Synthesis of novel 5-amino-thiazolo[4,5-d]pyrimidines as E. coli and S. aureus SecA inhibitors

Article abstract of DOI:10.1016/j.bmc.2010.10.027

An efficient synthesis of a library of 5-amino-thiazolo[4,5-d]pyrimidines is reported. Regioselective displacements of chlorines, as well as regioselective diazotation reactions are described, which allow the introduction of structural diversity on the sc

Full text of DOI:10.1016/j.bmc.2010.10.027

Bioorganic & Medicinal Chemistry 19 (2011) 702–714
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of novel 5-amino-thiazolo[4,5-d]pyrimidines as E. coli and S. aureus
SecA inhibitors
Mi-Yeon Jang a, Steven De Jonghe a, Kenneth Segers b, Jozef Anné b, Piet Herdewijn a,
⇑
a Katholieke Universiteit Leuven, Rega Institute for Medical Research, Laboratory of Medicinal Chemistry, Minderbroedersstraat 10, 3000 Leuven, Belgium
b Katholieke Universiteit Leuven, Rega Institute for Medical Research, Laboratory of Bacteriology, Minderbroedersstraat 10, 3000 Leuven, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 1 July 2010
Revised 11 October 2010
Accepted 12 October 2010
Available online 19 October 2010
An efficient synthesis of a library of 5-amino-thiazolo[4,5-d]pyrimidines is reported. Regioselective dis-
placements of chlorines, as well as regioselective diazotation reactions are described, which allow the
introduction of structural diversity on the scaffold by consecutive reactions. Screening of this focused
library led to the discovery of SecA inhibitors from Escherichia coli and Staphylococcus aureus.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Thiazolo[4,5-d]pyrimidines
SecA
Bacterial preprotein secretion
Inhibitor
ATPase
1. Introduction
of ATP to the stepwise translocation of preproteins across the bac-
terial cytoplasmic membrane.7,8 Because SecA is a conserved and
After the discovery of penicillin by Alexander Fleming, antibiot-
ics were regarded as wonder drugs for curing virtually all
infections. However, the careless use and overconsumption of anti-
biotics in both human and veterinary medicine has led to the
emergence of antibiotic-resistant bacterial strains. Of major con-
cern is the development of antibiotic resistance in Staphylococcus
aureus, primarily because S. aureus is frequently associated with
hospital and community-acquired infections. Infections with mul-
ti-drug resistant S. aureus have become responsible for huge
healthcare costs and are projected to be responsible for more
deaths this year in the United States than HIV/AIDS.1 Despite this
increasing problem of antibiotic resistance, the number of different
antibiotics available is dwindling and there are only a handful of
new antibiotics in the drug development pipeline.2 Therefore,
there is an urgent need for new antibacterial drugs preferably with
new modes of action to potentially avoid cross-resistance.3 In the
framework of the search for new therapies, new antibiotic targets
are currently being proposed and evaluated, including components
of the bacterial protein secretion pathways.4,5
essential protein in all bacteria but is absent in humans, it is con-
sidered as a promising antibacterial drug target.4,9
However, only a very limited number of SecA inhibitors have
been described so far. Sodium azide displays SecA inhibitory activ-
ity with an IC50 of 1 mM.10 The natural compound CJ-21058, iso-
lated from the fermentation broth of an unidentified fungus,
showed antibacterial activity against Gram-positive multi-drug
resistant bacteria by inhibiting the SecA translocation ATPase
activity.11 Researchers from Merck used a strain of S. aureus that
generates an antisense RNA against SecA to screen for novel anti-
bacterials. By this strategy, they were able to isolate a new cis-
decalin metabolite, which they named pannomycin.12 Scientists
from Wyeth developed an assay that makes use of a SecA-LacZ
fusion reporter construct in Escherichia coli, which is induced when
secretion is perturbed. Several compounds have been discovered
that turned out to be secretion inhibitors. However, they were also
found to have deleterious effects on membranes.13 In 2008,
researchers reported a structure-based virtual screening approach
using the published X-ray structure of E. coli SecA14 for the discov-
ery of small-molecule inhibitors of the intrinsic ATPase activity of
SecA.15 The most potent compounds had IC50 values of about
The major route for the transport of bacterial proteins across or
into the bacterial cytoplasmic membrane is provided by the Sec
pathway.6 A key component of the Sec transport system is the
peripheral membrane ATPase SecA, which couples the hydrolysis
100
optimization process in which they could improve the biological
activity yielding analogues with IC50 of 20–60
M.16
lM. Later on, the same research group reported a hit-to-lead
l
⇑
There is a very limited number of heterocyclic, drug-like scaf-
folds in literature known to be as SecA inhibitors. However, from
Corresponding author. Tel.: +32 (0)16 33 73 87; fax: +32 (0)16 33 73 40.
E-mail address: piet.herdewijn@rega.kuleuven.be (P. Herdewijn).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.10.027

M.-Y. Jang et al. / Bioorg. Med. Chem. 19 (2011) 702–714
703
a drug discovery standpoint of view, it is important to have access
to different heterocyclic structures. The availability of different
chemotypes offers a choice in terms of chemical accessibility and
prospects for lead optimization. As each heterocyclic scaffold has
its own characteristics regarding pharmacology, pharmacokinetics
and toxicology, it is important to have access to a wide variety of
different chemistries. In addition, backup compounds lower the
chance of attrition in later phases of drug development. A very
common strategy in order to find new chemistry starting point is
a high-throughput screening (HTS) campaign of commercially
available compound libraries against the target of interest.
