Evaluation of 2-thioxo-2,3,5,6,7,8-hexahydropyrimido[4,5-d]pyrimidin-4(1H)-one analogues as GAA activators

Article abstract of DOI:10.1016/j.ejmech.2010.01.027

Pompe disease is a lysosomal storage disease (LSD) caused by a deficiency in the lysosomal enzyme acid alpha-glucosidase. In several LSDs, enzyme inhibitors have been used as small molecule chaperones to facilitate and increase the translocation of mutant protein from the endoplasmic reticulum to the lysosome. Enzyme activators with chaperone activity would be even more desirable as they would not inhibit the enzyme after translocation and might potentiate the activity of the enzyme that is successfully translocated. Herein we report our initial findings of a new series of acid alpha-glucosidase activators.

Full text of DOI:10.1016/j.ejmech.2010.01.027

European Journal of Medicinal Chemistry 45 (2010) 1880–1897
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Evaluation of 2-thioxo-2,3,5,6,7,8-hexahydropyrimido[4,5-d]pyrimidin-4(1H)-one
analogues as GAA activators
,
a
a,
Juan J. Marugan a , Wei Zheng , Omid Motabar b, Noel Southall a, Ehud Goldin b, Ellen Sidransky b,
*
Ronald A. Aungst c, Ke Liu a, Subir Kumar Sadhukhan d, Christopher P. Austin b
a NIH Chemical Genomic Center, National Human Genome Research Institute, National Institutes of Heath, 9800 Medical Center Drive, Rockville, MD, USA
b Medical Genetics Branch, National Human Genome Research Institute, National Institutes of Heath, Bethesda, MD 20892, USA
c Albany Molecular Research, Inc., 26 Corporate Circle, Albany, NY 12203, USA
d Albany Molecular Research Hyderabad Research Center, SP Biotech Park, Turkapally, Shameerpet, R.R. District, Hyderabad 500 078, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Pompe disease is a lysosomal storage disease (LSD) caused by a deficiency in the lysosomal enzyme acid
alpha-glucosidase. In several LSDs, enzyme inhibitors have been used as small molecule chaperones to
facilitate and increase the translocation of mutant protein from the endoplasmic reticulum to the
lysosome. Enzyme activators with chaperone activity would be even more desirable as they would not
inhibit the enzyme after translocation and might potentiate the activity of the enzyme that is success-
fully translocated. Herein we report our initial findings of a new series of acid alpha-glucosidase
activators.
Received 18 December 2009
Received in revised form
13 January 2010
Accepted 14 January 2010
Available online 1 February 2010
Keywords:
Pompe disease
Published by Elsevier Masson SAS.
Lysosomal storage disorder
Acid alpha-glucosidase
Activation of GAA
1. Introduction
proteins accumulate in the endoplasmic reticulum (ER), presum-
ably due to an inability to fold properly or acquire the necessary
Pompe disease is an autosomal recessive disorder caused by the
deficiency or dysfunction of the lysosomal enzyme acid alpha-
glucosidase (GAA [1] EC 3.2.1.20/3). Epidemiological studies have
estimated its frequency to be 1 in every 40,000 births [1,2]. The
shape to be recognized for transport to the lysosome [7–10].
A general LSD strategy has been to search for small molecule
chaperones that are able to bind to mutant enzymes and assist with
the folding and transport process. These molecules have the ability
to improve the trafficking of the mutant protein between the ER
and the lysosome, resulting in lysosome size restoration and
phenotypic correction [11]. Paradoxically, all the small molecule
chaperones reported in the literature are inhibitors of the enzyme,
with the majority being iminosugars. One of these, 1-deoxynojir-
imycin (DNJ), is currently in phase II clinical trials for the treatment
of Pompe disease [12,13]. Iminosugars are problematic due to poor
selectivity and their small therapeutic window between improving
translocation and inhibiting enzyme activity [11,14]. Enzyme acti-
vators with chaperone capacity would be more desirable. This type
of molecule would not only improve the translocation of the
enzyme, but would also potentiate the activity of the lysosomal
protein, having a doubly beneficial effect. To our knowledge, Puli-
carside 1 is the only compound described as being an activator of
GAA [15]. Pulicarside 1 is able to enhance the hydrolysis of
function of GAA is to hydrolyze terminal a-1,4- and a-1,6-glucosidic
linkages of glycogen in the lysosome. Mutations in this enzyme
result in lysosomal enlargement due to glycogen accumulation. The
accumulation is especially severe in cardiac and skeletal muscle,
affecting breathing and mobility [1]. The only FDA treatment
currently approved for this disease is enzyme replacement therapy
(Myozyme), which is recombinant GAA produced in a Chinese
hamster ovary cell line [3]. Although Myozyme has been proven to
be clinically efficacious, the development of infusion related reac-
tions is common, and the majority of the patients (89%) test positive
for IgG antibodies to acid alpha-glucosidase [4]. These findings
reinforce the need to develop new treatments for Pompe disease.
More than 100 different GAA mutations induce Pompe disease
symptoms [5,6]. Many of these mutant proteins retain enzymatic
activity in vitro, but are not transported to the lysosome. These
p-nitrophenyl-
a-glucopyranoside by GAA in a dose-dependent
manner. To date, no data regarding its chaperone activity, or its
capacity to enhance the hydrolysis of glycogen, has been published.
* Corresponding author. Tel.: þ1 301 217 9198; fax: þ1 301 217 5736.
E-mail address: maruganj@mail.nih.gov (J.J. Marugan).
0223-5234/$ – see front matter Published by Elsevier Masson SAS.
doi:10.1016/j.ejmech.2010.01.027

J.J. Marugan et al. / European Journal of Medicinal Chemistry 45 (2010) 1880–1897
1881
a fluorescent emission at 590 nm when excited at 530 nm. In
addition, as a control for autofluorescence, we also used a second
substrate, 4-methylumbelliferyl-a-D-glucopyranoside (4MU-a-glu),
O
O
which liberates the blue dye 4-methylumbelliferyl (4MU) at an
emission wavelength of 440 nm when excited at 370 nm.
O
OH
O
O
2. Results
HO
HO
OH
In
a quantitative high-throughput screen [16] of 199,177
compounds, we found a compound,1-(3,4-dimethoxyphenethyl)-6-
propyl-2-thioxo-2,3,5,6,7,8-hexahydropyrimido[4,5-d]pyrimidin-
4(1H)-one 1, able to activate one and a half fold the hydrolysis of the
Fig. 1. Structure of Pulicarside 1.
A core goal of the NIH Chemical Genomics Center is to use
relevant, high-throughput biological assays to identify and develop
small molecule probes of biomedical interest, especially in the area
of rare and neglected diseases. As part of that effort, we have
developed several new screening methodologies to identify novel
non-iminosugar series with activity in LSD assays. We have focused
testing enzymes in as native a context as possible, including testing
the hydrolytic capacity of GAA in tissue homogenate [16]. Many
isolated glycosidases require allosteric activation to be functional
[17,18], so we wanted to avoid using purified enzyme preparations,
which depend upon the use of detergents to induce the active
conformation and functionality of the enzyme. We have observed
that it is common to find compounds that can inhibit isolated
enzymes but are inactive in cellular lysates. This is likely due to
enzymatic conformational differences between detergent-induced
conformation and cell lysate conformation and non-specific
protein binding. Another limitation of reconstituted assays is an
inability to detect enzyme activators, presumably because the
detergent used in reconstituted assays activates the enzyme in
a non-physiological way. One way to overcome these problems is to
screen the enzyme directly from tissue homogenate using a probe
red dye in tissue homogenate at 10 mM. Figs.1–3 show the activity of
this compound and other analogues in the confirmation assay. In
addition, we measured the activity of the lead compound 1 with the
blue dye and with a purified enzyme preparation. The results show
that the activation of the red dye hydrolysis can also be observed
using purified enzyme. Fig. 4 also shows that compound 1 activates
the hydrolysis of 4MU in a dose-dependent manner, both in purified
enzyme and tissue homogenate. Elimination of the dimethoxy
substituents of the phenethyl functional group reduces the activity
of the scaffold. Moreover, complete elimination of the hexahy-
dropyrimide ring almost erases the activity and its exchange by
a pyrrolopyrimidin core diminishes it.
Compound 1 is not auto-fluorescent under the assay conditions
and the hydrolysis reaction can be monitored using LC–MS. Fig. 5
shows the concentration-dependent increase in product (peak 2).
With these data in hand, we decided to embark on structure–
activity relationship studies. Scheme 1 shows the general method-
ology used for the synthesis of analogues with modifications at
positions 1 and 6 of the lead compound. Thus, primary amine 7 was
refluxed with ammonium thiocyanate in a suitable solvent such us
bromobenzene to yield the corresponding thiourea 8. This
compound was then reacted with ethyl cyanoacetate in the presence
of sodium ethoxide to produce the 2-thioxo-2,3-dihydropyrimidin-
specific for GAA activity, resorufin
a-D-glucopyranoside [16]. Upon
hydrolysis, the red dye resorufin is liberated, producing
Fig. 2. Hydrolytic reactions of the red and blue dyes.

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J.J. Marugan et al. / European Journal of Medicinal Chemistry 45 (2010) 1880–1897
O
S
F
O
N
O
N
F
F
H
N
F
F
O
N
O
N
H
N
F
O
N
HN
N
HN
O
N
HN
HN
N
O
NH
HN
O
O
S
N
H
S
NH2
O
HN
N
O
S
N
H
N
H
O
H
NH2
O
OMe
OMe
OMe
OMe
1
OMe
5
OMe
OMe
4
OMe
3
2
6
Activity= 356.03 Activity= 142.07
Activity= 121.14
Activity= 64.19
Activity= 32.08
Activity= Inactive
Fig. 3. Activity of the lead compound and analogues in the primary screen. The activity here disclosed corresponds with the percentage of signal increase observed after adding
100 M of activator compound to the hydrolysis reaction using resorufin -D-glucopyranoside (red dye) as hydrolytic substrate and spleen homogenate as reaction conditions.
m
a
4(1H)-one derivative 9. Last, a primary amine 10 and formaldehyde
were added to a solution of amine 9 in ethanol and refluxed for 3 h to
produce target 2-thioxo-2,3,5,6,7,8-hexahydropyrimido[4,5-d]pyr-
imidin-4(1H)-one 11.
We also synthesized a number of analogues with modifications
in the molecular core of the molecule. We were especially eager to
test the necessity of the thiocarbonyl functional group within the
template. In addition, we produced a number of compounds with
diverse modifications in the hexahydropyrimide ring with the aim
of testing the possibility of introducing aromaticity and the
necessity of maintaining the hydrogen donor. Schemes 2 and 3
describe their synthesis.
The synthesis of the 2-thioxo-2,3-dihydro-1H-purin-6(9H)-one
14 analogue started with the nitrosilation of the previously
described amine 5, followed by a reduction of intermediate 12 with
sodium dithionite to produce compound 13. Cyclization in the
presence of formamide yields the final purine 14. For the synthesis
of compound 18, intermediate 13 was reacted with 1,4-dioxane-
2,3-diol 17 in ethanol. Last, compound 16 was obtained refluxing
compound 5 with pentane-2,4-dione 15.
Regarding the pyrido[4,3-d]pyrimidine series, triazine and ethyl
acetoacetate reacted in the presence of a sodium ethoxide to yield
the main core 20. This compound was iodinized and dehydrated in
the presence of phosphorus oxychloride to produce intermediate
22. Sonogashira coupling, hydrolysis of the chloride in the presence
of ammonium acetate and acetylene reduction with palladium in
carbon gave the final products.
in a similar fashion than previous compounds replacing the thio-
urea moiety by a urea.
For evaluating activity improvements, both the efficacy
(percentage of activatory response compared to base line) and AC50
(concentration necessary to obtain 50% of maximal activation)
should be analyzed. To generate a proper AC50 the maximum
possible activation must be reached at the highest concentration,
but at the tested concentrations (twelve points between 650 pM
and 100 mM) we were not always able to reach a response plateau.
Due to solubility problems, we did not evaluate the activity at even
higher concentrations, so we have decided to report here the
maximum efficacy reach with the compound as initial evaluation
for tracking SAR. Full concentration–% response curves of every
compound can be found in PubChem.
Tables 1–4 show the SAR of our compounds.
Fig. 7 shows the concentration–% response curves of our best
compounds in several assays. In addition, Fig. 6 disclosed the red
dye-tissue homogenate AC50 obtained with the same compounds
as well as their corresponding log P and total Polar Surface Area
calculated using ChemDraw program. Corresponding efficacy
values can be seen in Tables 2–4.
In order to further evaluate our best activators, we measured
their specific ability to activate the hydrolysis of 4MU-a-glu. Fig. 8
shows how compounds 1, 64, 71, 48, and 49 increase the produc-
tion of 4MU in a dose-dependent manner when they are in the
presence of recombinant human GAA but not in the presence of
alpha-galactosidase A.
Another modification evaluated was the replacement of the
thiocarbonyl functional group by a carbonyl substituent. Scheme 4
shows that the synthesis of this compound, 32, was accomplished
In addition, we evaluated the effect of our lead compounds on
the hydrolysis of the natural substrate glycogen. Fig. 9 shows that
none of our compounds improved the hydrolysis of glycogen.
3. Discussion
O
5
4
6
400
300
200
100
0
The aim of this project was to identify small molecule activators
of GAA, and to characterize their potential to overcome the GAA
deficiency in Pompe disease. Several new assays were developed
for high-throughput screening to address the perceived short-
comings of the traditional purified enzyme assay. First, a red-
shifted fluorogenic substrate was developed to avoid auto-
fluorescent false positives. Second, the protocol was adjusted to
use tissue homogenate instead of purified enzyme with the hope of
assaying enzyme activity in a more relevant context, and to elimi-
nate compounds that might bind proteins non-specifically. These
conditions led to the identification of compound 1. As shown in
Fig. 4, this compound has an extraordinary capacity for activation,
being able to increase hydrolysis rates several times over.
Compound 1 is not auto-fluorescent under the assay conditions and
is able to activate the hydrolysis of both blue and red dyes.
HN
N
blue-purified
blue-spleen
red-purified
red-spleen
3
8
N
H
7
S
N
2
1
O
O
-8
-7
-6
-5
-4
Compound Concentration (Log M)
Compound 1
Fig. 4. Activation of substrate hydrolysis by lead compound using two different
substrates (resorufin -D-
a
-D-glucopyranoside ¼ red dye and 4-methylumbelliferyl-
a
glucopyranoside ¼ blue dye) and two different conditions (spleen homogenated and
purified enzyme).

