogen bonds in black. The Van der
Waals volume of 9d is displayed as a transparent yellow surface.
Figure 3. UV–vis scans of incubations containing rat brain homogenate, oxyHb,
NADPH and -arginine. The increase in absorbance intensity at a wavelength of
401 nm is indicative of metHb production as a result of NO formation by NOS
present in the homogenate fraction.
L

L. H. A. Prins et al. / Bioorg. Med. Chem. 17 (2009) 7523–7530
7527
Figure 6. Putative binding mode of 10d to the human MAO-B active site.
Hydrogens are hidden, except those involved in hydrogen bonds, and selected
amino acid residues are displayed in dark green. The FAD cofactor is indicated in
red, interacting water molecules in blue and hydrogen bonds in black. The Van der
Waals volume of 10d is displayed as a transparent yellow surface.
Figure 8. Putative binding mode of CSC to the human MAO-B active site. Hydrogens
are hidden, except those involved in hydrogen bonds, and selected amino acid
residues are displayed in dark green. The FAD cofactor is indicated in red,
interacting water molecules in blue and hydrogen bonds in black. The Van der
Waals volume of CSC is displayed as a transparent yellow surface.
accommodate the pyrimidine and pteridine rings while the phenyl
and styryl moieties would be better stabilised within the hydro-
phobic entrance cavity. In addition, hydrogen bonding between
the carbonyl oxygens of the pyrimidine and pteridine rings and
integral water molecules within the substrate cavity would further
promote these binding orientations.
For structures 9b–d the possibility of hydrogen bond formation
with the amino functional group on C6 of the pyrimidine ring also
exists as demonstrated in Figure 7. CSC adopts a similar binding
mode to those observed for the pyrimidine (9b–d) and pteridine
(10b, d) analogues and the caffeine ring of CSC is located in the
substrate cavity of the MAO-B active site model while the styryl
side chain extends into the entrance cavity (Fig. 8). As observed
for the pyrimidine and pteridine analogues, the caffeine carbonyl
oxygens can also act as potential hydrogen bond acceptors within
the substrate cavity.
The log IC50 values of the pyrimidine (9c–d) and pteridine (10d)
analogues and of CSC correlated with the DockScore value with a
correlation coefficient (r2 value) of 0.97 (Table 2). Although it is ex-
tremely difficult to accurately rank compounds based on their
binding affinity in a docking study, this observation may serve as
additional evidence that this docking protocol is effective for
examining the molecular interactions between the inhibitors stud-
ied here and the active site of MAO-B. This docking protocol also
appears to have predictive value and may in the future be devel-
oped as a predictive model for MAO-B inhibition.
Table 2
Comparison of calculated affinities (DockScore values) of compounds 9c, 9d, 10d and
CSC with experimental MAO-B inhibitory activities (log IC50 values)
Ligand
DockScore
log IC50 (lM)
CSC
10d
9d
60.171
59.898
57.842
54.249
ꢀ0.836a
ꢀ0.503
ꢀ0.220
1.081
9c
a
Value obtained from the literature.35
3. Discussion
The current study has identified pteridine-2,4-diones as prom-
ising novel inhibitors of MAO-B. Analogue 10d which is substituted
with a 3-chlorostyryl functional group at C6 of the pteridine-2,4-
dione ring was found to be the most potent inhibitor with an
IC50 value of 0.314 lM. This inhibition potency is comparable to
that of CSC, a potent reversible MAO-B inhibitor.20,21,35 Interest-
ingly, the pteridine-2,4-diones were found to be more potent
MAO-B inhibitors than their corresponding pyrimidine precursors.
For example pyrimidine 9d, the precursor of 10d, has an IC50 value
of 0.602 lM compared to the 0.314 lM of 10d. The same trend is
observed for pteridine-2,4-dione 10b, which is more potent than
pyrimidine 9b. A possible reason for this observation is that the
amine functional group at C6 of the pyrimidine ring might be pro-
tonated at physiological pH. This may hinder access to the active
site or prevent effective binding within the site.