Although the number of available compounds for HTS has dramat-
ically increased over the last few years, large-scale random combi-
natorial libraries have contributed proportionally less to the
identification of novel hits or leads. Especially for antibacterial
HTS campaigns, the success rate is four to fivefold lower than for
targets from other therapeutic areas.17 The results of these HTS
campaign were disappointing. An often cited reason for this lack
of productivity is the lack of chemical diversity in the available
compound libraries. The only way to address this issue involves
the synthesis of novel (i.e., non-commercially available) compound
libraries, based on unexplored chemistry. The structures of most
marketed antibiotics do not obey general rules formulated for
drug-likeness (such as the Lipinski rules). This is due to the com-
plex structure of the cell wall of bacteria, the particular structure
of the bacterial targets and the mode of action which is needed
to generate a bacteriostatic or bactericidal effect. SecA is a periph-
eral membrane ATPase: it adheres only temporarily with the
bacterial membrane (i.e., when SecA is actively involved in prepro-
tein translocation). Up to now, it seems to be refractory to inhibitor
design. Therefore, we have conducted the synthesis (and the use
for SecA screening) of novel scaffolds that have been hardly
explored in antibacterial drug discovery, primarily based on the
chemistry of thiazolo[4,5-d]pyrimidines. Compounds based on this
scaffold are not very well represented in commercial compound
libraries and, although this scaffold is not frequently used in drug
discovery programs, some interesting biological activity has been
associated with this scaffold. Thiazolo[4,5-d]pyrimidine deriva-
tives, prepared as guanine mimics, showed potent in vitro activity
against human cytomegalovirus (HCMV).18 Thiazolo[4,5-d]pyrimi-
dine-5,7-dione analogues have been reported as having
anti-inflammatory activities, due to TNF inhibition.19 2-Oxo-3-
aryl-thiazolo[4,5-d]pyrimidine analogues have been synthesized
as antagonists of the corticotrophin releasing hormone (CRH) R1
receptor.20 2-Thio-3-aryl-thiazolo[4,5-d]pyrimidine derivatives
have been described as having anticancer,21 anti-inflammatory
and antimicrobial activity.22 2-Aminothiazolo[4,5-d]pyrimidines,
that act as CXCR2 receptor antagonists are also known.23 Recently,
2,7-substituted-thiazolo[4,5-d]pyrimidines have been described as
ATP-competitive inhibitors of several protein kinases (EGFR, cSrc,
HER-2, Lyn and c-Abl).24
These initial data demonstrate that thiazolo[4,5-d]pyrimidines
display a plethora of biological activities and this scaffold can
therefore be considered as a privileged structure.25 Priviliged struc-
tures represent a class of molecules capable of binding to multiple
target proteins (receptors or enzymes) with high affinity. The
exploitation of these molecules allows the medicinal chemist to
rapidly discover biologically active compounds across a broad
range of therapeutic areas. In order to fully explore the biological
activity of this scaffold, it is necessary to have access to synthetic
schemes that can be used for the construction of compound
libraries. To our knowledge, there are no systematic studies done
on how to elaborate in a systematic way the chemistry of this com-
pound class. Therefore, the main goal of this research is to develop
synthetic schemes that can be easily adapted for use in parallel
chemistry, and hence are suitable to establish structure–activity
relationship (SAR) studies. We introduced a broad structural vari-
ety into the thiazolo[4,5-d]pyrimidine scaffold in which different
substitution sites can be varied in one synthetic cycle. In this paper,
we wish to report our findings towards the elaboration of this type
of chemistry and its application for the discovery of novel SecA
inhibitors.
Scheme 1. Synthesis of 5-amino-thiazolo[4,5-d]pyrimidines. Reagents and conditions: (a) alcohol, NaH, reflux; (b) amine, water, reflux; (c) KSCN, pyridine, Br2, DMF, 65–5 °C;
(d) DMF/water (4:1 v/v), 140 °C; (e) isoamyl nitrite, TMSBr, CH3CN, rt; (f) NaNO2, concd H2SO4, 80 °C then concd HCl, CuCl, 40 °C; (g) boronic acid, K2CO3, Pd(PPh3)4, dioxane/
water (3:1 v/v), 100 °C; (h) tributylphenylstannane, Pd(PPh3)4, reflux; (i) phenylacetylene, Pd(PPh3)2Cl2, CuI, NEt3, dioxane, 80 °C; (j) amine, NEt3, dioxane, rt; (k) 5% HCl,
100 °C; (l) m-tolyl isocyanate, DMF, rt; (m) p-chlorophenoxyacetyl chloride, pyridine, DMF, rt.; (n) H2, 10% Pd/C, THF/MeOH (2:1 v/v), rt.

704
M.-Y. Jang et al. / Bioorg. Med. Chem. 19 (2011) 702–714
thiocyanate, bromine and pyridine in N,N-dimethylformamide
2. Chemistry
(DMF), followed by refluxing in DMF/water, afforded the 2,5-diami-
no-7-substituted-thiazolo[4,5-d]pyrimidine analogues 3a–c.28 For
the N-acetyl-piperazine analogue 1d, a direct formation of the thia-
zole ring was observed (yielding compound 3d), without the need of
heating at 140 °C. In order to introduce structural variety at position
2, the 2-amino group was converted to bromine via diazotation of
3a–c by treatment with isoamyl nitrite and bromotrimethylsilane
in acetonitrile.23a For the 7-N-acetylpiperazine derivative 4d, we
opted for the introduction of a chlorine using diazotation, followed
by chlorination with CuCl (Sandmeyer reaction). The 2-halo (bro-
mine or chlorine) substituted heterocycle is a versatile starting
material for a wide variety of palladium catalyzed cross-coupling
reactions to form new C–C bonds. Standard reaction conditions for
In order to have access to synthetic strategies that can be used in
high-throughput medicinal chemistry labs, it is desirable to use a
multi-halogenated thiazolo[4,5-d]pyrimidine as a key intermediate
from which, in several consecutive steps, structural variety can be
introduced.Theycan react witha widerange ofsuitable nucleophiles,
but can also be used for a variety of palladium-mediated cross-
coupling reactions such as Hartwig–Buchwald, Heck, Negishi,
Sonogashira, Stille and Suzuki couplings. Therefore, halogenated het-
erocycles are valuablescaffolds forthedesignofheterocycliclibraries
and in addition, they are useful tools to investigate chemical
reactivity.