J.J. Marugan et al. / European Journal of Medicinal Chemistry 45 (2010) 1880–1897
1883
Reaction with no activator
1
mAU
100
50
0
2
0
1
1
1
2
2
2
3
3
3
4
5
5
5
6
7
8 min
Reaction with 5 µM activator
1
mAU
80
40
0
2
4
6
7
8 min
Reaction with 37.5 µM activator
1
mAU
80
2
40
0
3
4
6
7
8 min
Fig. 5. Evaluation of the hydrolysis of 4-methylumbelliferyl-
a-D-glucopyranoside (blue dye) in the presence of several concentrations of compound 1 by direct measurement using
LC–MS. Peak 1: 4MU- -glu, peak 2: 4MU, peak 3: compound 1.
a
Moreover, activation of substrate hydrolysis was confirmed by LC–
MS, demonstrating that compound 1 is able to increase the amount
of 4MU produced in a concentration-dependent manner.
and 7 present the red dye-tissue homogenate AC50 and the titra-
tion curves of our most potent compounds. It can be seen that while
maintaining the high efficacy of our initial lead we were able to
improve over 16 times the AC50 of the series. Our best compound 49
SAR structural changes show that all the core modifications
synthesized failed to further activate hydrolysis of our fluorogenic
substrate and even replacement of the thiocarbonyl functional
group by a carbonyl group diminished activity (Table 1, compounds
32 and 1). Replacement of the pendant aromatic ring at position 1
with various alkyl chains, Table 3, also abolished activity. Several
heteroaromatic rings pendant to position 1 were poorly active or
greatly reduced the activity of the lead compound. Shortening the
linker from two carbons to one, Table 3 compounds 1 and 64,
produces a small increment in efficacy. Moreover, adding one more
methylene unit to the length of the alkyl linker, Table 3 compounds
2 and 65, also increased efficacy. Optimization of the substitution
pattern for the phenethyl group at position 1, Table 2, showed that
electron donating groups in the meta- or para-positions demon-
strated increased efficacy, while chloro substitution provided better
results in the ortho- followed by the meta- position. Similarly,
electron donating di-substituted compounds tend to provide better
efficacy when substituted in the 3,4-positions. In contrast, bis-
halogenated compounds were most efficacious when substituted
at the 2,6- or the 2,3-positions. We also studied the influence of
substituents at position 6. Table 4 shows that propyl 1, isobutyl 70
and benzyl 72 were the most efficacious compounds.
had an AC50 of 1.00
mM when it was tested in red dye-tissue
homogenate, and 355 nM when it was tested in red dye isolate
enzyme conditions. In addition, it can be seen that the most active
molecules have very reasonable calculated log P and tPSA, and in
general increments of log P and reduction of tPSA correlated with
improvements in activity. This activity is GAA-dependent, as shown
in Fig. 8, and enzyme specific because the series does not activate
the hydrolysis of substrates for other glycosidase enzymes, such us
alpha-Galactosidase A or Glucocerebrosidase (data not shown).
Unfortunately, this series’ activity is also substrate dependent.
Although the data demonstrate that the series can activate the
hydrolysis of both red and blue dyes, Fig. 9 shows that these
compounds do not activate the hydrolysis of the natural substrate,
glycogen. We speculate that the conformation adopt by GAA for
cleaving the polymeric natural substrate may be different than the
one adopted in the presence of a small substrate such us resorufin
a-D-glucopyranoside or 4MU-a-glu. Other lysosomal enzymes such
as beta-glucosidase, require the binding of an allosteric modulator
[17]. It is therefore possible that the isolated enzyme assay is not an
adequate model to test for the hydrolysis of glycogen in situ. This
demonstrates the need for the development of assays that would be
able to predict the efficacy and activity of new molecules under
more physiologically relevant conditions. Currently, our group is
Overall, the data shows a quite narrow SAR in which most
modifications yielded diminished activation of hydrolysis. Figs. 6
O
N
O
N
NH2
R2-NH2
R2
OEt
N
NH2
HN
NC
HN
10
NH4SCN
HN
R1
S
O
N
R1
S
NH2
S
PhBr, reflux
H
HCHO, EtOH, reflux
NaOEt, EtOH, reflux
R1
R1
11
7
8
9
Scheme 1. General methodology for the synthesis of analogues at position 1 and 6.

1884
J.J. Marugan et al. / European Journal of Medicinal Chemistry 45 (2010) 1880–1897
O
N
O
N
O
N
O
N
NO
NH2
NH2
N
HN
HN
HN
HN
S
NH2
S
NH2
S
S
N
H
10 % AcOH
NaNO2
NH4OH
Na2S2O4
HCONH2
reflux
OMe
OMe
OMe
OMe
OMe
OMe
12
OMe
13
OMe
14
5
O
O
OH
OH
O
O
EtOH, rt
17
15
O
N
O
N
N
N
HN
HN
S
S
N
OMe
OMe
OMe
OMe
18
16
Scheme 2. Modifications at the molecular core.
working on the development of a new HTS-adaptable assay that is
able to measure the glycogen hydrolysis by tissue homogenate
GAA. In addition, the glycogen-based assay involves the cleavage of
both 1,4 and 1,6 glycosidic bonds, and it is possible that this series
selectively activates only 1,4 bond cleavage and thus this assay’s
results may be misleading. More importantly, we expect this series
to directly interact with the protein, and as such may still possess
chaperone activity; this will require further testing.
4. Conclusions
In conclusion, we have developed the SAR of an initial lead
compound 1 with modifications in all the main areas of the
molecule using a tissue homogenate assay. Activation of hydrolysis
by this compound is enzyme and substrate dependent. It is not
obvious what the reason for this substrate selectivity is but we
speculate that the conformation adopted by GAA for cleaving the
O
I
Cl
I
O
O
CO2Et
H3C
N
N
I2
POCl3
HN
N
N
N
13
HN
N
N
NaOH
NaOEt, EtOH
N
N
N
19
20
21
22
Pd(PPh3)2Cl2
THF
R
O
R
Cl
O
NH4OAc
AcOH
HN
MeOH/CH2Cl2
N
N
N
N
Pd/C
HN
R
N
N
N
R
23: R = Ph
24: R = 3,4-(OMe)2Ph
27: R = Ph
28: R = 3,4-(OMe)2Ph
25: R = Ph
26: R = 3,4-(OMe)2Ph
Scheme 3. Additional template modifications.

J.J. Marugan et al. / European Journal of Medicinal Chemistry 45 (2010) 1880–1897
1885
O
O
N
NH2
Me
N
NH2
HN
HN
HN
O
OEt
NC
N
H
O
N
NH2
O
n-PrNH2
O
Urea
NaOEt, EtOH, reflux
HCHO, EtOH, reflux
H2O, cat. Conc.HCl
OMe
OMe
OMe
OMe
OMe
OMe
OMe
31
OMe
29
30
32
Scheme 4. Synthesis of compound 32.
(continued)
polymeric natural substrate may be different than the one adopted
in the presence of a small substrate such us resorufin
a
-
D
-gluco-
Compound number Identity of R on Table 2 Yield (%) MS ESI, m/z [M þ H]þ
pyranoside or 4MU- -glu. We are in the process of testing the
a
8l
2,6-Dimethylbenzyl
2,3-Dimethylbenzyl
3,4-Dimethylbenzyl
2,4-Dimethylbenzyl
2,3-Dichlorobenzyl
3,5-Dimethylbenzyl
2-Chlorobenzyl
3-Chlorobenzyl
4-Chlorobenzyl
2-Methylbenzyl
3-Methylbenzyl
4-Methylbenzyl
2-Methoxybenzyl
3-Methoxybenzyl
4-Methoxybenzyl
Methyl
18
22
29
36
30
25
17
22
17
20
20
20
21
20
17
20
21
29
20
24
Crude
209
209
209
209
250
209
215
215
215
195
195
195
211
211
211
119
147
161
195
187
182
8m
8n
8o
8p
8q
8r
8s
8t
8u
8v
8w
8x
chaperone capacity of our molecules [28,29]. Independently of the
inhibitory or activatory activity of a series, improving the binding
might result in an increment in the capacity of the molecule to
stabilize folding. Further results of that work will be presented in
a separate paper.
5. Experimental section
5.1. Chemistry
8y
8z
The reagents and solvents were used as commercial anhydrous
grade without further purification. The column chromatography
was carried out over silica gel (100–200 mesh). 1H NMR spectra
were recorded with a Bruker 400 MHz spectrometer from solutions
in CDCl3 and DMSO-d6. Chemical shifts in 1H NMR spectra are
8aa
8ab
8ac
8ad
8ae
8af
Isopropyl
t-Butyl
Phenethyl
2-Thiophene
reported in parts per million (ppm,
standard Me4Si (TMS,
was performed using an Agilent Time-Of-Flight Mass Spectrometer
(TOF, Agilent Technologies, Santa Clara, CA). A 3 min gradient from
4 to 100% acetonitrile (0.1% formic acid) in water (0.1% formic acid)
was used with a 4 min run time at a flow rate of 1 mL/min. A Zorbax
d) downfield from the internal
¼ 0 ppm). Molecular weight confirmation
2-Pyridyl
d
5.1.2. General procedure for the preparation of 2-thioxo-2,3-
dihydropyrimidin-4(1H)-one derivative 9 [20]
Thiourea8 (2.08 mmol) andethylcyanoacetate(2.28 mmol) were
added to a solution of sodium (2.08 mmol) in ethanol (5 mL). The
reaction mixture was heated at reflux for 4 h. The reaction mixture
was cooled to room temperature and concentrated under reduced
pressure. The crude residue was purified by silica-gel column
chromatography (1–3% MeOH in CH2Cl2) to afford pyrimidinone 9.
The products were characterized by 1H NMR and MS analysis.
SB-C18 column (3.5
m
m, 2.1 ꢀ 30 mm) was used at a temperature of
50 ꢁC. Confirmation of molecular formula was confirmed using
electrospray ionization in the positive mode with the Agilent
Masshunter software (version B.02).
5.1.1. General procedure for the preparation of thiourea 8 [19]
Ammonium thiocyanate (27.58 mmol) was added to a stirred
solution of amine 7 (27.58 mmol) in bromobenzene (10 mL) at room
temperature. The reaction mixturewas heated at reflux temperature
for 6 h. The reaction mixture was cooled to room temperature and
concentrated under reduced pressure. The crude residue was puri-
fied by silica-gel column chromatography (2–10% MeOH in CH2Cl2)
to afford thiourea 8. The products were characterized by MS analysis
and subjected to the next step without further purification.
5.1.2.1. Compound 5 (R ¼ 3,4-di(OMe)benzyl). Yield ¼ 31%; 1H NMR
(400 MHz, DMSO-d6)
d 11.86 (s, 1H, NH), 7.06 (s, 2H, Ar–H), 6.97
(s, 1H, Ar–H), 6.88–6.82 (m, 2H, NH2), 4.85 (s, 1H, C(5)-H), 4.48 (br s,
2H, N–CH2), 3.73 (s, 3H, OCH3), 3.70 (s, 3H, OCH3), 2.84 (t, J ¼ 8 Hz, 2H,
N–CH2–CH2); HRMS (ESI) m/z calculated for [C14H17N3O3S þ H]þ
307.0991, found 307.0993.
5.1.2.2. Compound 9b (R ¼ 2,3-di(OMe)benzyl). Yield ¼ 30%; 1H
NMR (400 MHz, DMSO-d6)
d
11.83 (s, 1H, NH), 7.01 (t, J ¼ 8.0 Hz, 1H,
Ar–H), 6.99–6.85 (m, 4H, Ar–H and NH2), 4.89 (s, 1H, C(5)-H), 4.54
(br s, 2H, N–CH2), 3.78 (s, 3H, OCH3), 3.73 (s, 3H, OCH3), 2.91
(t, J ¼ 8.0 Hz, 2H, N–CH2–CH2); MS (ESI) m/z 308 [C14H17N3O3S þ H]þ.
Compound number Identity of R on Table 2 Yield (%) MS ESI, m/z [M þ H]þ
8a
8b
8c
8d
8e
8f
8g
8h
8i
3,4-Di(OMe)benzyl
2,3-Di(OMe)benzyl
2,4-Di(OMe)benzyl
2,5-Di(OMe)benzyl
2,6-Di(OMe)benzyl
3,5-Di(OMe)benzyl
3,4-Di(OEt)benzyl
33
26
23
20
21
24
22
241
241
241
241
241
241
269
238
248
248
248
5.1.2.3. Compound 9c (R ¼ 2,4-di(OMe)benzyl). Yield ¼ 30%; 1H
NMR (400 MHz, DMSO-d6)
d
11.83 (s, 1H, NH), 7.01 (t, J ¼ 8.0 Hz, 1H,
Ar–H), 6.99–6.85 (m, 4H, Ar–H and NH2), 4.89 (s, 1H, C(5)-H), 4.54
(br s, 2H, N–CH2), 3.78 (s, 3H, OCH3), 3.73 (s, 1H, OCH3), 2.91 (t,
J ¼ 8.0 Hz, 2H, N–CH2–CH2); MS (ESI) m/z 308 [C14H17N3O3S þ H]þ.
3,4-(OCH2CH2O)benzyl 20
2,4-Dichlorobenzyl
3,4-Dichlorobenzyl
2,6-Dichlorobenzyl
20
23
23
8j
8k
5.1.2.4. Compound 9d (R ¼ 2,5-di(OMe)benzyl). Yield ¼ 32%; 1H
(continued on next page)
NMR (400 MHz, DMSO-d6)
d
11.83 (s, 1H, NH), 7.01 (t, J ¼ 8.0 Hz,

Table 1
Analogues with modifications at the core template.
Compound number
Core structure
R1
Maximum response
Inactive
Compound number
Core structure
R1
Maximum response
Inactive
O
O
HN
HN
N
N
N
N
OMe
OMe
inactive
inactive
inactive
inactive
9a
25
S
N
NH2
N
N
N
N
R1
R1
O
O
N
OMe
OMe
HN
HN
OMe
OMe
14
16
18
23
24
Inactive
Inactive
Inactive
Inactive
Inactive
26
27
28
32
1
Inactive
Inactive
Inactive
297.37
365.03
S
N
N
H
R1
R1
O
O
HN
HN
OMe
OMe
S
N
N
R1
R1
O
O
N
N
HN
HN
OMe
OMe
OMe
OMe
inactive
inactive
inactive
S
N
R1
R1
Cl
O
Me
N
N
N
HN
OMe
OMe
N
N
N
N
O
H
R1
R
Cl
O
Me
OMe
OMe
N
N
N
HN
OMe
OMe
1
N
N
S
H
R1
R1