The current study also shows that styryl substitution at C6 of
the pteridine-2,4-dione results in better MAO-B inhibitors than
phenyl substitution. For example 10d was approximately 16-fold
more potent as a MAO-B inhibitor than was 10b. Chlorine substitu-
tion at C3 of the styryl ring was again found to be important for
binding to the active site of MAO-B19,20 and throughout both the
pyrimidine and pteridine series, the chlorine substituted com-
pounds exhibited higher activity as MAO-B inhibitors. This concurs
with the reported findings that CSC is approximately 22-fold more
potent than the corresponding unsubstituted (E)-8-styrylcaffe-
ines.21 This data suggests that the inhibitors examined here have
a similar binding mode as CSC. In general, substitution of the styryl
ring appears to improve the binding interactions within the en-
trance cavity.
Figure 7. Putative binding mode of 9c to the human MAO-B active site. Hydrogens
are hidden, except those involved in hydrogen bonds, and selected amino acid
residues are displayed in dark green. The FAD cofactor is indicated in red,
interacting water molecules in blue and hydrogen bonds in black. The Van der
Waals volume of 9c is displayed as a transparent yellow surface.
As has been the case in numerous other enzymatic studies, the
limited solubility of the test compounds in this study restricted
the full exploration of their biological profile. In the MAO-B assay

7528
L. H. A. Prins et al. / Bioorg. Med. Chem. 17 (2009) 7523–7530
for example, compound 10a and its pyrimidine precursor 9a were
insoluble in the buffer medium at 100 and 30 M, respectively.
5.2. Synthesis
l
Another factor specifically relating to the NOS oxyHb assay,
which hindered biological evaluation, was the UV absorption
properties of the test inhibitors. Compounds 9c and 10b were
both shown to absorb UV light in the assay detection range
stretching from 390 to 430 nm. As a result, NOS inhibition data
could not be calculated for either of these compounds. As none
of the pteridine analogues showed promise as NOS inhibitors, nei-
ther the obtained results nor the assay procedure were optimised
any further.
General procedure for the synthesis of pyrimidines 9a–d: 1,3-
Dimethyl-5,6-diaminouracil (17.6 mmol) was suspended in 10 mL
ethanol (anhydrous). The appropriate aldehyde (20.6 mmol) was
dissolved in 5 mL ethanol (anhydrous) and added to the above
suspension. After heating the mixture under reflux for 4 h it was al-
lowed to cool to room temperature. The reaction was cooled on an
ice bath for 30 min and the precipitate was collected by filtration
and washed with ethanol. Recrystallisation from DMF/H2O gave
the pure intermediary compounds (9a–d) in excellent yield.
General procedure for the synthesis of pteridine-2,4-dione ana-
logues 10a–d: The appropriate pyrimidine compound (9a–b, d,
8.5 mmol) was dissolved in 10 mL N,N0-dimethylformamide at
ambient temperature and triethyl orthoformate (43 mmol) was
added. The solution was refluxed for 10 h and then allowed to cool
to room temperature. The reaction was incubated on an ice bath
for 30 min and the resulting precipitate was collected by filtration.
The pteridine-2,4-diones were recrystallised from N,N0-dimethyl-
formamide (10a, 10b) or acetone (10d).
4. Conclusion
Although multifunctional drugs are a promising strategy for the
treatment of neurodegenerative diseases,36,37,1 designing drugs
that act at multiple targets remains a challenge. Designing pteri-
dine analogues to target the BH4 binding site of NOS was not suc-
cessful in this study and it is hypothesised that the styryl phenyl
may be sterically hindering binding of the test compounds to NOS.