The synthesis of thiazolo[4,5-d]pyrimidine analogues starts from
commercially available 2,6-diamino-6-chloro-pyrimidine. The 2,6-
diamino-4-substituted pyrimidine analogues 1a–d are easily
obtained from commercially available 2,6-diamino-6-chloro-
pyrimidine by known procedures (Scheme 1). The desired alkoxy
groups were introduced by refluxing 2,6-diamino-4-chloropyrimi-
dine in the appropriate alcohol with sodium hydride,26 whereas
for the introduction of morpholine and N-acetylpiperazine, water
was chosen as solvent.27 Thiocyanation of the 2,6-diamino-
4-substituted pyrimidines 1a–d by treatment with potassium
a Suzuki coupling (method g),29 a Stille coupling (method h),30
a
Sonogashira coupling (method i)31 and a nucleophilic aromatic
substitution reaction (method j)32 of compounds 4a–d afforded
the desired compounds 5a–i in moderate to good yields (Table 1).
The acetyl group of 7-((N-acetyl)piperazin-1-yl)thiazolo[4,5-d]
pyrimidine 5i was cleaved off under acidic conditions, and the
piperazine moiety 6 was coupled with m-tolyl isocyanate (yielding
urea 7a) or with p-chlorophenoxyacetyl chloride (affording amide
7b).
Table 1
Overview of the thiazolo[4,5-d]pyrimidines
R
S
N
R2
N
H2N
N
Reactant
Method
g
Product
R
R2
Yield (%)
88
O
F
4a
5a
N
O
4a
4a
4a
4a
4a
g
h
i
5b
5c
5d
5e
5f
32
50
89
86
93
N
O
N
O
N
O
N
O
j
N
O
H
N
OCH3
j
N
OCH2CH3
4b
4c
g
g
5g
5h
F
F
F
86
76
O
Ac
N
4d
g
5i
73
N

M.-Y. Jang et al. / Bioorg. Med. Chem. 19 (2011) 702–714
705
The presence of two amino groups for the diazotation procedure
presents a potential liability, regarding regioselectivity. Using
compound 4a as representative example, the selective diazotation
of the 2-amino group was proven. The bromine was cleaved off by
catalytic hydrogenation, yielding 5-amino-7-N-morpholinothiazol-
o[4,5-d]pyrimidine 8. The assignment of C(7) at 159 ppm in the
13C NMR spectrum was staightforward by the observation of a
HMBC correlation (3J) between the protons of the morpholino moi-
ety and C(7) (Fig. 1). A clear HMBC cross-peak was seen between
C(7a) (96 ppm) and the proton at 9.5 ppm (3J), which is only possi-
ble if this signal is due to a proton at position 2 of the scaffold 8.
This confirms the presence of the hydrogen at position 2 of the
scaffold and hence, proves that the diazotation preferentially takes
place at the 2-amino group. This observation is in clear agreement
with previous results in which a selective diazotation of the amino
group at position 2 of 2,5-diaminothiazolo[4,5-d]pyrimidin-7-
(6H)one has been described and was proven by chemical
transformation.33
pyrimidin-7(6H)one 9 (Scheme 2).33 The lactam functionality of
compound was chlorinated by refluxing in POCl3 with
N,N-dimethylaniline,34 yielding
5-amino-2,7-dichlorothiazol-
9
a
o[4,5-d]pyrimidine 10. It was expected that the chlorine at position
7 would be more reactive. It is indeed known that for similar
heterocycles, such as pyrido[3,2-d]pyrimidines35 and pyrido[2,3-
d]pyrimidines,36 the chlorine at position 4 of the scaffold is extre-
mely labile and can be easily displaced by nucleophiles. Moreover,
when a closely related 6,8-dichloropurine was subjected to a Stille
coupling, selective coupling at the 6 position was achieved.37
However, reaction of 10 with 1 equiv of a nucleophile allowed
selective substitution of chlorine at position 2. Two representative
examples of nucleophiles were selected:
a primary amine
(methoxyethylamine) and a secondary amine (N-Boc-piperazine).
The subsequent Suzuki coupling of 11a and 11b with 4-fluor-
ophenylboronic acid afforded compounds 12a and 12b. The Boc
group of 12b was cleaved off under acidic conditions, and the
piperazine moiety 13 was further derivatised with m-tolyl isocya-
nate or p-chlorophenoxyacetyl chloride affording compounds 14a
and 14b, respectively.
In order to prove the regiochemistry of the nucleophilic aro-
matic substitution reaction, the remaining chlorine of compound
11b was cleaved off catalytically using hydrogen and palladium
as catalyst. The obtained compound turned out to be very insoluble
in common organic solvents, and therefore, the amino group was
acetylated yielding compound 15, which is easier to handle. The
structure of compound 15 was solved by Heteronuclear Multiple
Bond Correlation (HMBC) spectroscopy (Fig. 1). The 13C signal at
d = 170 ppm, arising from C(5), was used as a starting point in
the structural elucidation. A clear HMBC cross-peak was seen be-
tween C(5) and the proton at 8.7 ppm, which is only possible if this
signal is due to a proton at position 7 of the scaffold. A direct cou-
pling (1J) of H(7) with C(7) is observed, making the assignment of
C(7) at d = 149 ppm possible. Also, clear HMBC correlations are
found between H(7) and C(7a) (2J) and C(3a) (3J). These findings
To introduce a variety of substituents at positions 2 and 7 of the
5-amino-thiazolo[4,5-d]pyrimidine scaffold, a 2,7-dihalo-thiazol-
o[4,5-d]pyrimidine was considered as a ideal key intermediate. If
the two halogen atoms show different reactivity, this characteristic
can then be exploited for sequential introduction of different sub-
stituents. The 5-amino-2,7-dichlorothiazolo[4,5-d]pyrimidine was
synthesized from the known 5-amino-2-chlorothiazolo[4,5-d]
Figure 1. Structure elucidation of compounds 8 and 15.