Table 2
Analogues with modifications at the aromatic substituent.
O
N
HN
N
S
N
H
R1
Compound
number
R1
Maximum
response
Compound
number
R1
Maximum
response
Compound
number
R1
Maximum
response
Cl
Cl
33
135.96
43
300.10
53
315.83
MeO
34
35
36
295.22
299.89
310.68
44
45
46
244.98
322.93
337.60
54
55
56
226.60
262.27
327.26
MeO
MeO
OMe
OMe
262.27
MeO
MeO
Cl
Cl
37
38
197.44
120.29
47
48
218.24
332.48
57
OMe
308.06
356.03
Cl
OMe
OMe
Cl
1
Cl
Cl
OMe
OMe
Cl
MeO
39
40
242.70
301.87
49
50
345.62
325.11
58
331.78
142.07
Cl
Cl
OMe
2
Cl
O
OMe
41
42
308.72
112.26
51
52
Cl 321.42
Cl 88.81
59
60
O
328.21
305.52
OEt
OEt
Cl

1888
J.J. Marugan et al. / European Journal of Medicinal Chemistry 45 (2010) 1880–1897
Table 3
Table 4
Additional analogues with modifications in the functional group at position 1.
Analogues with modifications in the functional group at position 6.
O
O
R1
HN
N
HN
N
S
N
N
H
S
N
R1
N
H
Compound number R1
Maximum response
356.03
MeO
OMe
OMe
OMe
inactive
1
Compound number
R1
Maximum response
356.03
1
N
61
62
30.59
69
70
282.39
338.17
S
192.02
S
63
64
221.14
365.30
71
72
233.61
334.61
OMe
OMe
73
274.75
OMe
OMe
2
142.07
5.1.2.5. Compound 9e (R ¼ 2,6-di(OMe)benzyl). Yield ¼ 34%; 1H
NMR (400 MHz, DMSO-d6)
d
11.68 (s, 1H, NH), 7.16 (d, J ¼ 8.4 Hz, 1H,
Ar–H), 6.74 (s, 2H, NH2), 6.60 (d, J ¼ 8.4 Hz, 2H, Ar–H), 4.85 (s, 1H,
C(5)-H), 4.47 (br s, 2H, N–CH2), 3.73 (s, 6H, OCH3), 2.93 (br s, 2H,
N–CH2–CH2); MS (ESI) m/z 308 [C14H17N3O3S þ H]þ.
65
66
165.72
5.1.2.6. Compound 9f (R ¼ 3,5-di(OMe)benzyl). Yield ¼ 39%; 1H
NMR (400 MHz, DMSO-d6) d 11.88 (s,1H, NH), 7.07 (s, 2H, NH2), 6.53
(d, J ¼ 2.4 Hz, 2H, Ar–H), 6.35 (t, J ¼ 2 Hz, 1H, Ar–H), 4.86 (s, 1H,
C(5)-H), 4.55 (br s, 2H, N–CH2), 3.71 (s, 6H, OCH3), 2.83 (t, J ¼ 8 Hz,
2H, N–CH2–CH2); MS (ESI) m/z 308 [C14H17N3O3S þ H]þ.
Inactive
5.1.2.7. Compound 9g (R ¼ 3,4-di(OEt)benzyl). Yield ¼ 27%; 1H NMR
(400 MHz, DMSO-d6)
d 11.85 (s, 1H, NH), 7.05 (s, 2H, NH2), 6.97
(s, 1H, Ar–H), 6.86 (d, J ¼ 8.0 Hz, 1H, Ar–H), 6.81 (dd, J ¼ 8.0, 1.2 Hz,
1H, Ar–H), 4.85 (s, 1H, C(5)-H), 4.50 (br s, 2H, N–CH2), 4.01–3.93
(m, 4H, O–CH2), 2.82 (t, J ¼ 8.0 Hz, 2H, N–CH2–CH2), 1.33–1.27
(m, 6H, O–CH2–CH3); MS (ESI) m/z 336 [C16H21N3O3S þ H]þ.
67
68
Inactive
Inactive
5.1.2.8. Compound 9h (R ¼ 3,4-(OCH2CH2O)benzyl). Yield ¼ 42%; 1H
NMR (400 MHz, DMSO-d6) d 11.86 (s,1H, NH), 7.07 (s, 2H, NH2), 6.88
(s, 1H, Ar–H), 6.81–6.76 (m, 2H, Ar–H), 4.85 (s, 1H, C(5)-H), 4.38 (br
s, 2H, N–CH2), 4.19 (s, 4H, O–CH2CH2–O), 2.77 (t, J ¼ 8.4 Hz, 2H,
N–CH2–CH2); MS (ESI) m/z 306 [C14H15N3O3S þ H]þ.
1H, Ar–H), 6.99–6.85 (m, 4H, Ar–H and NH2), 4.89 (s, 1H), 4.54 (br
s, 2H, N–CH2), 3.78 (s, 3H, OCH3), 3.73 (s, 3H, OCH3), 2.91 (t,
J
¼
8.0 Hz, 2H, N–CH2–CH2); MS (ESI) m/z 308
5.1.2.9. Compound 9i (R ¼ 2,4-dichlorobenzyl). Yield ¼ 32%; 1H
[C14H17N3O3S þ H]þ.
NMR (400 MHz, DMSO-d6) d 11.78 (s, 1H, NH), 7.55 (s, 1H, Ar–H),

J.J. Marugan et al. / European Journal of Medicinal Chemistry 45 (2010) 1880–1897
1889
O
N
O
N
O
N
O
N
O
N
HN
N
HN
N
HN
N
HN
N
HN
N
S
N
H
S
N
H
S
N
H
S
N
H
S
N
H
Cl
Cl
Cl
Cl
O
O
O
O
O
O
70
48
49
64
1
Log P: 3.2
Log P: 4.17
tPSA: 47.61
Log P: 4.17
tPSA: 47.61
Log P: 2.52
Log P: 2.8
tPSA: 66.07
AC50= 7.08 uM
tPSA: 66.07
AC50= 6.31 uM
tPSA: 66.07
AC50= 16.79 uM
AC50= 5.01 uM
AC50= 1.00 uM
Fig. 6. Structure of best activators.
7.38 (s, 2H, Ar–H), 6.97 (s, 2H, NH2), 4.90 (s, 1H, C(5)-H), 4.87–4.66
(m, 2H, N–CH2), 3.03 (t, J ¼ 7.6 Hz, 2H, N–CH2–CH2); MS (ESI) m/z
316 [C12H11Cl2N3OS þ H]þ.
Ar–H), 6.98–6.92 (m, 4H, Ar–H and NH2), 4.89 (s, 1H, C(5)-H), 4.49
(br s, 2H, N–CH2), 2.83 (t, J ¼ 8.0 Hz, 2H, N–CH2–CH2), 2.30 (s, 3H,
CH3), 2.21 (s, 3H, CH3); MS (ESI) m/z 276 [C14H17N3OS þ H]þ.
5.1.2.10. Compound 9j (R ¼ 3,4-dichlorobenzyl). Yield ¼ 39%; 1H
5.1.2.14. Compound 9n (R ¼ 3,4-dimethylbenzyl). Yield ¼ 30%; 1H
NMR (400 MHz, DMSO-d6)
d
11.90 (s,1H, NH), 7.64 (s,1H, Ar–H), 7.58
NMR (400 MHz, DMSO-d6) d 11.84 (s, 1H, NH), 7.12–7.05 (m, 5H,
(d, J ¼ 8.0 Hz, 1H, Ar–H), 7.33 (d, J ¼ 8.0 Hz, 1H, Ar–H), 7.12 (s, 2H,
NH2), 4.85 (s, 1H, C(5)-H), 4.47 (br s, 2H, N–CH2), 2.92 (t, J ¼ 8.0 Hz,
2H, N–CH2–CH2); MS (ESI) m/z 316 [C12H11Cl2N3OS þ H]þ.
Ar–H and NH2), 4.87 (s, 1H, C(5)-H), 4.47 (br s, 2H, N–CH2), 2.83
(t, J ¼ 8 Hz, 2H, N–CH2–CH2), 2.18 (s, 3H, CH3), 2.17 (s, 3H, CH3); MS
(ESI) m/z 276 [C14H17N3OS þ H]þ.
5.1.2.11. Compound 9k (R ¼ 2,6-dichlorobenzyl). Yield ¼ 32%; 1H
5.1.2.15. Compound 9o (R ¼ 2,4-dimethylbenzyl). Yield ¼ 45%; 1H
NMR (400 MHz, DMSO-d6)
d
11.90 (s, 1H, NH), 7.64 (s, 1H, Ar–H),
NMR (400 MHz, DMSO-d6)
d
11.86 (s, 1H, NH), 7.16 (d, J ¼ 7.6 Hz, 1H,
7.58 (d, J ¼ 8.0 Hz, 1H, Ar–H), 7.33 (d, J ¼ 8.0 Hz, 1H, Ar–H), 7.12
Ar–H), 6.98–6.92 (m, 4H, Ar–H and NH2), 4.89 (s, 1H, C(5)-H), 4.49
(br s, 2H, N–CH2), 2.83 (t, J ¼ 8.0 Hz, 2H, N–CH2–CH2), 2.30 (s, 3H,
CH3), 2.21 (s, 3H, CH3); MS (ESI) m/z 276 [C14H17N3OS þ H]þ.
(s, 2H, NH2), 4.85 (s, 1H, C(5)-H), 4.47 (br s, 2H, N–CH2), 2.92
(t,
J
¼
8.0 Hz, 2H, N–CH2–CH2); MS (ESI) m/z 316
[C12H11Cl2N3OS þ H]þ.
5.1.2.16. Compound 9p (R ¼ 2,3-dichlorobenzyl). Yield ¼ 29%; 1H
5.1.2.12. Compound 9l (R ¼ 2,6-dimethylbenzyl). Yield ¼ 30%; 1H
NMR (400 MHz, DMSO-d6)
1H, Ar–H), 7.52–7.50 (m, 1H, Ar–H), 7.32–7.28 (m, 1H, Ar–H), 6.99
(s, 2H, NH2), 4.88 (s, 1H, C(5)-H), 4.22–4.19 (m, 2H, N–CH2), 3.10
d
11.80 (s, 1H, NH), 7.67 (d, J ¼ 11.2 Hz,
NMR (400 MHz, DMSO-d6)
d
11.91 (s, 1H, NH), 6.99–6.98 (m, 3H,
Ar–H and NH2), 6.82 (s, 2H, Ar–H), 4.94 (s, 1H, C(5)-H), 4.47 (br s,
2H, N–CH2), 2.92 (t, J ¼ 7.2 Hz, 2H, N–CH2–CH2), 2.38 (s, 6H, CH3);
MS (ESI) m/z 276 [C14H17N3OS þ H]þ.
(t,
J
¼
7.6 Hz, 2H, N–CH2–CH2); MS (ESI) m/z 316
[C12H11Cl2N3OS þ H]þ.
5.1.2.13. Compound 9m (R ¼ 2,3-dimethylbenzyl). Yield ¼ 32%; 1H
5.1.2.17. Compound 9q (R ¼ 3,5-dimethylbenzyl). Yield ¼ 50%; 1H
NMR (400 MHz, DMSO-d6)
d
11.86 (s, 1H, NH), 7.16 (d, J ¼ 7.6 Hz, 1H,
NMR (400 MHz, DMSO-d6) d 11.83 (s, 1H, NH), 7.03 (s, 2H, Ar–H),
400
400
300
200
100
0
a
b
1
48
49
71
1
48
49
71
64
300
200
64
100
0
-8
-7
-6
-5
-4
-8
-7
-6
-5
-4
Concentration (log [M])
Concentration (log [M])
400
300
200
100
0
c
d
1
400
300
200
100
0
1
48
49
71
64
48
49
71
64
-8
-7
-6
-5
-4
-8
-7
-6
-5
-4
Concentration (log [M])
Concentration (log [M])
Fig. 7. Titration curves of best activators. a. Red purified enzyme; b. blue purified enzyme; c. red spleen; d. blue spleen.

1890
J.J. Marugan et al. / European Journal of Medicinal Chemistry 45 (2010) 1880–1897
a
1
20000
15000
10000
5000
0
64
70
48
49
0
2
6 25100
0
2
6 25100
0
2
6 25100
0
2
6 25100
0 2 6 25100
Compound, uM
50
40
30
20
10
b
1
64
70
48
49
0
0
2
6 25100
0
2
6 25100
0
2
6 25100
0
2
6 25100
0 2 6 25100
Compound, uM
c
1
20000
15000
10000
5000
0
64
70
48
49
0
2
6 25100
0
2
6 25100
0
2
6 25100
0
2
6 25100
0 2 6 25100
Compound, uM
Fig. 8. a. Concentration-dependent effect of most active compounds on the hydrolysis of 4-methylumbelliferyl-
b. Concentration-dependent effect of most active compounds on the hydrolysis of 4-methylumbelliferyl- -D-glucopyranoside (4MU-
c. Concentration-dependent effect of most active compounds on the hydrolysis of 4-methylumbelliferyl- -D-galactosidase (4MU- -gal) in the presence of purified alpha-galactosidase A.
a-D-glucopyranoside (4MU-a-glu) in the presence of purified GAA.
a
a-glu) in the absence of purified GAA.
a
a