Comparing the calculated MAO-B Ki values for the synthesised
pteridines (Table 1) with those experimentally determined for a
series of benzimidazoles,38 reveals that pteridines may be better
scaffolds for MAO-B inhibition than benzimidazoles and in general
exhibit MAO-B inhibition only slightly less potent than that ob-
served with the corresponding caffeine structures. These com-
pounds thus show potential for the design of potent reversible
MAO-B inhibitors and might, with optimisation, be attractive alter-
natives to the caffeine analogues.
5.2.1. 6-Amino-1,3-dimethyl-5-{[1-phenylmeth-(E)-ylidene]-
amino}-1H-pyrimidine-2,4-dione (9a)
Compound 9a was prepared from 1,3-dimethyl-5,6-diamino-
uracil (8) and benzaldehyde in a yield of 59.2%. C13H14N4O2; mp:
225 °C (DMF/H2O). The measured melting point was in accordance
with the literature value (225 °C).26
5.2.2. 6-Amino-5-{[1-(3-chlorophenyl)-meth-(E)-ylidene]-
amino}-1,3-dimethyl-1H-pyrimidine-2,4-dione (9b)
Compound 9b was prepared from 1,3-dimethyl-5,6-diamino-
5. Experimental
uracil (8) and 3-chloro-benzaldehyde in
a yield of 78.6%.
C13H13ClN4O2; mp: 253 °C (DMF/H2O); 1H NMR (600 MHz,
DMSO-d6) dH: 9.71–9.67 (s, 1H), 8.07–7.71 (m, 2H), 7.46–7.33 (m,
4H), 3.42–3.37 (s, 3H), 3.19–3.14 (s, 3H); 13C NMR (150 MHz,
DMSO-d6) dC: 157.70 (C), 154.20 (C), 150.00 (CH), 148.00 (C),
141.20 (C), 134.10 (C), 130.60 (CH), 129.00 (CH), 126.90 (CH),
126.20 (CH), 99.10 (C), 30.90 (CH3), 27.50 (CH3); MS (EI, 70 eV)
Caution: MMTP is a structural analogue of the nigrostriatal neu-
rotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and
it should be handled using disposable latex gloves and protective
eyewear. Procedures for the safe handling of neurotoxic compounds
were followed as described previously.39
m/z: 292 (M+); IR (KBr)
mmax: 3414, 3297, 3081, 1690, 1551,
5.1. Materials and instrumentation
1593, 1518, 1449, 1263, 1226, 1207, 1067, 876, 789, 760, 748,
711 cmꢀ1; Anal. Calcd for C13H13ClN4O2: C, 53.34; H, 4.48; N,
19.14. Found: C, 54.20; H, 4.30; N, 19.40.
All chemicals and reagents were purchased from commercial
sources and solvents were dried using standard methods. 1,3-Di-
methyl-5,6-diaminouracil (8),40 trans-3-chloro-cinnamaldehyde41
and the oxalate salt of MMTP42 were prepared according to
previously reported methods. Because of chemical instability, 8
was used within 24 h of preparation. Thin-layer chromatography
was performed on 0.20 mm thick aluminium silica gel sheets
(AlugramÒ SIL G/UV254, Kieselgel 60, Macherey-NagelÒ Düren,
Germany) with an appropriate mobile phase and visualisation
was achieved using UV light (254 and 366 nm) and iodine vapour.
All melting points (mp) were obtained using a StuartÒ SMP10 melt-
ing point apparatus and are uncorrected. 1H and 13C NMR spectra
were recorded using a BrukerÒ Avance III 600 spectrometer at a
frequency of 600 and 150 MHz, respectively. Chemical shifts are
expressed in parts per million (d) relative to the signal from tetra-
methylsilane (Me4Si), added to an appropriate deuterated solvent
(CDCl3 or DMSO-d6). Spin multiplicities are given as s (singlet), d
(doublet), dd (doublet of doublets) and m (multiplet). Mass spectra
were obtained by direct insertion electron impact ionisation mass
spectrometry (EI-MS) using an analytical VG 7070E mass spec-
5.2.3. 6-Amino-1,3-dimethyl-5-[(E)-3-phenyl-prop-2-en-(E)-
ylideneamino]-1H-pyrimidine-2,4-dione (9c)
Compound 9c was prepared from 1,3-dimethyl-5,6-diamino-
uracil (8) and trans-cinnamaldehyde in
a yield of 76.1%.