Scheme 2. Synthesis of 5-amino-7-(4-fluorophenyl)thiazolo[4,5-d]pyrimidines. Reagents and conditions: (a) POCl3, N,N-dimethylaniline, reflux; (b) amine, NEt3, dioxane,
100 °C; (c) 4-fluorophenylboronic acid, K2CO3, Pd(PPh3)4, dioxane/water (3:1 v/v), 100 °C; (d) TFA, CH2Cl2, rt; (e) m-tolylisocyanate, DMF, rt; (f) p-chlorophenoxyacetyl
chloride, pyridine, DMF, rt; (g) H2, 10% Pd/C, THF/MeOH (2:1 v/v), rt; (h) Ac2O, reflux.
Scheme 3. Determination of regioselectivity via chemical synthesis. Reagents and conditions: (a) NaH, EtOH, reflux; (b) 2-methoxyethylamine, dioxane, 60 °C.

706
M.-Y. Jang et al. / Bioorg. Med. Chem. 19 (2011) 702–714
prove that the N-Boc-piperazinyl group is present at position 2,
whereas C(7) bears a hydrogen atom.
Having established a reliable synthetic scheme for the prepara-
tion of 5-amino-2,7-disubstituted thiazolo[4,5-d]pyrimidine ana-
logues, we envisioned to use this know-how to determine the
regiochemistry of the nucleophilic aromatic substitution on the
2,7-dichlorothiazolo[4,5-d]pyrimidine scaffold (as explained in
Schemes 1 and 2) through chemical synthesis (Scheme 3). We
opted for the simpler substituents on the heterocyclic scaffold,
making the interpretation of NMR spectra easier. Therefore, com-
pound 11a was reacted with ethanol in basic condition, yielding
compound 16. The same compound has been made independently
starting from 5-amino-2-bromo-7-ethoxythiazolo[5,4-d]pyrimi-
dine 4b. In this sequence, the ethoxy group is undoubtedly fixed
at position 7 (as it is introduced in the first step of the synthesis),
and the methoxyethylamine side chain is positioned at position 2
(as has been proven that diazotation preferentially takes place at
position 2). As the compounds obtained in both ways were com-
pletely identical (NMR, TLC), it indicates that the chlorine at posi-
tion 2 is more reactive than the one at position 7 and it supports
our earlier findings, described in Scheme 2.
Figure 3. Time courses of intrinsic, membrane and translocation ATP hydrolysis by
ecSecA. EcSecA (1 g/50 l) was incubated with 100 M ATP in the presence of
l
l
l
IMVs containing overexpressed ecSecYEG and the preprotein ProPhoA (4; trans-
location ATPase), in the presence of IMVs alone (5; membrane ATPase) or in the
absence of IMVs and ProPhoA (s; intrinsic ATPase). At several time points after the
start of the ATPase reaction, released Pi was quantified using the malachite green
method as described in ‘Section 5’. Data represent the average of three independent
experiments, and standard deviations were less <5%.
3. Biological evaluation
Compounds were assayed in an in vitro colorimetric assay
employing recombinant E. coli or S. aureus SecA, isolated inner
membrane vesicles containing overexpressed SecYEG and the
E. coli preprotein AlkProPhoA(Cys-). The SecA proteins and the
AlkProPhoA preprotein have been purified via Ni-NTA-affinity
chromatography. The purity of the proteins has been verified via
SDS–PAGE and this did not reveal any contaminating proteins
(Fig. 2).
Compounds were evaluated at an initial concentration of 200 lM
for their ability to inhibit the intrinsic ATPase activity of E. coli and S.
aureus SecA, using the malachite green colorimetric method for the
detection of free inorganic phosphate.38 Because the intrinsic ATP-
ase activityof SecA representsa restingnon-activestate, compounds
were also evaluated for their ability to inhibit the preprotein-
stimulated translocation ATPase activity of SecA.
Figure 4. Time courses of intrinsic, membrane and translocation ATP hydrolysis by
ecSecA. The linear phase of the progress curves in Figure 3 is shown, indicating that
the intrinsic, membrane and translocation ATPase activity of ecSecA is linear during
the first 30 min of the reaction.
Before setting up these assays, time curves of intrinsic, mem-
brane and translocation ATP hydrolysis by E. coli SecA have been
generated (Figs. 3 and 4). Under the assay conditions (see ‘Section
5’), the intrinsic and membrane ATPase activities are linear up to
60 min, whereas for the translocation ATPase assay, ATP hydrolysis
follows a linear time course during the first 30 min of the reaction.
Under the experimental conditions used, the intrinsic ATPase
activity is about twofold stimulated by E. coli membrane vesicles
containing overexpressed SecYEG, and approximately 10-fold
when also the E. coli preproteine AlkProPhoA(Cys-) is present.
In order to define the experimental conditions for final screen-
ing assays, we have generated time curves of intrinsic ATP hydro-
lysis at different enzyme concentrations. As can be derived from
Figure 5, the time course of intrinsic E. coli SecA ATPase activity
is linear up to 120 min for E. coli SecA concentrations between
0.5 and 2.5
l
g ecSecA/50
l
l. Based on these results, we have de-
g/50 l and an
fined a fixed E. coli SecA concentration of 2.5
l
l
incubation time of 2 h for screening assays. As the hydrolysis of
ATP under these experimental conditions is linear up to
120 min, the rate of ATP hydrolysis can be determined by dividing
the amount of Pi formed by 120 (yielding nmol Pi/min). Similar
experiments have been performed for S. aureus SecA (Fig. 6) from
which the final conditions for the S. aureus SecA screening assay
have been established (28 °C, 20 lg saSecA1/50 ll, 2 h incubation
time).