J.J. Marugan et al. / European Journal of Medicinal Chemistry 45 (2010) 1880–1897
1891
8000
7000
6000
5000
4000
3000
2000
1000
0
1
64
70
48
49
0
2
6
25 100
0
2
6
25 100
0
2
6
25 100
0
2
6
25 100
0
2
6 25 100
No
No
6 uM
NB-DNJ
Cpd Enzyme
Compound, uM
Fig. 9. Effect of lead compounds on rhGAA using glycogen as the substrate.
6.96 (s, 2H, NH2), 6.85 (s, 1H, Ar–H), 4.88 (s, 1H, C(5)-H), 4.47 (br s,
2H, N–CH2), 2.83 (t, J ¼ 8.0 Hz, 2H, N–CH2–CH2), 2.24 (s, 6H, CH3);
MS (ESI) m/z 276 [C14H17N3OS þ H]þ.
Ar–H), 7.05 (s, 2H, NH2), 6.95–6.91 (m, 2H, Ar–H), 6.79 (d, J ¼ 8.0 Hz,
1H, Ar–H), 4.87 (s, 1H, C(5)-H), 4.50 (br s, 2H, N–CH2), 3.73 (s, 3H,
OCH3), 2.89 (t, J ¼ 8.0 Hz, 2H, N–CH2–CH2); MS (ESI) m/z 277
[C13H15N3O2S þ H]þ.
5.1.2.18. Compound 9r (R ¼ 2-chlorobenzyl). Yield ¼ 43%; 1H NMR
(400 MHz, DMSO-d6)
d
11.87 (s, 1H, NH), 7.37 (s, 4H, Ar–H), 7.09 (s,
5.1.2.26. Compound 9z (R ¼ 4-methoxybenzyl). Yield ¼ 21%; 1H
2H, NH2), 4.86 (s, 1H, C(5)-H), 4.49 (br s, 2H, N–CH2), 2.90 (t,
NMR (400 MHz, DMSO-d6)
d
11.84 (s, 1H, NH), 7.22 (t, J ¼ 8.0 Hz, 1H,
J ¼ 8 Hz, 2H, N–CH2–CH2); MS (ESI) m/z 282 [C12H12ClN3OS þ H]þ.
Ar–H), 7.05 (s, 2H, NH2), 6.95–6.91 (m, 2H, Ar–H), 6.79 (d, J ¼ 8.0 Hz,
1H, Ar–H), 4.87 (s, 1H, C(5)-H), 4.50 (br s, 2H, N–CH2), 3.73 (s, 3H,
OCH3), 2.89 (t, J ¼ 8.0 Hz, 2H, N–CH2–CH2); MS (ESI) m/z 277
[C13H15N3O2S þ H]þ.
5.1.2.19. Compound 9s (R ¼ 3-chlorobenzyl). Yield ¼ 60%; 1H NMR
(400 MHz, DMSO-d6)
d 11.87 (s, 1H, NH), 7.37 (s, 4H, Ar–H), 7.09 (s,
2H, NH2), 4.86 (s, 1H, C(5)-H), 4.49 (br s, 2H, N–CH2), 2.90 (t,
J ¼ 8 Hz, 2H, N–CH2–CH2); MS (ESI) m/z 282 [C12H12ClN3OS þ H]þ.
5.1.2.27. Compound 9aa (R ¼ methyl). Yield ¼ 45%; 1H NMR
(400 MHz, DMSO-d6)
d 11.78 (s, 1H, NH), 7.02 (s, 2H, NH2), 4.81 (s,
5.1.2.20. Compound 9t (R ¼ 4-chlorobenzyl). Yield ¼ 25%; 1H NMR
1H, C(5)-H), 4.15 (br s, 2H, N–CH2), 1.65 (t, J ¼ 8.0 Hz, 2H, N–CH2–
(400 MHz, DMSO-d6)
d
11.87 (s, 1H, NH), 7.37 (s, 4H, Ar–H), 7.09 (s,
CH2), 0.85 (t,
J
¼
8.0 Hz, 3H, CH3); MS (ESI) m/z 214
2H, NH2), 4.86 (s, 1H, C(5)-H), 4.49 (br s, 2H, N–CH2), 2.90 (t,
[C9H15N3OS þ H]þ.
J ¼ 8 Hz, 2H, N–CH2–CH2); MS (ESI) m/z 282 [C12H12ClN3OS þ H]þ.
5.1.2.28. Compound 9ab (R ¼ isopropyl). Yield ¼ 48%; 1H NMR
5.1.2.21. Compound 9u (R ¼ 2-methylbenzyl). Yield ¼ 28%; 1H NMR
(400 MHz, DMSO-d6) d 11.78 (s, 1H, NH), 6.79 (s, 2H, NH2), 4.88 (s,
(400 MHz, DMSO-d6)
d
11.87 (s, 1H, NH), 7.29–6.99 (m, 6H, Ar–H
1H, C(5)-H), 4.39 (br s, 2H, N–CH2), 1.68–1.61 (m,1H, CH(CH3)2) 1.49
(t, J ¼ 7.6 Hz, 2H, N–CH2–CH2), 0.94–0.91 (m, 6H, CH(CH3)2); MS
(ESI) m/z 214 [C9H15N3OS þ H]þ.
and NH2), 4.90 (s, 1H, C(5)-H), 4.49 (br s, 2H, N–CH2), 2.91–2.87
(m, 2H, N–CH2–CH2), 2.35 (s, 3H); MS (ESI) m/z 262
[C13H15N3OS þ H]þ.
5.1.2.29. Compound 9ac (R ¼ t-butyl). Yield ¼ 29%; 1H NMR
5.1.2.22. Compound 9v (R ¼ 3-methylbenzyl). Yield ¼ 29%; 1H NMR
(400 MHz, DMSO-d6) d 11.78 (s, 1H, NH), 6.79 (s, 2H, NH2), 4.88 (s,
(400 MHz, DMSO-d6)
d
11.87 (s, 1H, NH), 7.29–6.99 (m, 6H, Ar–H
1H, C(5)-H), 4.39 (br s, 2H, N–CH2), 1.49 (t, J ¼ 7.6 Hz, 2H, N–CH2–
and NH2), 4.90 (s, 1H, C(5)-H), 4.49 (br s, 2H, N–CH2), 2.91–2.87
(m, 2H, N–CH2–CH2), 2.35 (s, 3H); MS (ESI) m/z 262
[C13H15N3OS þ H]þ.
CH2), 0.93 (s, 9H, C(CH3)3); MS (ESI) m/z 228 [C10H17N3OS þ H]þ.
5.1.2.30. Compound 9ad (R ¼ phenethyl). Yield ¼ 33%; 1H NMR
(400 MHz, DMSO-d6)
d 11.78 (s, 1H, NH), 7.28–7.14 (m, 5H, Ar–H),
5.1.2.23. Compound 9w (R ¼ 4-methylbenzyl). Yield ¼ 30%; 1H NMR
7.05 (s, 2H, NH2), 4.83 (s, 1H, C(5)-H), 4.33–4.32 (m, 2H, N–CH2),
2.62 (t, J ¼ 8 Hz, 2H, C6H5–CH2), 1.90 (t, J ¼ 8 Hz, 2H, N–CH2–CH2);
MS (ESI) m/z 262 [C13H15N3OS þ H]þ.
(400 MHz, DMSO-d6)
d
11.87 (s, 1H, NH), 7.29 (d, J ¼ 8.0 Hz, 2H,
Ar–H), 7.13 (t, J ¼ 8.0 Hz, 2H, Ar–H), 6.99 (s, 2H, NH2), 4.90 (s, 1H,
C(5)-H), 4.51 (br s, 2H, N–CH2), 2.91 (t, J ¼ 8.0 Hz, 2H, N–CH2–CH2),
2.35 (s, 3H); MS (ESI) m/z 261 [C13H15N3OS þ H]þ.
5.1.2.31. Compound 9ae (R ¼ 2-thiophene). Yield ¼ 36%; 1H NMR
(400 MHz, DMSO-d6)
d 11.87 (s, 1H, NH), 7.70–7.64 (m, 1H, Ar–H),
5.1.2.24. Compound 9x (R ¼ 2-methoxybenzyl). Yield ¼ 80%; 1H
7.08 (s, 2H, NH2), 6.98–6.95 (m, 2H, Ar–H), 4.86 (s, 1H, C(5)-H), 4.85
(br s, 2H, N–CH2), 3.13 (t, J ¼ 8 Hz, 2H, N–CH2–CH2); MS (ESI) m/z
254 [C10H11N3OS2 þ H]þ.
NMR (400 MHz, DMSO-d6)
d
11.84 (s, 1H, NH), 7.22 (t, J ¼ 8.0 Hz, 1H,
Ar–H), 7.05 (s, 2H, NH2), 6.95–6.91 (m, 2H, Ar–H), 6.79 (d, J ¼ 8.0 Hz,
1H, Ar–H), 4.87 (s, 1H, C(5)-H), 4.50 (br s, 2H, N–CH2), 3.73 (s, 3H,
OCH3), 2.89 (t, J ¼ 8.0 Hz, 2H, N–CH2–CH2); MS (ESI) m/z 277
[C13H15N3O2S þ H]þ.
5.1.2.32. Compound 9af (R ¼ 2-pyridyl). Yield ¼ 32%; 1H NMR
(400 MHz, DMSO-d6)
d
11.84 (s, 1H, NH), 8.49 (d, J ¼ 4.4 Hz, 1H),
7.79–7.70 (m, 1H, Ar–H), 7.35–7.23 (m, 2H, Ar–H), 7.11 (s, 2H, NH2),
4.87 (s, 1H, C(5)-H), 4.72 (br s, 2H, N–CH2), 3.12 (t, J ¼ 8 Hz, 2H,
N–CH2–CH2); MS (ESI) m/z 249 [C11H12N4OS þ H]þ.
5.1.2.25. Compound 9y (R ¼ 3-methoxybenzyl). Yield ¼ 59%; 1H
NMR (400 MHz, DMSO-d6)
d
11.84 (s, 1H, NH), 7.22 (t, J ¼ 8.0 Hz, 1H,