C15H16N4O2; mp: 235 °C (DMF/H2O); 1H NMR (600 MHz, DMSO-
d6) dH: 9.48–9.45 (d, 1H, J = 8.21 Hz), 7.55–7.52 (d, 2H,
J = 8.62 Hz), 7.39–7.25 (m, 5H), 7.00–6.97 (d, 2H, J = 8.00 Hz), 3.36
(s, 3H), 3.15 (s, 3H); 13C NMR (150 MHz, DMSO-d6) dC: 156.81
(C), 153.26 (C), 151.32 (CH), 149.63 (2 ꢁ C), 136.53 (CH), 131.62
(CH), 128.73 (2 ꢁ CH), 128.12 (2 ꢁ CH), 126.59 (CH), 99.39 (C),
30.21 (CH3), 26.99 (CH3); MS (EI, 70 eV) m/z: 285 (M+); IR (KBr)
m
max: 3498, 3374, 3061, 3032, 2996, 1680, 1608, 1585, 1518,
1448, 1226, 1162, 1068, 973, 916, 840, 766, 747, 694 cmꢀ1; Anal.
Calcd for C15H16N4O2: C, 63.37; H, 5.67; N, 19.71. Found: C,
62.60; H, 5.60; N, 18.80.
5.2.4. 6-Amino-5-[(E)-3-(3-chloro-phenyl)-prop-2-en-(E)-
ylideneamino]-1,3-dimethyl-1H-pyrimidine-2,4-dione (9d)
Compound 9d was prepared from 1,3-dimethyl-5,6-diamino-
uracil (8) and trans-3-chlorocinnamaldehyde in a yield of 59.9%.
C15H15ClN4O2; mp: 222 °C (DMF/H2O); 1H NMR (600 MHz,
DMSO-d6) dH: 9.44–9.42 (d, 1H, J = 8.76 Hz), 7.61–7.49 (m, 2H),
7.42–7.28 (m, 4H), 7.10–7.02 (dd, 1H, J = 8.79, 16.13 Hz), 6.98–
trometer. IR spectra were recorded in KBr on a NicoletÒ Nexus
TM
470-FT IR spectrometer over the range 400–4000 cmꢀ1 employing
the diffuse reflectance method. Elemental analyses were done
using a Perkin–ElmerÒ 2400 Series II CHNS elemental analyser
with argon gas as carrier.

L. H. A. Prins et al. / Bioorg. Med. Chem. 17 (2009) 7523–7530
7529
6.92 (d, 1H, J = 16.18 Hz), 3.40–3.36 (s, 3H), 3.19–3.14 (s, 3H); 13C
NMR (150 MHz, DMSO-d6) dC: 157.20 (C), 154.00 (C), 151.10
(CH), 150.09 (C), 139.50 (C), 135.00 (C), 134.10 (CH), 133.70 (CH),
131.10 (CH), 128.10 (CH), 125.75 (CH), 100.00 (CH), 30.90 (CH3),
potassium phosphate buffer (10 mM, pH 7–7.2) containing sucrose
(0.25 M) and ethylenediaminetetra-acetic acid (EDTA) (0.5 mM).
The homogenate was diluted to 620 mL with the above solution
and centrifuged at 600g for 15 min. The supernatant was decanted
through a cheese cloth and again centrifuged at 10,400g for 15 min.