Compounds 5i, 7b, 12a, 12b and 14a display around 50% inhibi-
tion of the intrinsic E. coli SecA ATPase activity at 200 M (Fig. 7).
l
They all have either a fluorophenyl or a piperazine substituent at
position 7 of the scaffold. Two compounds (5b and 14b) display
more pronounced E. coli SecA inhibition, with 70% and 76% inhibi-
tion, respectively. These compounds were selected for dose–
responses titration curves, yielding IC50 values of 197 and
Figure 2. SDS–Polyacrylamide gel electrophoresis of purified recombinant E. coli
SecA, S. aureus SecA1 and the E. coli preprotein ProPhoA. Lane M: molecular weight
marker; Lane 1: ecSecA; Lane 2: saSecA1; Lane 3: ProPhoA.
135 lM, respectively (Fig. 8).

M.-Y. Jang et al. / Bioorg. Med. Chem. 19 (2011) 702–714
707
Figure 8. Inhibition of the intrinsic ATPase activity of ecSecA by compound 5b and
14b. Initial rates of intrinsic ATP hydrolysis by ecSecA were measured at 37 °C in the
presence of 100 lM ATP and various concentrations (0–200 lM) of compound 5b
(.) and 14b (ꢀ). Rates were expressed relative to the rate of ATP hydrolysis
measured in the absence of compound. IC50 values were determined by fitting the
data by nonlinear regression analysis using GraphPad Prism. Values are the means
of three independent experiments, with error bars representing the standard
deviation.
Figure 5. Time courses of intrinsic ATP hydrolysis by ecSecA. EcSecA (0.5–10
lg/
50 l) was incubated at 37 °C with 100 M ATP in 50 mM Tris–HCl, 50 mM KCl,
l
l
5 mM MgCl2, 0.4 mg/ml BSA, pH 8.0). At several time points after the start of the
ATPase reaction, released Pi was quantified using the malachite green method as
described in experimental section. Data represent the average of three independent
experiments, and standard deviations were less <5%. Symbols used: h 0.5 lg
ecSecA/50
10 lg ecSecA/50
l
l; 4 1
l
l.
g ecSecA/50 ll; 5 2.5 lg ecSecA/50 ll; } 5 lg ecSecA/50 ll; s
l
By comparing the biological activity of compounds 12b, 13, 14a
and 14b, it is evident that by subtle structural variation of the
piperazine moiety the biological activity can be influenced. The
free piperazine moiety (compound 13) affords 35% inhibition,
whereas derivatisation as a carbamate (compound 12b) or a urea
(compound 14a) furnished compounds displaying 50% inhibition.
Reaction of the piperazine with an acyl chloride affords an amide
congener 14b with an IC50 value of 135 lM. In the 7-N-morpholino
series, the nature of the substituent at position 2 seems important
for E. coli SecA inhibition. The styryl group (compound 5b) is the
most potent compound (70% inhibition at 200 lM), whereas other
substituents, such as alkylamino groups (compounds 5e and 5f),
phenyl (compounds 5a and 5c) or acetylene moiety (compound
5d) are less active. The fact that compound 5c (which is structur-
ally very related to compound 5b) is totally devoid of SecA inhibi-
tory activity, points towards a specific inhibitory activity of the
compounds.
In general, the compounds were less efficient in inhibiting the
translocation E. coli SecA ATPase activity when compared to their
effect on the intrinsic ATPase activity of E. coli SecA. However,
the two most potent analogues (compounds 5b and 14b) had also
Figure 6. Time courses of intrinsic ATP hydrolysis by saSecA1. saSecA1 (2.5–50
lg/
50 l) was incubated at 28 °C with 100 M ATP in 50 mM HEPES, 50 mM KCl, 5 mM
l
l
MgCl2, 0.4 mg/ml BSA, pH 7.5). At several time points after the start of the ATPase
reaction, released Pi was quantified using the malachite green method as described
in Materials and Methods. Data represent the average of three independent
experiments, and standard deviations were less <5%. Symbols used: j 2.5
saSecA1/50 l; .5 g saSecA1/50 g saSecA1/50 l; } 20 g saSecA1/
l; 5 10
50 l; s 30 g saSecA1/50 l; h 40 g saSecA1/50 g saSecA1/50 l.
l; 4 50
lg
l
l
l
l
l
l
l
l
l
l
l
l
l
Figure 7. Inhibition of the intrinsic ATPase activity of saSecA and ecSecA and of the translocation ATPase activity of ecSecA by different thiazolo[4,5-d]pyrimidines. Initial
rates of intrinsic ATP hydrolysis by saSecA (dark grey bars) and ecSecA (white bars) and the translocation ATPase activity of ecSecA (light grey bars) were measured in the
presence of 100
lM ATP and 200 lM compound. Rates were expressed relative to the rate of ATP hydrolysis measured in the absence of compound. Values are the means of
three independent experiments, with error bars representing the standard deviation.

708
M.-Y. Jang et al. / Bioorg. Med. Chem. 19 (2011) 702–714
60
(Fig. 9B).
Overall, it seems that the compounds are less active against
lM for inhibition of the SecA translocation ATPase activity
SecA of S. aureus than of E. coli. An explanation for this observation
cannot be provided at this moment however, but local structural
differences in the compound binding site of E. coli and S. aureus
SecA might explain this apparent discrepancy. At a concentration
of 200 lM, the most potent compounds (5b, 5d, 7a and 14a) dis-
play around 40% inhibition of the S. aureus SecA ATPase activity.