1892
J.J. Marugan et al. / European Journal of Medicinal Chemistry 45 (2010) 1880–1897
5.1.3. General procedure for the preparation of 10 [21–23]
n-Propylamine (4.55 mmol) and formaldehyde (6.50 mmol)
were added to a solution of amine 9 (3.25 mmol) in ethanol
(20 mL). The reaction mixture was heated at reflux for 3 h, cooled to
room temperature, and filtered through a sintered glass funnel to
afford amine 10. The compound was characterized by 1H NMR and
MS analysis, and HRMS.
2.34 (t, J ¼ 7.2 Hz, 2H, CH2CH2CH3), 1.44 (q, J ¼ 7.2 Hz, 2H,
CH2CH2CH3), 0.87 (t, J ¼ 7.2 Hz, 3H, CH2CH2CH3); HRMS (ESI) m/z
calculated for [C17H21ClN4OS þ H]þ 364.1125, found 364.1128.
5.1.3.8. Compound 38 (R ¼ 4-chlorobenzyl). Yield ¼ 40%; 1H NMR
(400 MHz, DMSO-d6)
d 12.00 (s, 1H, N(3)-H), 7.42–7.34 (m, 5H,
N(8)-H and Ar–H), 4.46 (br s, 2H, N–CH2), 3.98 (s, 2H, C(7)-H2), 3.37
(s, 2H, C(5)-H2), 2.91 (t, J ¼ 8.00 Hz, 2H, N–CH2–CH2), 2.34
(t, J ¼ 7.2 Hz, 2H, CH2CH2CH3), 1.45 (q, J ¼ 7.2 Hz, 2H, CH2CH2CH3),
0.87 (t, J ¼ 7.2 Hz, 3H, CH2CH2CH3); HRMS (ESI) m/z calculated for
[C17H21ClN4OS þ H]þ 364.1125, found 364.1130.
5.1.3.1. Compound 1 (R ¼ 3,4-di(OMe)benzyl). Yield ¼ 51%; 1H NMR
(400 MHz, DMSO-d6)
d 12.00 (s, 1H, N(3)-H), 7.37 (s, 1H, N(8)-H),
6.92–6.81 (m, 3H, Ar–H), 4.47 (br s, 2H, N–CH2), 3.98 (s, 2H, C(7)-
H2), 3.73 (s, 3H, OCH3), 3.70 (s, 3H, OCH3), 3.36 (s, 2H, C(5)-H2), 2.85
(t, J ¼ 8 Hz, 2H, N–CH2–CH2), 2.33 (t, J ¼ 7.2 Hz, 2H, CH2CH2CH3),
1.44 (q, J ¼ 7.2 Hz, 2H, CH2CH2CH3), 0.86 (t, J ¼ 7.2 Hz, 3H,
CH2CH2CH3); HRMS (ESI) m/z calculated for [C19H26N4O3S þ H]þ
390.1726, found 390.1733.
5.1.3.9. Compound 39 (R ¼ 2-methoxybenzyl). Yield ¼ 35%; 1H NMR
(400 MHz, DMSO-d6) d 11.91 (s,1H, N(3)-H), 7.22–7.14 (m, 2H, N(8)-H
and Ar–H), 7.07 (s,1H, Ar–H), 6.94–6.85 (m, 2H, Ar–H), 4.57 (br s, 2H,
N–CH2), 3.94 (s, 2H, C(7)-H2), 3.75 (s, 3H, OCH3), 3.36 (s, 2H, C(5)-H2),
2.90 (t, J ¼ 7.2 Hz, 2H, N–CH2–CH2), 2.32 (t, J ¼ 7.2 Hz, 2H,
CH2CH2CH3), 1.44 (q, J ¼ 7.2 Hz, 2H, CH2CH2CH3), 0.86 (t, J ¼ 7.2 Hz,
3H, CH2CH2CH3); HRMS (ESI) m/z calculated for [C18H24N4O2S þ H]þ
360.1620, found 360.1623.
5.1.3.2. Compound 2 (R ¼ 3,5-dimethylbenzyl). Yield ¼ 50%; 1H
NMR (400 MHz, DMSO-d6) d 12.02 (s, 1H, N(3)-H), 7.40 (s, 1H, N(8)-
H), 7.10 (s, 1H, Ar–H), 7.06 (s, 2H, Ar–H), 4.44 (br s, 2H, N–CH2), 3.98
(s, 2H, C(7)-H2), 3.37 (s, 2H, C(5)-H2), 2.82 (t, J ¼ 8 Hz, 2H, N–CH2–
CH2), 2.34 (t, J ¼ 7.2 Hz, 2H, CH2CH2CH3), 2.19 (s, 3H, Ar–CH3), 2.17
(s, 3H, Ar–CH3), 1.50–1.41 (m, 2H, CH2CH2CH3), 0.86 (t, J ¼ 7.2 Hz,
3H, CH2CH2CH3) HRMS (ESI) m/z calculated for 330.1514
[C19H26N4OS þ H]þ, found 330.1515.
5.1.3.10. Compound 40 (R ¼ 3-methoxybenzyl). Yield ¼ 57%; 1H
NMR (400 MHz, DMSO-d6)
d 12.03 (s, 1H, N(3)-H), 7.41 (s, 1H,
N(8)-H), 7.22 (t, J ¼ 7.6 Hz, 1H, Ar–H), 6.91 (s, 2H, Ar–H), 6.80
(d, J ¼ 7.6 Hz, 1H, Ar–H), 4.48 (br s, 2H, N–CH2), 3.98 (s, 2H, C(7)-
H2), 3.73 (s, 3H, OCH3), 3.37 (s, 2H, C(5)-H2), 2.88 (t, J ¼ 8.0 Hz,
2H, N–CH2–CH2), 2.34 (t, J ¼ 7.2 Hz, 2H, CH2CH2CH3), 1.45 (m,
2H, CH2CH2CH3), 0.86 (t, J ¼ 7.2 Hz, 3H, CH2CH2CH3); HRMS
(ESI) m/z calculated for [C18H24N4O2S þ H]þ 360.1620, found
360.1621.
5.1.3.3. Compound 33 (R ¼ 2-methylbenzyl). Yield ¼ 45%; 1H NMR
(400 MHz, DMSO-d6)
d 12.03 (s, 1H, N(3)-H), 7.32 (s, 1H, N(8)-H),
7.27 (t, J ¼ 7.2 Hz, 1H, Ar–H), 7.13 (s, 3H, Ar–H), 4.51 (br s, 2H, N–
CH2), 3.97 (s, 2H, C(7)-H2), 3.37 (s, 2H, C(5)-H2), 2.89 (t, J ¼ 7.2 Hz,
2H, N–CH2–CH2), 2.34 (m, 5H, CH2CH2CH3 and Ar–CH3), 1.45 (q,
J ¼ 7.2 Hz, 2H, CH2CH2CH3), 0.86 (t, J ¼ 7.2 Hz, 3H, CH2CH2CH3);
HRMS (ESI) m/z calculated for [C18H24N4OS þ H]þ 344.1671, found
344.1672.
5.1.3.11. Compound 41 (R ¼ 4-methoxybenzyl). Yield ¼ 35%; 1H
NMR (400 MHz, DMSO-d6) d 12.00 (s, 1H, N(3)-H), 7.39 (s, 1H, N(8)-
H), 7.25 (d, J ¼ 8.4 Hz, 2H, Ar–H), 6.89 (d, J ¼ 8.4 Hz, 2H, Ar–H), 4.43
(br s, 2H, N–CH2), 3.99 (s, 2H, C(7)-H2), 3.71 (s, 3H, OCH3), 3.37 (s,
2H, C(5)-H2), 2.84 (t, J ¼ 8.0 Hz, 2H, N–CH2–CH2), 2.35 (t, J ¼ 7.2 Hz,
2H, CH2CH2CH3), 1.45 (q, J ¼ 7.2 Hz, 2H, CH2CH2CH3), 0.86 (t,
J ¼ 7.2 Hz, 3H, CH2CH2CH3); HRMS (ESI) m/z calculated for
[C18H24N4O2S þ H]þ 360.1620, found 360.1623.
5.1.3.4. Compound 34 (R ¼ 3-methylbenzyl). Yield ¼ 45%; 1H NMR
(400 MHz, DMSO-d6)
d 12.02 (s, 1H, N(3)-H), 7.41 (s, 1H, N(8)-H),
7.22–7.12 (m, 3H, Ar–H), 7.40 (d, J ¼ 7.2 Hz, 1H, Ar–H), 4.47 (br s,
2H, N–CH2), 3.99 (s, 2H, C(7)-H2), 3.37 (s, 2H, C(5)-H2), 2.87 (t,
J ¼ 8.0 Hz, 2H, N–CH2–CH2), 2.32 (t, J ¼ 7.2 Hz, 2H, CH2CH2CH3),
2.28 (s, 3H, Ar–CH3), 1.45 (q, J ¼ 7.2 Hz, 2H, CH2CH2CH3), 0.87 (t,
J ¼ 7.2 Hz, 3H, CH2CH2CH3); HRMS (ESI) m/z calculated for
[C18H24N4OS þ H]þ 344.1671, found 344.1672.
5.1.3.12. Compound 42 (R ¼ 2,6-dimethylbenzyl). Yield ¼ 42%; 1H
NMR (400 MHz, DMSO-d6) d 12.08 (s, 1H, N(3)-H), 7.13 (s, 1H, N(8)-
H), 6.98 (s, 3H, Ar–H), 4.32 (br s, 2H, N–CH2), 3.94 (s, 2H, C(7)-H2),
3.37 (s, 2H, C(5)-H2), 2.92 (s, 2H, N–CH2–CH2), 2.37–2.32 (m, 8H,
CH2CH2CH3 and Ar–CH3), 1.45 (q, J ¼ 7.2 Hz, 2H, CH2CH2CH3), 0.85
(t, J ¼ 7.2 Hz, 3H, CH2CH2CH3); HRMS (ESI) m/z calculated for
[C19H26N4OS þ H]þ 358.1827, found 358.1833.
5.1.3.5. Compound 35 (R ¼ 4-methylbenzyl). Yield ¼ 42%; 1H NMR
(400 MHz, DMSO-d6)
d 11.99 (s, 1H, N(3)-H), 7.38 (s, 1H, N(8)-H),
7.22 (d, J ¼ 7.6 Hz, 2H, Ar–H), 7.12 (d, J ¼ 8.0 Hz, 2H, Ar–H), 4.45
(br s, 2H, N–CH2), 3.99 (s, 2H, C(7)-H2), 3.37 (s, 2H, C(5)-H2), 2.86 (t,
J ¼ 8.0 Hz, 2H, N–CH2–CH2), 2.34 (t, J ¼ 7.2 Hz, 2H, CH2CH2CH3),
2.26 (s, 3H, Ar–CH3),1.45 (m, 2H, CH2CH2CH3), 0.87 (t, J ¼ 7.2 Hz, 3H,
CH2CH2CH3); HRMS (ESI) m/z calculated for [C18H24N4OS þ H]þ
344.1671, found 344.1672.
5.1.3.13. Compound 43 (R ¼ 2,3-dimethylbenzyl). Yield ¼ 33%; 1H
NMR (400 MHz, DMSO-d6) d 12.03 (s, 1H, N(3)-H), 7.25 (s, 1H, N(8)-
H), 7.19 (t, J ¼ 1.2 Hz, 1H, Ar–H), 7.02 (s, 2H, Ar–H), 4.48 (br s, 2H, N–
CH2), 3.97 (s, 2H, C(7)-H2), 3.37 (s, 2H, C(5)-H2), 2.91 (t, J ¼ 8.0 Hz,
2H, N–CH2–CH2), 2.35 (t, J ¼ 7.2 Hz, 2H, CH2CH2CH3), 2.24 (s, 3H,
Ar–CH3), 2.22 (s, 3H, Ar–CH3), 1.49–1.40 (m, 2H, CH2CH2CH3), 0.86
(t, J ¼ 1.72 Hz, 3H, CH2CH2CH3); HRMS (ESI) m/z calculated for
[C19H26N4OS þ H]þ 358.1827, found 358.1833.
5.1.3.6. Compound 36 (R ¼ 2-chlorobenzyl). Yield ¼ 45%; 1H NMR
(400 MHz, DMSO-d6) d 11.98 (s,1H, N(3)-H), 7.41–7.25 (m, 5H, N(8)-H
and Ar–H), 4.64 (s, 2H, N–CH2), 3.93 (s, 2H, C(7)-H2), 3.36 (s, 2H, C(5)-
H2), 3.05 (t, J ¼ 7.6 Hz, 2H, N–CH2–CH2), 2.32 (t, J ¼ 7.2 Hz, 2H,
CH2CH2CH3), 1.46 (q, J ¼ 7.2 Hz, 2H, CH2CH2CH3), 0.86 (t, J ¼ 7.6 Hz,
3H, CH2CH2CH3); HRMS (ESI) m/z calculated for [C17H21ClN4OS þ H]þ
364.1125, found 364.1125.
5.1.3.14. Compound 44 (R ¼ 2,5-dimethylbenzyl). Yield ¼ 32%; 1H
NMR (400 MHz, DMSO-d6) d 12.02 (s, 1H, N(3)-H), 7.40 (s, 1H, N(8)-
H), 7.10 (s, 1H, Ar–H), 7.06 (s, 3H, Ar–H), 4.44 (br s, 2H, N–CH2), 3.98
(s, 2H, C(7)-H2), 3.37 (s, 2H, C(5)-H2), 2.82 (t, J ¼ 8.0 Hz, 2H, N–CH2–
CH2), 2.34 (t, J ¼ 7.2 Hz, 2H, CH2CH2CH3), 2.19 (s, 3H, Ar–CH3), 2.17
(s, 3H, Ar–CH3), 1.50–1.41 (m, 2H, CH2CH2CH3), 0.86 (t, J ¼ 7.2 Hz,
3H, CH2CH2CH3); HRMS (ESI) m/z calculated for [C19H26N4OS þ H]þ
358.1827, found 358.1832.
5.1.3.7. Compound 37 (R ¼ 3-chlorobenzyl). Yield ¼ 40%; 1H NMR
(400 MHz, DMSO-d6) d 12.03 (s, 1H, N(3)-H), 7.43 (s, 2H, N(8)-H and
Ar–H), 7.37–7.28 (m, 3H, Ar–H), 4.47 (br s, 2H, N–CH2), 3.99 (s, 2H,
C(7)-H2), 3.37 (s, 2H, C(5)-H2), 2.93 (t, J ¼ 8.0 Hz, 2H, N–CH2–CH2),