The pellet obtained was homogenised in a small volume of a potas-
sium phosphate buffer (10 mM, pH 7–7.2) containing sucrose
(0.25 M) and diluted to a final volume of 124 mL with the same
buffer. The homogenate was centrifuged at 7500g for 15 min and
the resulting pellet was homogenised in a small volume of a buffer
containing tris (10 mM, pH 7–7.2) and potassium chloride
(0.15 M). The homogenate was diluted to a final volume of
52 mL, centrifuged at 7500g for 15 min and the pellet obtained
27.80 (CH3); MS (EI, 70 eV) m/z: 318 (M+); IR (KBr)
mmax: 3411,
3323, 3125, 3054, 3029, 1684, 1611, 1559, 1509, 1438, 1280,
1218, 1165, 1068, 898, 871, 791, 749, 702 cmꢀ1; Anal. Calcd for
C15H15ClN4O2: C, 56.52; H, 4.74; N, 17.58. Found: C, 56.20; H,
4.40; N, 17.90.
5.2.5. 1,3-Dimethyl-6-phenyl-1H-pteridine-2,4-dione (10a)
Compound 10a was prepared from 6-amino-1,3-dimethyl-5-
{[1-phenylmeth-(E)-ylidene]-amino}-1H-pyrimidine-2,4-dione (9a)
and triethyl orthoformate in a yield of 29.3%. C14H12N4O2; mp:
258 °C (DMF); 1H NMR (600 MHz, DMSO-d6) dH: 8.16–8.12 (m, 1H),
7.53–7.50 (m, 2H), 7.50–7.48 (m, 2H), 7.48–7.45 (m, 1H), 3.52–
3.50 (s, 3H), 3.28–3.26 (s, 3H); 13C NMR (150 MHz, DMSO- d6) dC:
155.00 (2 ꢁ C), 151.80 (CH), 150.10 (C), 148.90 (C), 142.00 (2 ꢁ C),
130.10 (CH), 129.25 (2 ꢁ CH), 127.00 (2 ꢁ CH), 30.10 (CH3), 28.20
was stored at ꢀ70 °C in 300
l
L aliquots. Before use, the mitochon-
drial isolate was suspended in 300
l
L of sodium phosphate buffer
(100 mM, pH 7.4) containing glycerol (50%, w/v) and the protein
concentration was determined by the Bradford method44 using bo-
vine serum albumin as reference standard. As noted earlier, the
mitochondrial fraction obtained from baboon liver tissue is devoid
of MAO-A activity27 and therefore inactivation of the enzyme with
clorgyline was unnecessary. The MAO-A and -B mixed substrate
(CH3); MS (EI, 70 EV) m/z: 268 (M+); IR (KBr)
mmax: 3165, 1697,
1659, 1561, 1525, 1472, 1356, 1298, 1229, 1060, 786, 758, 746,
707 cmꢀ1; Anal. Calcd for C14H12N4O2: C, 62.68; H, 4.51; N, 20.88.
Found: C, 62.00; H, 4.70; N, 20.30.
MMTP (Km = 60.9 l
M for baboon liver MAO-B)25 was used as sub-
strate for the inhibition studies. All incubations were performed in
sodium phosphate buffer (100 mM, pH 7.4) and contained MMTP
5.2.6. 6-(3-Chlorophenyl)-1,3-dimethyl-1H-pteridine-2,4-dione
(10b)
Compound 10b was prepared from 6-amino-5-{[1-(3-chloro-
phenyl)-meth-(E)-ylidene]-amino}-1,3-dimethyl-1H-pyridine-2,4-
(50
ious concentrations of the test inhibitors, producing a final incuba-
tion volume of 500 L. Stock solutions of the test inhibitors were
prepared in DMSO and added to the incubation mixtures to yield
a final DMSO concentration of 4% (v/v). DMSO concentrations
higher than 4% are reported to inhibit MAO-B.45 Samples were
incubated at 37 °C for 10 min, a time period for which dihydropy-
ridinium metabolite formation is reported to be linear.27 The
lM), the mitochondrial isolate (0.15 mg protein/mL), and var-
l
dione (9b) and triethyl orthoformate in
a yield of 27.1%.