To the best of our knowledge, these compounds represent the first
examples of S. aureus SecA inhibitors.
Although the activity is weak, they can be used as starting
points for further optimization. The chemistry described in this pa-
per will enable to rapidly explore the SAR with respect to SecA
inhibition and to improve the potency. In addition, compound 5b
has a molecular weight of only 339 and can be considered as a frag-
ment, rather than a hit. Hence, fragment based drug discovery
could be employed as the X-ray crystal structure of SecA from
E. coli is available.14
4. Conclusion
In summary, the investigation of regioselective reactions on a
2,7-dichlorothiazolo[4,5-d]pyrimidine scaffold has shown that
the chlorine at position 2 is more reactive than the one at position
7. In addition, regioselective diazotations on a 2,5-diamino-thiazol-
o[4,5-d]pyrimidine leads selectively to the 2-halo-5-amino-thiaz-
olo[4,5-d]pyrimidine. These findings can be exploited for the
sequential introduction of substituents at positions 2 and 7 and al-
low for the construction of a highly diverse 5-amino-thiazolo[4,5-
d]pyrimidine library. Screening of this compound library against
SecA led to the discovery of hits with activity against SecA from
E. coli and S. aureus. Currently, we are exploiting this chemistry
in order to improve the biological activity of the hits.
Figure 9. Inhibition of the ecSecA translocation ATPase activity by compound 14b.
(A) Initial rates of translocation ATP hydrolysis by ecSecA were measured in the
presence of different ATP (0–2 mM) and compound concentrations (0–200 lM), as
described in ‘Section 5’. Apparent Km and Vmax values were determined by fitting
the data by nonlinear regression analysis to the Michaelis–Menten equation. Data
represent the average of three independent experiments, with error bars repre-
senting the standard deviation. Symbols used: s no inhibitor; 4 25
50 M inhibitor; h 100 M inhibitor. (B) Dixon plot of the data
M inhibitor; 5 200
in Figure 4A. Linear regression of the data yielded a Ki value of 60 M for inhibition
of the ecSecA translocation ATPase. Symbols used: j 5 M ATP; N 10 M ATP;
.50 M ATP; ꢀ 100 M ATP; s 500 M ATP; h 1000 M ATP; } 2000 M ATP.
lM inhibitor; }
5. Experimental section
5.1. General
l
l
l
l
l
l
l
l
l
l
l
For all reactions, analytical grade solvents were used. All mois-
ture-sensitive reactions were carried out in oven-dried glass-ware
(135 °C). 1H and 13C NMR spectra were recorded with a Bruker
Advance 300 (1H NMR: 300 MHz, 13C NMR: 75 MHz), using
tetramethylsilane as internal standard for 1H NMR spectra and
DMSO-d6 (39.5 ppm) or CDCl3 (77.2 ppm) for 13C NMR spectra.
Abbreviations used are: s = singlet, d = doublet, t = triplet, q = quar-
tet, m = multiplet, br s = broad singlet. Coupling constants are ex-
pressed in Hertz. Mass spectra are obtained with a Finnigan LCQ
advantage Max (ion trap) mass spectrophotometer from Thermo
Finnigan, San Jose, CA, USA. Exact mass measurements are
performed on a quadrupole time-of-flight mass spectrometer
(Q-tof-2, Micromass, Manchester, UK) equipped with a standard
electrospray-ionization (ESI) interface. Samples were infused in
pronounced effect on the translocation ATPase activity of SecA
(more than 50% inhibition at 200 M).
The most promising analogue (compound 14b) was selected for
Ki-determination. Initial rates of ATP hydrolysis were measured in
the E. coli SecA translocation ATPase activity assay at different ATP
l
(0–2 mM) and inhibitor (0–200 lM) concentrations (Fig. 9A). Data
were fit by nonlinear regression analysis to the Michaelis–Menten
equation and the apparent Km and Vmax values were determined for
each inhibitor concentration (Table 2). It is clear that compound
14b increases Km and decreases Vmax, which is indicative of
‘mixed-type’ inhibition. In other words, inhibition of the ecSecA
translocation ATPase activity by compound 14b has both a non-
competitive and a competitive component, resulting from a combi-
nation of a decreased turnover number and decreased affinity of
the active site for ATP. Dixon plot analysis yielded a Ki value of
i-PrOH/H2O (1:1) at 3 ll/min. Melting points are determined on a
Barnstead IA 9200 and are uncorrected. Precoated aluminum sheets
(Fluka Silica gel/TLC-cards, 254 nm) were used for TLC. Column
chromatography was performed on ICN silica gel 63–200, 60 Å.
Table 2
5.2. General procedure for the preparation of compounds 1–16
5.2.1. 6-Morpholinopyrimidine-2,4-diamine (1a)
Apparent Km and Vmax values for the ecSecA translocation ATPase activity in the
presence of different concentrations of compound 14b
l
M Inhibitor
50
To
a suspension of 2,6-diamino-4-chloropyrimidine (3.0 g,
0
25
100
86.5
484.0
200
54.2
567.4
20.7 mmol) in water (100 ml) was added morpholine (7.26 ml,
83.0 mmol). The mixture was refluxed overnight. After cooling
down to room temperature, the solution was treated with a 1 N
Vmax,app
Km,app
136.1
288.1
115.8
298.4
96.1
319.8

M.-Y. Jang et al. / Bioorg. Med. Chem. 19 (2011) 702–714
709
NaOH solution to lead a precipitate. The resulting solid was filtered
off, washed with water and dried over P2O5, furnishing the title
compound as a pale yellow solid (3.3 g, 81%). 1H NMR (300 MHz,
DMSO, 25 °C): d = 5.78 (s, 2H, NH2), 5.59 (s, 2H, NH2), 5.03 (s, 1H,
CH), 3.60 (br s, 4H, O(CH2)2), 3.31 (br s, 4H, N(CH2)2) ppm. 13C
NMR (75 MHz, DMSO, 25 °C): d = 165.2, 164.1, 162.6, 74.1, 65.9,
44.2 ppm. MS: 196.06 [M+H]+
5.2.6. 6-Ethoxy-5-thiocyanatopyrimidine-2,4-diamine (2b)
This compound was synthesized from 1b according to the pro-
cedure for the preparation of compound 2a. The crude residue was
purified by flash chromatography on silica (CH2Cl2/MeOH 50:1) to
yield the title compound as a pale yellow solid (89%). Mp 182 °C.