J.J. Marugan et al. / European Journal of Medicinal Chemistry 45 (2010) 1880–1897
1893
5.1.3.15. Compound 45 (R ¼ 2,4-dimethylbenzyl). Yield ¼ 32%; 1H
HRMS (ESI) m/z calculated for [C17H20Cl2N4OS þ H]þ 398.0735,
NMR (400 MHz, DMSO-d6)
d
12.06 (s, 1H, N(3)-H), 7.40 (br s, 1H,
found 398.0741.
N(8)-H), 7.15 (s,1H, Ar–H), 6.95 (s, 2H, Ar–H), 4.48 (br s, 2H, N–CH2),
3.97 (s, 2H, C(7)-H2), 3.37 (s, 2H, C(5)-H2), 2.84 (t, J ¼ 8.0 Hz, 2H,
N–CH2–CH2), 2.36–2.30 (m, 5H,, CH2CH2CH3 and Ar–CH3), 2.21
(s, 3H, Ar–CH3), 1.50–1.41 (m, 2H, CH2CH2CH3), 0.86 (t, J ¼ 7.2 Hz,
3H, CH2CH2CH3); HRMS (ESI) m/z calculated for [C19H26N4OS þ H]þ
358.1827, found 358.1844.
5.1.3.23. Compound 53 (R ¼ 3,5-dichlorobenzyl). Yield ¼ 40%; 1H
NMR (400 MHz, DMSO-d6) d 12.05 (s, 1H, N(3)-H), 7.49 (s, 1H, N(8)-
H), 7.44 (s,1H, Ar–H), 7.39 (s, 2 H, Ar–H), 4.47 (br s, 2H, N–CH2), 4.00
(s, 2H, C(7)-H2), 3.38 (s, 2H, C(5)-H2), 2.96 (t, J ¼ 7.2 Hz, 2H, N–CH2–
CH2), 2.36 (t, J ¼ 7.2 Hz, 2H, CH2CH2CH3), 1.50 (m, 2H, CH2CH2CH3),
0.89 (t, J ¼ 7.2 Hz, 3H, CH2CH2CH3); HRMS (ESI) m/z calculated for
[C17H20Cl2N4OS þ H]þ 398.0735, found 398.0743.
5.1.3.16. Compound 46 (R ¼ 3,4-dimethylbenzyl). Yield ¼ 32%; 1H
NMR (400 MHz, DMSO-d6) d 12.02 (s, 1H, N(3)-H), 7.40 (s, 1H, N(8)-
H), 7.10 (s, 1H, Ar–H), 7.06 (s, 3H, Ar–H), 4.44 (br s, 2H, N–CH2), 3.98
(s, 2H, C(7)-H2), 3.37 (s, 2H, C(5)-H2), 2.82 (t, J ¼ 8 Hz, 2H, N–CH2–
CH2), 2.34 (t, J ¼ 7.2 Hz, 2H, CH2CH2CH3), 2.19 (s, 3H, Ar–CH3), 2.17
(s, 3H, Ar–CH3), 1.50–1.41 (m, 2H, CH2CH2CH3), 0.86 (t, J ¼ 7.2 Hz,
3H, CH2CH2CH3); HRMS (ESI) m/z calculated for [C19H26N4OS þ H]þ
358.1827, found 358.1833.
5.1.3.24. Compound 54 (R ¼ 2,6-di(OMe)benzyl). Yield ¼ 51%; 1H
NMR (400 MHz, DMSO-d6)
d
11.75 (s, 1H, N(3)-H), 7.12 (t, J ¼ 8.4 Hz,
1H, Ar–H), 6.79 (s, 1H, N(8)-H), 6.55 (d, J ¼ 8.0 Hz, 2H, Ar–H), 4.50
(br s, 2H, N–CH2), 3.88 (s, 2H, C(7)-H2), 3.70 (s, 6H, OCH3), 3.33 (s,
2H, C(5)-H2), 2.92 (t, J ¼ 6.0 Hz, 2H, N–CH2–CH2), 2.31 (t, J ¼ 7.2 Hz,
2H, CH2CH2CH3), 1.43 (q, J ¼ 7.2 Hz, 2H, CH2CH2CH3), 0.85 (t,
J ¼ 7.2 Hz, 3H, CH2CH2CH3); HRMS (ESI) m/z calculated for
[C19H26N4O3S þ H]þ 390.1726, found 390.1730.
5.1.3.17. Compound 47 (R ¼ 3,5-dimethylbenzyl). Yield ¼ 50%; 1H
NMR (400 MHz, DMSO-d6) d 12.03 (s, 1H, N(3)-H), 7.32 (s, 1H, N(8)-
H), 7.27 (t, J ¼ 7.2 Hz, 1H, Ar–H), 7.13 (s, 3H, Ar–H), 4.51 (br s, 2H, N–
CH2), 3.97 (s, 2H, C(7)-H2), 3.37 (s, 2H, C(5)-H2), 2.89 (t, J ¼ 7.2 Hz,
2H, N–CH2–CH2), 2.34 (t, J ¼ 7.2 Hz, 2H, CH2CH2CH3), 2.33 (s, 6H,
Ar–CH3), 1.45 (q, J ¼ 7.2 Hz, 2H, CH2CH2CH3), 0.86 (t, J ¼ 7.2 Hz, 3H,
CH2CH2CH3); HRMS (ESI) m/z calculated for [C19H26N4OS þ H]þ
358.1827, found 358.1831.
5.1.3.25. Compound 55 (R ¼ 2,3-di(OMe)benzyl). Yield ¼ 55%; 1H
NMR (400 MHz, DMSO-d6) d 12.06 (s, 1H, N(3)-H), 7.24 (s, 1H, N(8)-
H), 7.08 (t, J ¼ 8.0 Hz, 1H, Ar–H), 7.00 (d, J ¼ 8.4 Hz, 1H, Ar–H), 6.93
(d, J ¼ 8.4 Hz,1H, Ar–H), 4.59 (br s, 2H, N–CH2), 4.06 (s, 2H, C(7)-H2),
3.85 (s, 3H, OCH3), 3.80 (s, 3H, OCH3), 3.45 (s, 2H, C(5)-H2), 2.99 (t,
J ¼ 8.0 Hz, 2H, N–CH2–CH2), 2.41 (t, J ¼ 7.2 Hz, 2H, CH2CH2CH3), 1.45
(q, J ¼ 7.2 Hz, 2H, CH2CH2CH3), 0.94 (t, J ¼ 7.2 Hz, 3H, CH2CH2CH3);
HRMS (ESI) m/z calculated for [C19H26N4O3S þ H]þ 390.1726, found
390.1734.
5.1.3.18. Compound 48 (R ¼ 2,6-dichlorobenzyl). Yield ¼ 40%; 1H
NMR (400 MHz, DMSO-d6) d 11.79 (s, 1H, N(3)-H), 7.37 (s, 1H, N(8)-
H), 7.35 (s, 1H, Ar–H), 7.24–7.20 (m, 2H, Ar–H), 4.80 (br s, 2H, N–
CH2), 3.90 (s, 2H, C(7)-H2), 3.23 (m, 4H,, N–CH2–CH2 and C(5)-H2),
2.33 (t, J ¼ 7.2 Hz, 2H, CH2CH2CH3), 1.43 (q, J ¼ 7.2 Hz, 2H,
CH2CH2CH3), 0.85 (t, J ¼ 7.2 Hz, 3H, CH2CH2CH3); HRMS (ESI) m/z
calculated for [C17H20Cl2N4OS þ H]þ 398.0735, found 398.0741.
5.1.3.26. Compound 56 (R ¼ 2,5-di(OMe)benzyl). Yield ¼ 40%; 1H
NMR (400 MHz, DMSO-d6)
d 12.00 (s, 1H, N(3)-H), 7.37 (s, 1H, N(8)-
H), 6.92–6.81 (m, 3H, Ar–H), 4.47 (br s, 2H, N–CH2), 3.98 (s, 2H, C(7)-
H2), 3.73 (s, 3H, OCH3), 3.70 (s, 3H, OCH3), 3.36 (s, 3H, C(5)-H2), 2.85
(t, J ¼ 8 Hz, 2H, N–CH2–CH2), 2.33 (t, J ¼ 7.2 Hz, 2H, CH2CH2CH3),1.44
(q, J ¼ 7.2 Hz, 2H, CH2CH2CH3), 0.86 (t, J ¼ 7.2 Hz, 3H, CH2CH2CH3);
HRMS (ESI) m/z calculated for [C19H26N4O3S þ H]þ 390.1726, found
390.1730.
5.1.3.19. Compound 49 (R ¼ 2,3-dichlorobenzyl). Yield ¼ 29%; 1H
NMR (400 MHz, DMSO-d6) d 11.96 (s, 1H, N(3)-H), 7.52–7.50 (m, 1H,
N(8)-H), 7.32–7.28 (m, 3H, Ar–H), 4.66 (br s, 2H, N–CH2), 3.93 (s, 2H,
C(7)-H2), 3.36 (s, 2H, C(5)-H2), 3.11 (t, J ¼ 7.2 Hz, 2H, N–CH2–CH2),
2.32 (t, J ¼ 7.2 Hz, 2H, CH2CH2CH3), 1.44 (q, J ¼ 7.2 Hz, 2H,
CH2CH2CH3), 0.86 (t, J ¼ 7.2 Hz, 3H, CH2CH2CH3); HRMS (ESI) m/z
calculated for [C17H20Cl2N4OS þ H]þ 398.0735, found 398.0741.
5.1.3.27. Compound 57 (R ¼ 2,4-di(OMe)benzyl). Yield ¼ 47%; 1H
NMR (400 MHz, DMSO-d6) d 11.92 (s, 1H, N(3)-H), 7.04–7.02 (m, 2H,
Ar–H and N(8)-H), 6.49–6.43 (m, 2H, Ar–H), 4.51 (br s, 2H, N–CH2),
3.95 (s, 2H, C(7)-H2), 3.73 (s, 3H, OCH3), 3.71 (s, 3H, OCH3), 3.35 (s,
2H, C(5)-H2), 2.81 (t, J ¼ 7.6 Hz, 2H, N–CH2–CH2), 2.31 (t, J ¼ 7.2 Hz,
2H, CH2CH2CH3), 1.44 (q, J ¼ 7.6 Hz, 2H, CH2CH2CH3), 0.85 (t,
J ¼ 7.2 Hz, 3H, CH2CH2CH3); HRMS (ESI) m/z calculated for
[C19H26N4O3S þ H]þ 390.1726, found 390.1731.
5.1.3.20. Compound 50 (R ¼ 2,5-dichlorobenzyl). Yield ¼ 21%; 1H
NMR (400 MHz, DMSO-d6)
d 11.96 (s, 1H, N(3)-H), 7.47 (s, 1H, N(8)-
H), 7.45 (s, 1H, Ar–H), 7.34 (d, J ¼ 2.4 Hz, 2H, Ar–H), 4.69 (br s, 2H,
N–CH2), 3.94 (s, 2H, C(7)-H2), 3.37 (s, 2H, C(5)-H2), 3.07 (t, J ¼ 7.2 Hz,
2H, N–CH2–CH2), 2.35 (t, J ¼ 6.8 Hz, 2H, CH2CH2CH3), 1.49–1.40
(m, 2H, CH2CH2CH3), 0.88 (t, J ¼ 7.2 Hz, 3H, CH2CH2CH3); HRMS (ESI)
m/z calculated for [C17H20Cl2N4OS þ H]þ 398.0735, found 398.0743.
5.1.3.28. Compound 58 (R ¼ 3,5-di(OMe)benzyl). Yield ¼ 39%; 1H
NMR (400 MHz, DMSO-d6) d 12.03 (s, 1H, N(3)-H), 7.38 (s, 1H, N(8)-
H), 6.50 (s, 2H, Ar–H), 6.36 (s, 1H, Ar–H), 4.51 (br s, 2H, N–CH2), 3.98
(s, 2H, C(7)-H2), 3.71 (s, 6H, OCH3), 3.36 (s, 2H, C(5)-H2), 2.84 (t,
J ¼ 8.0 Hz, 2H, N–CH2–CH2), 2.33 (t, J ¼ 7.2 Hz, 2H, CH2CH2CH3),
1.49–1.40 (m, 2H, CH2CH2CH3), 0.86 (t, J ¼ 7.2 Hz, 3H, CH2CH2CH3);
HRMS (ESI) m/z calculated for [C19H26N4O3S þ H]þ 390.1726, found
390.1728.
5.1.3.21. Compound 51 (R ¼ 2,4-dichlorobenzyl). Yield ¼ 39%; 1H
NMR (400 MHz, DMSO-d6) d 11.94 (s, 1H, N(3)-H), 7.55 (s, 1H, Ar–H,
N(8)-H), 7.38 (s, 2H, Ar–H), 7.28 (s,1H, Ar–H), 4.66 (br s, 2H, N–CH2),
3.94 (s, 2H, C(7)-H2), 3.36 (s, 2H, C(5)-H2), 3.04 (t, J ¼ 7.2 Hz, 2H, N–
CH2–CH2), 2.32 (t. J ¼ 7.2 Hz, 2H, CH2CH2CH3), 1.44 (q, J ¼ 7.2 Hz, 2H,
CH2CH2CH3), 0.86 (t, J ¼ 7.2 Hz, 3H, CH2CH2CH3); HRMS (ESI) m/z
calculated for [C17H20Cl2N4OS þ H]þ 398.0735, found 398.0735.
5.1.3.29. Compound 59 (R ¼ 3,4-(OCH2CH2O)benzyl). Yield ¼ 33%;
1H NMR (400 MHz, DMSO-d6)
d 12.02 (s, 1H, N(3)-H), 7.39 (s, 1H,
5.1.3.22. Compound 52 (R ¼ 3,4-dichlorobenzyl). Yield ¼ 50%; 1H
N(8)-H), 6.86 (s, 1H, Ar–H), 6.78 (s, 2H, Ar–H), 4.34 (br s, 2H, N–
CH2), 4.20 (s, 4H, –OCH2CH2O–), 3.99 (s, 2H, C(7)-H2), 3.37 (s, 2H,
C(5)-H2), 2.78 (t, J ¼ 8.0 Hz, 2H, N–CH2–CH2), 2.34 (t, J ¼ 7.2 Hz, 2H,
CH2CH2CH3), 1.45 (q, J ¼ 7.2 Hz, 2H, CH2CH2CH3), 0.86 (t, J ¼ 7.2 Hz,
3H, CH2CH2CH3). HRMS (ESI) m/z calculated for [C19H24N4O3S þ H]þ
388.1569, found 388.1571.
NMR (400 MHz, DMSO-d6) d 12.04 (s, 1H, N(3)-H), 7.61–7.58 (m, 2H,
N(8)-H and Ar–H), 7.44 (s, 1H, Ar–H), 7.32–7.30 (m, 1H, Ar–H), 4.48
(br s, 2H, N–CH2), 3.99 (s, 2H, C(7)-H2), 3.37 (s, 2H, C(5)-H2), 2.93 (t,
J ¼ 8.0 Hz, 2H, N–CH2–CH2), 2.33 (t, J ¼ 7.2 Hz, 2H, CH2CH2CH3), 1.45
(q, J ¼ 7.2 Hz, 2H, CH2CH2CH3), 0.87 (t, J ¼ 7.2 Hz, 3H, CH2CH2CH3);