C14H11ClN4O2; mp: 233–236 °C (DMF); 1H NMR (600 MHz, CDCl3)
dH: 9.06–9.04 (s, 1H), 8.11–8.09 (s, 1H), 7.99–7.95 (m, 1H), 7.48–
7.45 (m, 2H), 3.79–3.72 (s, 3H), 3.60–3.55 (s, 3H); 13C NMR
(150 MHz, CDCl3) dC: 160.00 (C), 150.02 (C), 147.00 (C, CH),
144.90 (C), 136.30 (C), 135.10 (C), 130.02 (CH), 130.00 (CH),
127.00 (CH), 126.80 (CH), 125.00 (C), 29.22 (CH3), 29.00 (CH3);
enzyme reactions were terminated by the addition of 10 lL per-
chloric acid (70%) and centrifuged at 16,000g for 10 min. The
supernatant fractions were removed and the concentrations of
the MAO-B generated product, MMDP+, were measured spectro-
photometrically at 420 nm using a ShimadzuÒ MultiSpec-1501
MS (EI, 70 eV) m/z: 302 (M+); IR (KBr)
mmax: 3066, 1717, 1669,
1545, 1505, 1424, 1336, 1218, 1105, 1010, 908, 794, 750, 739,
687 cmꢀ1; Anal. Calcd for C14H11ClN4O2: C, 55.55; H, 3.66; N,
18.51. Found: C, 56.20; H, 3.60; N, 18.60.
spectrophotometer (e
= 25,000 Mꢀ1). The IC50 values were deter-
mined from plots of the rate of MAO-B catalysed MMDP+ formation
versus the logarithm of the inhibitor concentration. For this pur-
pose the PrismÒ 4.02 (GraphPad, Sorrento Valley, CA) software
package was employed. At least eight different concentrations of
the test inhibitor, spanning 3 orders of magnitude were used to
construct the sigmoidal dose–response curve. The IC50 values are
reported as mean standard error of the mean (SEM) of duplicate
determinations.
5.2.7. 6-[(E)-2-(3-Chlorostyryl)]-1,3-dimethyl-1H-pteridine-2,4-
dione (10d)
Compound 10d was prepared from 6-amino-5-[(E)-3-(3-chloro-
phenyl)-prop-2-en-(E)-ylideneamino]-1,3-dimethyl-1H-pyrimi-
dine-2,4-dione (9d) and triethyl orthoformate in a yield of 7.2%.
C16H13ClN4O2; mp: 270 °C (acetone); 1H NMR (600 MHz, CDCl3)
dH: 8.65 (s, 1H), 7.65 (d, 1H, J = 15.97 Hz), 7.61–7.36 (m, 4H), 7.27
(d, 1H, J = 15.29 Hz), 3.70 (s, 3H), 3.50 (s, 3H); 13C NMR (150
MHz, CDCl3) dC: 159.06 (C), 149.68 (C), 146.43 (CH), 145.14 (C),
138.23 (C), 135.57 (C), 133.78 (C), 129.84 (2 ꢁ CH), 127.51
(2 ꢁ CH), 125.63 (C), 123.49 (CH), 122.24 (CH), 29.06 (CH3), 27.00
5.3.2. NOS inhibition study
Brain tissue (1 g/5 mL) of male Sprague-Dawley rats were
homogenised in a solution consisting of 4-(2-hydroxyethyl)pipera-
zine-1-ethanesulfonic acid (HEPES) (100 mM, pH 7.4), sucrose
(CH3); MS (EI, 70 eV) m/z: 328 (M+); IR (KBr)
mmax: 3096, 3062,
(320 mM), EDTA (1 mM),
(10 g/mL), soybean-trypsin inhibitor (10
(2 g/mL).