1H NMR (300 MHz, DMSO, 25 °C): d = 6.88 (s, 2H, NH2), 6.61 (s,
2H, NH2), 4.31 (q, J = 7.1 Hz, 2H, CH2), 1.30 (t, J = 7.1 Hz, 3H, CH3)
ppm. 13C NMR (75 MHz, DMSO, 25 °C): d = 169.1, 165.6, 163.1,
112.1, 66.5, 62.0, 14.6 ppm. HRMS: calcd for C7H10N5OS [M+H]+
212.06061, found 212.05973.
5.2.2. 6-Ethoxypyrimidine-2,4-diamine (1b)
To a solution of NaH (2.77 g, 69.2 mmol) in ethanol (150 ml)
was added 2,6-diamino-4-chloropyrimidine (5.0 g, 34.6 mmol).
The mixture was refluxed for 6 h. After cooling, the solution was
neutralized with a 5–6 N HCl solution in isopropyl alcohol. After
removing the solvents in vacuo, the residue was purified by chro-
matography on silica gel (CH2Cl2/MeOH 40:1), yielding the title
compound as a white solid (4.0 g, 75%). Mp 164–165 °C. 1H NMR
(300 MHz, DMSO, 25 °C): d = 6.00 (s, 2H, NH2), 5.88 (s, 2H, NH2),
5.03 (s, 1H, CH), 4.13 (q, J = 7.1 Hz, 2H, CH2), 1.22 (t, J = 7.1 Hz,
3H, CH3) ppm. 13C NMR (75 MHz, DMSO, 25 °C): d = 170.0, 165.9,
162.9, 76.1, 60.2, 14.7 ppm. HRMS: calcd for C6H11N4O [M+H]+
155.09329, found 155.09260.
5.2.7. 6-(Isopentyloxy)-5-thiocyanatopyrimidine-2,4-diamine
(2c)
This compound was synthesized from 1c, according to the pro-
cedure for the preparation of compound 2a. The crude residue was
purified by flash chromatography on silica (CH2Cl2/MeOH 50:1) to
yield the title compound as a pale yellow solid (76%). Mp 152 °C.
1H NMR (300 MHz, DMSO, 25 °C): d = 6.87 (s, 2H, NH2), 6.59 (s,
2H, NH2), 4.29 (t, J = 6.6 Hz, 2H, OCH2), 1.74 (sixtet, J = 6.6 Hz, 1H,
CH), 1.59 (q, J = 6.7 Hz, 2H, CH2), 0.93 (d, J = 6.6 Hz, 6H, CH3) ppm.
13C NMR (75 MHz, DMSO, 25 °C): 169.1, 165.6, 163.1, 112.1, 66.6,
64.6, 37.2, 24.6, 22.4 ppm. HRMS: calcd for C10H16N5OS [M+H]+
254.10756, found 254.10689.
5.2.3. 6-(Isopentyloxy)pyrimidine-2,4-diamine (1c)
This compound was synthesized according to the procedure for
the preparation of compound 1b, using isoamyl alcohol. The crude
residue was purified by flash chromatography on silica (CH2Cl2/
MeOH 40:1) to yield the title compound as a pale yellow oil
(95%). 1H NMR (300 MHz, CDCl3, 25 °C): d = 5.23 (s, 1H, C@CH),
4.69 (s, 2H, NH2), 4.51 (s, 2H, NH2), 4.18 (t, J = 6.7 Hz, 2H, OCH2),
1.77 (m, 1H, CH), 1.60 (q, J = 6.7 Hz, 2H, CH2), 0.94 (d, J = 6.6 Hz,
6H, CH3) ppm. 13C NMR (75 MHz, CDCl3, 25 °C): d = 170.6, 165.3,
162.3, 76.6, 63.8, 37.1, 24.3, 21.9 ppm. HRMS: calcd for C9H17N4O
[M+H]+ 197.14024, found 197.13955.
5.2.8. 7-Morpholinothiazolo[4,5-d]pyrimidine-2,5-diamine (3a)
6-Morpholino-5-thiocyanatopyrimidine-2,4-diamine 2a (3.5 g,
14.3 mmol) was heated at 140 °C in a DMF/water solution (4:1,
50 ml) for two days. After cooling, the resulting solid was filtered
off, washed with water and ethyl acetate, furnishing the title com-
pound as a yellow solid (2.1 g, 58%). Mp 242 °C. 1H NMR (300 MHz,
DMSO, 25 °C): d = 7.95 (s, 2H, NH2), 6.01 (s, 2H, NH2), 3.66 (br s, 4H,
O(CH2)2), 3.58 (br s, 4H, N(CH2)2) ppm. 13C NMR (75 MHz, DMSO,
25 °C): d = 171.2, 170.6, 160.9, 157.8, 90.8, 65.9, 45.7 ppm. HRMS:
calcd for C9H13N6OS [M+H]+ 253.08715, found 253.08621.