1894
J.J. Marugan et al. / European Journal of Medicinal Chemistry 45 (2010) 1880–1897
5.1.3.30. Compound 60 (R ¼ 3,4-di(OEt)benzyl). Yield ¼ 33%; 1H
CH2CH2CH3); HRMS (ESI) m/z calculated for [C15H26N4OS þ H]þ
NMR (400 MHz, DMSO-d6)
d
12.01 (s, 1H, N(3)-H), 7.37 (s, 1H, N(8)-
310.1827, found 310.1829.
H), 6.91–6.79 (m, 3H, Ar–H), 4.44 (br s, 2H, N–CH2), 4.01–3.93 (m,
6H, C(7)-H2 and OCH2CH3), 3.36 (s, 2H, C(5)-H2), 2.82 (t, J ¼ 8.0 Hz,
2H, N–CH2–CH2), 2.33 (t, J ¼ 7.2 Hz, 2H, CH2CH2CH3), 1.45 (t,
J ¼ 7.2 Hz, 2H, CH2CH2CH3), 1.33–1.27 (m, 6H, OCH2CH3), 0.86 (t,
J ¼ 7.2 Hz, 3H, CH2CH2CH3); HRMS (ESI) m/z calculated for
[C21H30N4O3S þ H]þ 418.2039, found 418.2048.
5.1.3.38. Compound 68 (R
(400 MHz, DMSO-d6)
¼
methyl). Yield ¼ 32%; 1H NMR
d
11.92 (s, 1H, N(3)-H), 7.35 (s, 1H, N(8)-H),
4.20 (br s, 2H, N–CH2), 4.00 (s, 2H, C(7)-H2), 3.36 (s, 2H, C(5)-H2),
2.34 (t, J ¼ 7.2 Hz, 2H, CH2CH2CH3), 1.65–1.58 (m, 2H, N–CH2–CH2),
1.56–1.40 (m, 2H, CH2CH2CH3), 0.85 (m, 6H, NCH2CH2CH3 and
CH2CH2CH3); HRMS (ESI) m/z calculated for [C12H20N4OS þ H]þ
268.1358, found 268.1360.
5.1.3.31. Compound 61 (R ¼ 2-pyridyl). Yield ¼ 32%; 1H NMR
(400 MHz, DMSO-d6)
d 12.01 (s, 1H, N(3)-H), 7.38 (s, 1H, N(8)-H),
6.92–6.81 (m, 3H, Ar–H), 4.48 (br s, 2H, N–CH2), 3.98 (s, 2H, C(7)-
H2), 3.36 (s, 2H, C(5)-H2), 2.85 (t, J ¼ 8.0 Hz, 2H, N–CH2–CH2), 2.33
(t, J ¼ 7.2 Hz, 2H, CH2CH2CH3), 1.45 (q, J ¼ 7.2 Hz, 2H, CH2CH2CH3),
0.86 (t, J ¼ 7.2 Hz, 3H, CH2CH2CH3); HRMS (ESI) m/z calculated for
[C16H21N5OS þ H]þ 331.1467, found 331.1468.
5.1.4. General procedure for the preparation of compounds 69–73
The amine (4.55 mmol) and formaldehyde (6.50 mmol) were
added to a solution of 5 (3.25 mmol) in ethanol (20 mL). The
reaction mixture was heated at reflux for 3 h, then cooled to room
temperature. The resulting precipitate was filtered through a sin-
tered glass funnel to afford compounds 69–73. The final
compounds were characterized by 1H NMR and MS analysis.
5.1.3.32. Compound 62 (R ¼ 3-thienyl). Yield ¼ 37%; 1H NMR
(400 MHz, DMSO-d6) d 12.03 (s, 1H, N(3)-H), 7.49 (d, J ¼ 4.4 Hz, 1H,
Ar–H), 7.41 (s, 1H, N(8)-H), 7.27 (s, 1H, Ar–H), 7.10 (d, J ¼ 4.8 Hz, 1H,
Ar–H), 4.51 (s, 2H, N–CH2), 3.98 (s, 2H, C(7)-H2), 3.32 (s, 2H, C(5)-
H2), 2.96 (t, J ¼ 8 Hz, 2H, N–CH2–CH2), 2.36 (t, J ¼ 7.2 Hz 2H,
CH2CH2CH3), 1.50–1.41 (m, 2H, CH2CH2CH3), 0.88 (t, J ¼ 7.2 Hz, 3H,
CH2CH2CH3); HRMS (ESI) m/z calculated for [C15H20N4OS2þH]þ
336.1079, found 336.1079.
5.1.4.1. Compound 69 (R
¼
methyl). Yield
¼
37%; 1H NMR
(400 MHz, DMSO-d6) 12.10 (s, 1H, N(3)-H), 7.50 (s, 1H, N(8)-H),
d
7.01–6.89 (m, 3H, Ar–H), 4.55 (br s, 2H, N–CH2), 4.00 (s, 2H, C(7)-
H2), 3.81 (s, 3H, OCH3), 3.78 (s, 3H, OCH3), 3.37 (s, 2H, C(5)-H2), 2.92
(t, J ¼ 8.0 Hz, 2H, N–CH2–CH2), 2.34 (s, 3H, NCH3); HRMS (ESI) m/z
calculated for [C17H22N4O3S þ H]þ 362.1413, found 362.1415.
5.1.3.33. Compound 63 (R ¼ 2-thienyl). Yield ¼ 40%; 1H NMR
5.1.4.2. Compound 70 (R ¼ isopropyl). Yield ¼ 33%; 1H NMR
(400 MHz, DMSO-d6)
d
12.02 (s, 1H, N(3)-H), 7.44 (s, 1H, N(8)-H),
(400 MHz, DMSO-d6) d 11.98 (s, 1H, N(3)-H), 7.35 (s, 1H, N(8)-H),
7.37–7.36 (m, 1H, Ar–H), 6.97 (s, 2H, Ar–H), 4.51 (br s, 2H, N–
CH2), 3.97 (s, 2H, C(7)-H2), 3.37 (s, 2H, C(5)-H2), 3.14 (t, J ¼ 8.0 Hz,
2H, N–CH2–CH2), 2.34 (t, J ¼ 7.2 Hz, 2H, CH2CH2CH3), 1.45 (q,
J ¼ 7.2 Hz, 2H, CH2CH2CH3), 0.86 (t, J ¼ 7.2 Hz, 3H, CH2CH2CH3);
HRMS (ESI) m/z calculated for [C15H20N4OS2þH]þ 336.1079, found
336.1078.
6.91–6.81 (m, 3H, Ar–H), 4.46 (br s, 2H, N–CH2), 4.03 (s, 2H, C(7)-
H2), 3.73 (s, 3H, OCH3), 3.71 (s, 3H, OCH3), 3.42 (s, 2H, C(5)-H2), 2.85
(t, J ¼ 8.0 Hz, 2H, N–CH2–CH2), 2.72–2.66 (m, 1H, CH(CH3)2), 1.03 (s,
6H, CH(CH3)2); HRMS (ESI) m/z calculated for [C19H26N4O3S þ H]þ
390.1726, found 390.1730.
5.1.4.3. Compound 71 (R
¼
isobutyl). Yield
¼
36%; 1H NMR
5.1.3.34. Compound 64 (R
¼
3,4-di(OMe)phenyl). 1H NMR
(400 MHz, DMSO-d6) 11.98 (s, 1H, N(3)-H), 7.35 (s, 1H, N(8)-H),
d
(400 MHz, DMSO-d6) 12.08 (s,1H, N(3)-H), 7.20 (s,1H, N(8)-H), 6.88
d
6.91–6.81 (m, 3H, Ar–H), 4.46 (br s, 2H, N–CH2), 4.03 (s, 2H, C(7)-
H2), 3.73 (s, 3H, OCH3), 3.71 (s, 3H, OCH3), 3.42 (s, 2H, C(5)-H2), 2.85
(t, J ¼ 8.0 Hz, 2H, N–CH2–CH2), 2.72–2.66 (m, 1H, CH(CH3)2), 2.15 (d,
J ¼ 8.0 Hz, 2H, CH2CH(CH3)2) 1.03 (s, 6H, CH(CH3)2); HRMS (ESI) m/z
calculated for [C20H28N4O3S þ H]þ 404.1882, found 404.1887.
(d, J ¼ 4.4 Hz, 2H, Ar–H), 6.65 (d, J ¼ 8.0 Hz, 1H, Ar–H), 5.51 (br s, 2H,
N–CH2), 3.92 (s, 2H, C(7)-H2), 3.70 (s, 6H, OCH3), 3.39 (s, 2H, C(5)-H2),
2.32 (t, J ¼ 7.2 Hz, 2H, CH2CH2CH3), 1.43 (q, J ¼ 7.2 Hz, 2H,
CH2CH2CH3), 0.84 (t, J ¼ 7.2 Hz, 3H, CH2CH2CH3); HRMS (ESI) m/z
calculated for [C18H24N4O3S þ H]þ 376.1569, found 376.1573.
5.1.4.4. Compound 72 (R ¼ phenyl). Yield ¼ 39%; 1H NMR (400 MHz,
5.1.3.35. Compound 65 (R ¼ phenethyl). Yield ¼ 33%; 1H NMR
DMSO-d6) d 12.09 (s, 1H, N(3)-H), 7.73 (s, 1H, N(8)-H), 7.24 (t,
(400 MHz, DMSO-d6)
d
11.94 (s, 1H, N(3)-H), 7.38 (s, 1H, N(8)-H),
J ¼ 8.0 Hz, 2H, Ar–H), 7.04 (d, J ¼ 8.0 Hz, 2H, Ar–H), 6.87–6.77 (m, 4 H,
Ar–H), 4.73 (s, 2H, C(7)-H2), 4.44 (br s, 2H, N–CH2), 4.12 (s, 2H, C(5)-
H2), 3.70 (s, 6H, OCH3), 2.78 (t, J ¼ 8.0 Hz, 2H, N–CH2–CH2); HRMS
(ESI) m/z calculated for [C22H24N4O3S þ H]þ 424.1569, found 424.1571
7.28–7.16 (m, 5H, Ar–H), 4.33 (br s, 2H, N–CH2), 4.00 (s, 2H, C(7)-
H2), 3.36 (s, 2H, C(5)-H2), 2.62 (t, J ¼ 8.0 Hz, 2H, N–CH2–CH2), 2.35
(t, J ¼ 7.2 Hz, 2H, CH2CH2CH3), 1.94–1.88 (m, 2H, C6H5–CH2), 1.45 (q,
J ¼ 7.2 Hz, 2H, CH2CH2CH3), 0.87 (t, J ¼ 7.2 Hz, 3H, CH2CH2CH3);
HRMS (ESI) m/z calculated for [C18H24N4OS þ H]þ 344.1671, found
344.1672.
5.1.4.5. Compound 73 (R ¼ benzyl). Yield ¼ 31%; 1H NMR (400 MHz,
DMSO-d6) d 12.01 (s,1H, N(3)-H), 7.43 (s,1H, N(8)-H), 7.34 (s, 4H, Ar–
H), 7.32–7.25 (m, 1H, Ar–H), 6.93–6.83 (m, 3H, Ar–H), 4.48 (br s, 2H,
N–CH2), 4.02 (s, 2H, C(7)-H2), 3.73 (s, 3H, OCH3), 3.70 (s, 3H, OCH3),
3.37 (s, 2H, C(5)-H2), 3.31 (s, 2H, NCH2Ph), 2.88 (t, J ¼ 8.0 Hz, 2H, N–
CH2–CH2); HRMS (ESI) m/z calculated for [C23H26N4O3S þ H]þ
438.1726, found 438.1734.
5.1.3.36. Compound 66 (R ¼ isopropyl). Yield ¼ 30%; 1H NMR
(400 MHz, DMSO-d6)
d 11.92 (s, 1H, N(3)-H), 7.26 (s, 1H, N(8)-H),
4.32 (br s, 2H, N–CH2), 3.97 (s, 2H, C(7)-H2), 3.35 (s, 2H, C(5)-H2),
2.34 (t, J ¼ 7.2 Hz, 2H, CH2CH2CH3), 1.68–1.61 (m, 1H CH(CH3)2),
1.49–1.40 (m, 4H, N–CH2–CH2 and CH2CH2CH3), 0.91–0.87 (m, 9H,
CH2CH2CH3 and CH(CH3)2); HRMS (ESI) m/z calculated for
[C14H24N4OS þ H]þ 296.1671, found 296.1673.
5.1.5. Synthesis of 6-amino-1-(3,4-dimethoxyphenethyl)-5-nitroso-
2-thioxo-2, 3-dihydro-pyrimidin-4(1H)-one 12
A suspension of 11 (110 mg, 0.36 mmol) in 10% aqueous acetic
acid (2 mL) was heated to 75 ꢁC. A solution of sodium nitrite (35 mg,
0.51 mmol) in water (1 mL) was added dropwise and heating was
continued for 1 h. Additional sodium nitrite (35 mg, 0.51 mmol) in
water (1 mL) was added dropwise and heating was continued for an
additional 1 h. After this time, the reaction mixture was poured into
5.1.3.37. Compound 67 (R
(400 MHz, DMSO-d6)
¼
t-butyl). Yield ¼ 34%; 1H NMR
d
11.95 (s, 1H, N(3)-H), 7.07 (s, 1H, N(8)-H),
4.46 (br s, 2H, N–CH2), 3.96 (s, 2H, C(7)-H2), 3.33 (s, 2H, C(5)-H2),
2.34 (t, J ¼ 7.2 Hz, 2H, CH2CH2CH3), 1.51–1.42 (m, 4H, t-Bu-CH2 and
CH2CH2CH3), 0.94 (s, 9H C(CH3)3), 0.86 (t, J ¼ 7.2 Hz, 3H,

J.J. Marugan et al. / European Journal of Medicinal Chemistry 45 (2010) 1880–1897
1895
ice/water and the resulting precipitate was filtered. The filter cake
3H, OCH3), 3.87 (s, 3H, OCH3), 3.07–3.03 (t, 2H, –NCH2CH2); HRMS
(ESI) m/z calculated for [C16H16N4O3S þ H]þ 344.0943, found
344.0944.
was washed with hexanes (5 mL) to afford 12 (70 mg, 58%) as a light
green solid: 1H NMR (400 MHz, DMSO-d6)
d 13.34 (br s,1H, N(3)-H),
12.73 (br s, 1H, NH2), 9.31 (br s, 1H, NH2), 6.91–6.79 (m, 3H, Ar–H),
4.51–4.50 (br s, 2H, N–CH2), 3.72 (s, 3H, OCH3), 3.70 (s, 3H, OCH3),
2.83–2.79 (t, 2H, NCH2CH2); MS (ESI) m/z 337 [C14H16N4O4S þ H]þ.
5.1.10. Synthesis of pyrido[4,3-d]pyrimidin-5(6H)-one 20 [25]
Ethyl acetoacetate 13 (4.32 mL, 61.65 mmol) and 1,3,5-triazine
19 (5.00 g, 61.65 mmol) were added to the stirring solution of
sodium (350 mg, 16.20 mmol) in ethanol (18 mL) at room
temperature. The reaction was heated at reflux for 1 h under
a nitrogen atmosphere. After this time, the reaction mixture was
concentrated under reduced pressure and the residue was diluted
with water (50 mL). The resulting aqueous solution was acidified
with concentrated hydrochloric acid and the resulting precipitate
was filtered. The filter cake was washed with cold acetone and
dried to afford compound 20 (400 mg, 4.5%) as a brown solid: 1H
5.1.6. Synthesis of 5,6-diamino-1-(3,4-dimethoxyphenethyl)-2-
thioxo-2,3-dihydro-pyrimidin-4(1H)-one 13
A suspension of 12 (70 mg, 0.21 mmol) in water (4 mL) and
ammonia (32% aq, 4 mL) was heated at 75 ꢁC for 20 min. Sodium
dithionite (90 mg, 0.52 mmol) was added portionwise and the
reaction was heated at 75 ꢁC for an additional 20 min. The reaction
was then cooled to room temperature and stirred for 1 h. At this
time, the pH of the solution was adjusted to 7 with an aqueous
solution of 1 M hydrochloric acid. The resulting precipitate was
collected by filtration and washed with water (5 mL) and hexanes
(5 mL). The crude product was purified by silica-gel column chro-
matography (2% MeOH in DCM) to afford 13 (20 mg, 30%) as an off-
NMR (400 MHz, DMSO-d6)
d 11.92 (s, 1H, NH), 9.40 (s, 1H, C(2)-H),
9.31 (s, 1H, C(4)-H), 7.70 (d, J ¼ 6.8 Hz, 1H, C(7)-H), 6.55 (d,
J ¼ 7.2 Hz, 1H, C(8)-H); MS (ESI) m/z 148 [C7H5N3O þ H]þ.
white solid: 1H NMR (400 MHz, DMSO-d6)
d
7.48 (br s, 1H, NH), 6.95
5.1.11. Synthesis of 8-iodopyrido[4,3-d]pyrimidin-5-(6H)-one 21
[26]
(br s, 1H, NH), 6.86–6.70 (m, 3H, Ar–H), 3.72 (s, 3H, OCH3), 3.70 (s,
3H, OCH3), 3.55–3.54 (m, 2H, –NCH2), 2.69–2.68 (m, 2H,
–NCH2CH2); MS (ESI) m/z 323 [C14H18N4O3S þ H]þ.
Iodine (1.10 g, 4.34 mmol) was added to a suspension of 20
(500 mg, 3.39 mmol) in 0.4 N aqueous sodium hydroxide (17 mL)
and the reaction mixture was heated at 80 ꢁC for 18 h. The reaction
mixture was cooled to room temperature and the resulting
precipitate was filtered. The filter cake was washed with water
(15 mL) and dried to afford 21 (550 mg, 59%) as a yellow solid: 1H
5.1.7. Synthesis of 3-(3,4-dimethoxyphenethyl)-2-thioxo-2,3-
dihydro-1H-purin-6(9H)-one 14
A mixture of 13 (400 mg, 1.24 mmol) and formamide (5 mL) was
heated at 165 ꢁC for 45 min and slowly allowed to cool to room
temperature. The resulting precipitate was filtered and washed
with formamide (10 mL), water (10 mL), and ethanol (10 mL) and
dried to afford 14 (290 mg, 70%) as a light yellow solid: 1H NMR
NMR (400 MHz, DMSO-d6)
d 12.15 (s, 1H, NH), 9.41 (s, 1H, C(2)-H),
9.31 (s, 1H, C(4)-H), 8.12 (s, 1H, C(7)-H); MS (ESI) m/z 274
[C7H4IN3O þ H]þ.
(400 MHz, DMSO-d6)
d
13.84 (br s,1H, N(7)-H), 12.48 (br s,1H, N(3)-
5.1.12. Synthesis of 5-chloro-8-iodopyrido[4,3-d]pyrimidine 22 [26]
A mixture of 21 (300 mg, 1.09 mmol) and POCl3 (9 mL) was
heated at reflux for 24 h. The reaction mixture was concentrated
under reduced pressure and the residue was poured into ice water.
The resulting mixture was made alkaline with K2CO3 and extracted
with CH2Cl2 (3 ꢀ 30 mL). The combined organic layer was dried
over Na2SO4 and concentrated under reduced pressure. The crude
residue was purified by silica-gel column chromatography (10%
EtOAc in petroleum ether) to afford 22 (200 mg, 65%) as a brown
solid: 1H NMR (400 MHz, DMSO-d6) 9.79 (s, 1H, C(2)-H), 9.67 (s, 1H,
C(4)-H), 9.19 (s, 1H, C(7)-H); MS (ESI) m/z 292 [C7H3ClN3 þ H]þ.
H), 8.18 (s, 1H, C(6)-H), 6.88–6.77 (m, 3H, Ar–H), 4.63–4.61 (t, 2H,
–NCH2CH2), 3.72 (s, 3H, OCH3), 3.70 (s, 3H, OCH3), 2.98–2.94 (t, 2H,
–NCH2CH2); HRMS (ESI) m/z calculated for [C15H16N4O3S þ H]þ
332.0943, found 332.0944.
5.1.8. Synthesis of 1-(3,4-dimethoxyphenethyl)-5,7-dimethyl-2-
thioxo-2,3-dihydropyrido [2,3-d]pyrimidin-4(1H)-one 16
A mixture of 9a (300 mg, 0.98 mmol), 15 (0.3 mL, 2.93 mmol)
and trifluoroacetic acid (15 mL) was heated at 60 ꢁC for 6 h, after
which time another portion of 15 (0.3 mL, 2.93 mmol) was added
and the reaction remained at reflux for 16 h. The reaction mixture
was cooled to room temperature, poured into ice/water and
extracted with EtOAc (2 ꢀ 25 mL). The organic layer was washed
with an aqueous saturated NaHCO3 solution, dried over Na2SO4,
and concentrated under reduced pressure. The resulting solid was
triturated with MTBE to afford 16 (120 mg, 33%) as an off-white
5.1.13. General procedure for Sonogashira coupling
A mixture of 22 (2.06 mmol) and anhydrous THF (18 mL) was
purged with argon gas for 30 min. Acetylene derivative (2.48 mmol)
was added to this solution followed by DIPEA (0.41 mmol), CuI
(0.10 mmol) and Pd(PPh3)2Cl2 (0.41 mmol). After stirring for 48 h
under an argon atmosphere, the reaction mixture was concentrated
under reduced pressure. The crude material was purified by silica-
gel column chromatography (10–30% EtOAc in petroleum ether) to
afford compounds 23 or 24.
solid: 1H NMR (400 MHz, DMSO-d6)
d 12.60 (br s, 1H, N(3)-H),
8.18 (s, 1H, C(6)-H), 6.88–6.86 (m, 2H, Ar–H), 6.80–6.78 (d,
J ¼ 8.4 Hz, 1H, Ar–H), 4.90–4.86 (t, J ¼ 8.0 Hz, 2H, –NCH2CH2), 3.74
(s, 3H, OCH3), 3.70 (s, 3H, OCH3), 2.92–2.88 (t, J ¼ 8.0 Hz, 2H,
–NCH2CH2), 2.67 (s, 3H, CH3), 2.54 (s, 3H, CH3); MS (ESI) m/z 372
[C19H21N3O3S þ H]þ.
5.1.13.1. 5-Chloro-8-(phenylethynyl)pyrido[4,3-d]pyrimidine 23. 1H
NMR (400 MHz, CDCl3)
d 9.87 (s, 1H, C(2)-H), 9.67 (s, 1H, C(4)-H),
5.1.9. Synthesis of 1-(3,4-dimethoxyphenethyl)-2-thioxo-2,3-
dihydropteridin-4(1H)-one 18 [24]
8.90 (s, 1H, C(7)-H), 7.70–7.69 (m, 2H, Ar–H), 7.43–7.42 (m, 3H,
Ar–H); HRMS (ESI) m/z calculated for [C15H8ClN3 þ H]þ 265.0407,
found 265.0403; 300 mg (45%) yield.
Diamine 13 (300 mg, 0.93 mmol) was added to the stirring
solution of 10 (0.150 g, 1.25 mmol) in ethanol (20 mL). After stirring
at room temperature for 3 h, the reaction was concentrated under
reduced pressure. The crude residue was purified by silica-gel
column chromatography (1–3% MeOH in DCM) to afford 18
(70 mg, 28%) as a light yellow solid: 1H NMR (400 MHz, CDCl3):
5.1.13.2. 5-Chloro-8-{(3,4-dimethoxyphenyl)ethynyl}pyrido[4,3-
d]pyrimidine 24. 1H NMR (400 MHz, CDCl3)
d 9.86 (s, 1H, C(2)-H),
9.67 (s, 1H, C(4)-H), 8.89 (s, 1H, C(7)-H), 7.31 (d, J ¼ 8.4 Hz, 1H,
Ar–H), 7.18 (s, 1H, Ar–H), 6.90 (d, J ¼ 8.4 Hz, 1H, Ar–H), 3.95 (s, 3H,
OCH3), 3.94 (s, 3H, OCH3); MS (ESI) m/z 326 [C17H12ClN3O2 þ H]þ;
400 mg (47%) yield.
d
9.68 (br s, 1H, N(3)-H), 8.75 (s, 1H, C(7)-H), 8.66 (s, 1H, C(6)-H),
6.91–6.80 (m, 3H, Ar–H), 4.97–4.93 (t, 2H, –NCH2CH2), 3.92 (s,