After 10 s of homogenation at 4 °C phenylmethylsulfonyl fluo-
ride (PMSF) (10 M/mL) was added and the mixture was homoge-
D/L
-dithiothretiol (1 mM), leupeptin
2954, 1719, 1670, 1539, 1501, 1460, 1346, 1287, 1220, 1010,
889, 794, 750, 727, 681 cmꢀ1; Anal. Calcd for C16H13ClN4O2: C,
58.46; H, 3.99; N, 17.04. Found: C, 59.80; H, 4.10; N, 16.70.
l
lg/mL) and aprotinin
l
l
5.3. Biological evaluation
nised for an additional 30 s. Thereafter the homogenate was
centrifuged at 12,000g for 10 min. The supernatant was collected
and divided into 2 mL aliquots, which were assayed immediately
or snap frozen and stored at ꢀ70 °C.
Approval for this study was obtained from the Ethics Commit-
tee for Research on Experimental Animals of the North-West Uni-
versity (Potchefstroom Campus).
Haemoglobin was converted to its oxygenated form as follows:
Crystallised haemoglobin (25 mg) was dissolved in 1 mL cold
HEPES buffer46 and reduced with an excess sodium dithionate
(0.958 mg). Oxygen was blown over the surface and the solution
gently swirled for 15 min. The gradual colour change from dark
red to bright red was indicative of the oxygenation of haemoglobin.
5.3.1. MAO-B inhibition study
Mitochondria from baboon liver tissue were isolated as fol-
lows:43 Baboon liver tissue (200 g) was grinded through a Foley
mill and homogenised with a glass/teflon homogeniser in 356 mL

7530
L. H. A. Prins et al. / Bioorg. Med. Chem. 17 (2009) 7523–7530
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formed using a SephadexÒ G-25 column.
The test compounds were dissolved in DMSO to yield a concen-
tration of 5% DMSO in the final incubation mixtures. The incuba-
tions contained HEPES (500 lM), test inhibitor, CaCl2 (250 lM)
and tissue homogenate (2.36 mg of the original brain tissue) and
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lM), NADPH (100 lM)
and -arginine (100 M). UV–vis scans (1 scan every 10 s) were re-
L
l
corded between 390 and 430 nm for a period of 10 min. The metHb
production was estimated by subtracting the absorbance at
411 nm from the absorbance at 401 nm. The IC50 values for the
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this purpose PrismÒ 4.02 was used.
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5.4. Molecular modeling
All computational studies were carried out with Discovery
StudioÒ 1.7 (Accelrys Software Inc., San Diego, CA). The crystal
structure of human MAO-B (PDB code: 2V5Z)32 recovered from
the Brookhaven Protein Database (www.rcsb.org/pdb) was applied
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crystal structure followed wherein the valences of the co-crystal-
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hydrogen atoms added. The receptor protein was then typed by
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adopted basis Newton–Rapheson), wherein the protein backbone
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plicit solvation model was used to account for the effects of the
aqueous environment. Minimisation of the receptor protein was
considered necessary since the protein X-ray structure might con-
tain residual energetic tensions from the crystallisation process.
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the energy-minimised receptor protein and the backbone con-
straint was removed, where after it was used as the starting model
for the docking simulation. The ligands to be docked were first con-
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ing simulations employing the Prepare Ligands application of
Discovery StudioÒ 1.7. The structures were docked into the receptor
model with the LigandFit application, which applied total ligand
flexibility whereby the final ligand conformations were determined
by the Monte Carlo conformation search method set to a variable
number of trial runs. The docked ligand conformations were further
refined using in situ ligand minimisation with the Smart Minimizer
algorithm. The best solution for each docked ligand was adjudged
by the DockScore scoring function of LigandFit. All the application
modules within Discovery StudioÒ 1.7 were set to their default val-
ues and 10 docking solutions were allowed for each ligand.
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Acknowledgement
The authors would like to express their gratitude towards the
National Research Foundation (South Africa) for financial support.
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References and notes
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