1896
J.J. Marugan et al. / European Journal of Medicinal Chemistry 45 (2010) 1880–1897
5.1.14. General procedure for hydrolysis [27]
for 4 h at this temperature. The reaction mixture was cooled to
room temperature and the solvent was evaporated under reduced
pressure. The crude residue was purified by silica-gel column
chromatography (1–3% MeOH in CH2Cl2) to afford 31 (400 mg, 46%)
NH4OAc (1.89 mmol) was added to a stirring suspension of 23 or
24 (0.18 mmol) in acetic acid (2 mL). The reaction mixture was
heated at 100 ꢁC for 2 h and then was concentrated under reduced
pressure. The resulting residue was purified by silica-gel column
chromatography (2% MeOH in CH2Cl2) to afford compound 25 or 26.
as an off-white solid: 1H NMR (400 MHz, DMSO-d6)
d 10.29 (s, 1H,
N(3)-H), 6.85–6.83 (m, 2H, Ar–H), 6.78 (s, 2H, NH2), 6.73 (d, J ¼ 8 Hz,
1H, Ar–H), 4.53 (s, 1H, C(5)-H), 3.92 (t, J ¼ 7.2 Hz, 2H, –NHCH2CH2),
3.72 (s, 3H, OCH3), 3.70 (s, 3H, OCH3), 2.72 (t, J ¼ 7.6 Hz, 2H,
–NHCH2CH2); MS (ESI) m/z 292 [C14H17N3O4 þ H]þ.
5.1.14.1. 8-(Phenylethynyl)pyrido[4,3-d]pyrimidin-5(6H)-one 25. 1H
NMR (400 MHz, DMSO-d6)
d 12.35 (s, 1H, NH), 9.46 (s, 1H, C(2)-H),
9.44 (s, 1H, C(4)-H), 8.15 (s, 1H, C(7)-H), 7.55–7.53 (m, 2H, Ar–H),
7.44–7.40 (m, 3H, Ar–H); HRMS (ESI) m/z calculated for
[C15H9N3O þ H]þ 247.0746, found 247.0746; 80 mg (42%) yield.
5.1.18. Synthesis of 1-(3,4-dimethoxyphenethyl)-6-propyl-5,6,7,8-
tetrahydropyrimido [4,5-d]pyrimidine-2,4(1H,3H)-dione 32
Propylamine (0.19 mL, 2.40 mmol) and formaldehyde
(0.27 mL, 3.42 mmol) were added to a solution of amine 31
(500 mg, 1.71 mmol) in EtOH (10 mL). The reaction mixture was
heated at reflux for 3 h. The reaction mixture was cooled and
concentrated under reduced pressure. The crude compound was
purified by silica-gel column chromatography (1%–3% MeOH in
CH2Cl2) to afford 32 (180 mg, 28%) as an off-white solid: 1H NMR
5.1.14.2. 8-{(3,4-Dimethoxyphenyl)ethynyl}pyrido[4,3-d]pyrimidin-
5(6H)-one 26. 1H NMR (400 MHz, DMSO-d6)
d 12.31 (s, 1H, NH),
9.46 (s, 1H, C(2)-H), 9.44 (s, 1H, C(4)-H), 8.10 (d, J ¼ 7.8 Hz, 1H, C(7)-
H), 7.11 (d, J ¼ 8.4 Hz, 1H, Ar–H), 7.06 (s, 1H, Ar–H), 6.99 (d,
J ¼ 8.4 Hz, 1H, Ar–H), 3.78 (s, 6H, OCH3); HRMS (ESI) m/z calculated
for [C17H13N3O3 þ H]þ 307.0957, found 307.0958; 80 mg (42%) yield.
(400 MHz, DMSO-d6)
d 10.48 (s, 1H, N(3)-H), 7.11 (s, 1H, N(8)-H),
5.1.15. General procedure for hydrogenation
6.84 (d, J ¼ 8.4 Hz, 1H, Ar–H), 6.79 (s, 1H, Ar–H), 6.72 (d, J ¼ 8.0 Hz,
1H, Ar–H), 3.93–3.89 (m, 4H, –NHCH2CH2 and C(7)-H2), 3.71
(s, 3H, OCH3), 3.69 (s, 3H, OCH3), 3.32 (s, 2H, C(5)-H2), 2.72
(t, J ¼ 7.6 Hz, 2H, CH2CH2CH3), 2.31 (t, J ¼ 6.8 Hz, 2H, NHCH2CH2),
Pd/C (10%, 50% wet, 20 mg dry weight) was added to a solution
of 25 or 26 (0.32 mmol) in 1:1 MeOH and CH2Cl2. The reaction-
mixture flask was attached to a Parr hydrogenator, evacuated,
charged with hydrogen gas to a pressure of 30 psi and shaken for
4–6 h. After this time, the hydrogen was evacuated, and nitrogen
was charged into the bottle. The reaction mixture was filtered
through a pad of Celite and the filtrate was concentrated under
reduced pressure. The crude material was purified by preparative
TLC plate (5% MeOH in CH2Cl2) to afford 27 or 28.
1.48–1.39 (m, 2H, CH2CH2CH3), 0.86 (t,
J
¼
7.2 Hz, 3H,
CH2CH2CH3); HRMS (ESI) m/z calculated for [C19H26N4O4 þ H]þ
374.1954, found 374.1956.
5.2. Biological experiments
4-Methylumbelliferyl-a-D-glucopyranoside (4MU-a-glu),
5.1.15.1. 8-(Phenylethyl)pyrido[4,3-d]pyrimidin-5(6H)-one 27. 1H
N-butyldeoxynojirimycin (NB-DNJ), glycogen from bovine liver, and
the buffer components were purchased from Sigma–Aldrich (St.
Louis, MO). Amplex Red reagent, DMEM, Opti-MEM, and other
medium components were purchased from Invitrogen (Eugene,
NMR (400 MHz, DMSO-d6)
d 11.74 (s, 1H, NH), 9.41 (s, 2H, C(2)-H
and C(4)-H), 7.43 (d, J ¼ 5.6 Hz, 1H, C(7)-H), 7.28–7.24 (m, 2H, Ar–
H), 7.19–7.14 (m, 3H, Ar–H), 2.99–2.96 (m, 2H, –NHCH2CH2), 2.90–
2.86 (m, 2H, –NHCH2CH2); HRMS (ESI) m/z calculated for
[C15H13N3O þ H]þ; 251.1059, found 251.1060; 20 mg (24%) yield.
OR). Resorufin a-D-glucopyranoside (Res-a-glu) was synthesized by
the Imaging Probe Development Center at the National Institutes of
Health. GAA was obtained from residual solution after clinical
infusions of MyozymeÒ (Genzyme). The enzyme solution was
mixed with 30% glycerol and small aliquots were stored at ꢂ80 ꢁC.
The human spleen tissue was homogenized using a food blender
at the maximal speed for 5 min, followed by 10 passes in a motor-
driven 50 mL glass-Teflon homogenizer. The homogenate was
centrifuged at 1000 ꢀ g for 10 min. The supernatant was then
5.1.15.2. 8-(3,4-Dimethoxyphenethyl)pyrido[4,3-d]pyrimidin-5(6H)-
one 28. 1H NMR (400 MHz, DMSO-d6)
d 11.74 (s, 1H, NH), 9.41 (s,
2H, C(2)-H and C(4)-H), 7.40 (s, 1H, C(7)-H), 6.81 (d, J ¼ 8.4 Hz, 1H,
Ar–H), 6.77 (s, 1H, Ar–H), 6.65 (d, J ¼ 7.6 Hz, 1H, Ar–H), 3.70 (s, 3H,
OCH3), 3.69 (s, 3H, OCH3), 2.96 (t, J ¼ 8 Hz, 2H, –NHCH2CH2), 2.81 (t,
J ¼ 8 Hz, 2H, –NHCH2CH2); HRMS (ESI) m/z calculated for
[C17H17N3O3 þ H]þ 311.1270, found 311.1276; 35 mg (23%) yield.
filtered using a 40
mm filter and aliquots of resultant spleen
homogenate were frozen at ꢂ80 ꢁC until use.
5.1.16. Synthesis of 1-(3,4-dimethoxyphenethyl)urea 30
The assay buffer was composed of 50 mM citric acid, 115 mM
K2PO4, 110 mM KCl, 10 mM NaCl, 1 mM MgCl2, and 0.01% Tween-20
at pH 5. It was stored at 4 ꢁC for up to 6 months. A solution of 1 M
NaOH, 1 M glycine at pH 10 was used as the stop solution for the
blue substrate assay. 1 M TRIS–HCl at pH 8.0 was used as the stop
solution for the red substrate assay.
Urea (2.60 g, 44.14 mmol) was added to a suspension of amine
29 (2.00 g, 11.03 mmol) in water (5 mL) and concentrated HCl
(0.13 mL,1.32 mmol). The reaction mixture was heated to reflux and
stirred for 6 h at this temperature. The reaction mixture was cooled
and left to stand at room temperature for 24 h. The resulting
precipitate was filtered, then washed with water (10 mL) and
acetone (10 mL) to afford 30 (1.10 g, 44%) as a white solid: 1H NMR
5.2.1. Enzyme assay in 1536-well plate format
(400 MHz, DMSO-d6)
d
6.84 (d, J ¼ 8.0 Hz, 1H, Ar–H), 6.77 (s, 1H,
In black 1536-well plates, 2 ml/well GAA enzyme solution was
added, followed by 23 nl/well compound in DMSO solution. After
5 min incubation at room temperature, the enzyme reaction was
Ar–H), 6.68 (d, J ¼ 12 Hz, 1H, Ar–H), 5.84 (t, J ¼ 5.2 Hz, 1H,
–NHCH2CH2), 5.40 (s, 2H, NH2), 3.72 (s, 3H, OCH3), 3.69 (s, 3H,
OCH3), 3.15 (q, J ¼ 6.8 Hz, 2H, –NHCH2CH2), 2.57 (t, J ¼ 7.2 Hz, 2H,
NHCH2CH2); MS (ESI) m/z 225 [C11H16N2O3 þ H]þ.
initiated by the addition of 2
m
l/well substrate. After 45 min incu-
bation at 37 ꢁC, the reaction was terminated by the addition of 2
ml/
well stop solution. The assay plate was then measured in the
Viewlux at a 573 nm excitation and 610 nm emission for the red
substrate, and a 365 nm excitation and 440 nm emission for the
blue substrate. The final concentrations of purified GAA, blue
5.1.17. Synthesis of 6-amino-1-(3,4-dimethoxyphenethyl)
pyrimidine-2,4(1H,3H)-dione 31
Urea 30 (1.00 g, 4.46 mmol) and ethyl cyanoacetate (0.71 mL,
6.69 mmol) were added to a solution of Na (160 mg, 6.69 mmol) in
EtOH (10 mL). The reaction mixture was heated to reflux and stirred
substrate, and red substrate were 5.5 nM, 75
respectively.
mM, and 15 mM,

J.J. Marugan et al. / European Journal of Medicinal Chemistry 45 (2010) 1880–1897
1897
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