5.2.4. 1-(4-(2,6-Diaminopyrimidin-4-yl)piperazin-1-yl)
ethanone (1d)
5.2.9. 7-Ethoxythiazolo[4,5-d]pyrimidine-2,5-diamine (3b)
This compound was synthesized from 2b according to the pro-
cedure for the preparation of compound 3a. The crude residue was
purified by flash chromatography on silica (CH2Cl2/MeOH 20:1) to
yield the title compound as a pale yellow solid (76%). Mp 224 °C.
1H NMR (300 MHz, DMSO, 25 °C): d = 8.05 (s, 2H, NH2), 6.28 (s,
2H, NH2), 4.37 (q, J = 7.1 Hz, 2H, CH2), 1.30 (t, J = 7.1 Hz, 3H, CH3)
ppm. 13C NMR (75 MHz, DMSO, 25 °C): d = 172.7, 171.6, 162.6,
162.2, 92.4, 61.4, 14.6 ppm. HRMS: calcd for C7H10N5OS [M+H]+
12.06061, found 212.05993.
To a solution of 2,6-diamino-4-chloropyrimidine (3.0 g, 20.8
mmol) in water (100 ml) was added N-acetylpiperazine (10.64 g,
83.0 mmol). The mixture was refluxed for 21 h. The orange solu-
tion was cooled down and made alkaline with a 10 M NaOH solu-
tion. The white precipitate was filtered off, washed with cold water
and dried over P2O5 in a vacuum desiccator to yield the title com-
pound as a white solid (3.3 g, 67%). Mp 274 °C 1H NMR (300 MHz,
DMSO, 25 °C): d = 5.74 (s, 2H, NH2), 5.51 (s, 2H, NH2), 5.04 (s, 1H,
CH), 3.33–3.46 (m, 8H, N(CH2)2, CON(CH2)2), 2.02 (s, 3H, CH3)
ppm. 13C NMR (75 MHz, DMSO, 25 °C): 168.3, 165.2, 163.5, 162.7,
74.1, 45.2, 43.7, 43.3, 40.5, 21.2 ppm. HRMS: calcd for C10H17N6O
[M+H]+ 237.1464, found 237.1454.
5.2.10. 7-(Isopentyloxy)thiazolo[4,5-d]pyrimidine-2,5-diamine
(3c)
This compound was synthesized from 2c in a yield of 91%,
according to a procedure for the preparation of compound 3a.
Mp 212 °C. 1H NMR (300 MHz, DMSO, 25 °C): d = 8.06 (s, 2H,
NH2), 6.30 (s, 2H, NH2), 4.35 (t, J = 6.7 Hz, 2H, OCH2), 1.69 (sixtet,
J = 6.5 Hz, 1H, CH), 1.59 (q, J = 6.6 Hz, 2H, CH2), 0.92 (d, J = 6.5 Hz,
6H, CH3) ppm. 13C NMR (75 MHz, DMSO, 25 °C): d = 172.5, 171.6,
162.7, 162.1, 92.4, 64.1, 37.2, 24.7, 22.4 ppm. HRMS: calcd for
5.2.5. 6-Morpholino-5-thiocyanatopyrimidine-2,4-diamine (2a)
A solution of 6-morpholinopyrimidine-2,4-diamine 1a (5.0 g,
25.6 mmol) and potassium thiocyanate (14.9 g, 0.15 mol) in DMF
(120 ml) was heated at 65 °C. Pyridine (4.14 ml, 51.2 mmol) was
added and the solution cooled to 5 °C. Bromine (1.31 ml,
25.6 mmol) was added slowly and the reaction mixture stirred
for 2 h at 5–10 °C. The reaction mixture was poured into ice-water
(100 ml) and stirred for 1 h. The volatiles were removed under re-
duced pressure and the resulting solid was diluted with water, fil-
tered and dried. The crude solid was purified by flash
chromatography on silica gel (CH2Cl2/MeOH 30:1) to yield the title
compound as white solid (3.61 g, 56%). Mp 161 °C. 1H NMR
(300 MHz, DMSO, 25 °C): d = 6.78 (s, 2H, NH2), 6.36 (s, 2H, NH2),
3.69 (br s, 4H, O(CH2)2), 3.41 (br s, 4H, N(CH2)2) ppm. 13C NMR
(75 MHz, DMSO, 25 °C): d = 168.1, 166.5, 162.6, 112.3, 69.1, 66.1,
49.2 ppm. HRMS: calcd for C9H13N6OS [M+H]+ 253.08715, found
253.08637.
C
10H16N5OS [M+H]+ 254.10756, found 254.10675.
5.2.11. 1-(4-(2,5-Diaminothiazolo[4,5-d]pyrimidin-7-yl)
piperazin-1-yl)ethanone (3d)
This compound was synthesized from 1d according to the pro-
cedure for the preparation of compound 2a. The crude residue was
triturated with hot methanol to yield the title compound as a pale
yellow solid (52%). Mp 263 °C. 1H NMR (300 MHz, DMSO, 25 °C):
d = 7.85 (s, 2H, NH2), 5.90 (s, 2H, NH2), 3.53–3.64 (m, 8H,
N(CH2)2, CON(CH2)2), 2.03 (s, 3H, CH3) ppm. 13C NMR (75 MHz,
DMSO, 25 °C): 172.2, 170.2, 168.5, 161.6, 157.5, 90.9, 45.3, 45.2,

710
M.-Y. Jang et al. / Bioorg. Med. Chem. 19 (2011) 702–714
44.9, 40.6, 21.2 ppm. HRMS: calcd for C11H16N7OS [M+H]+
294.1137, found 294.1111.
5.2.16. 2-(4-Fluorophenyl)-7-morpholinothiazolo[4,5-d]
pyrimidin-5-amine (5a)
To a solution of 2-bromo-7-morpholinothiazolo[4,5-d]pyrimi-
din-5-amine 4a (0.10 g, 0.32 mmol) in dioxane/water (3:1, 4 ml),
was added 4-fluorophenylboronic acid (53 mg, 0.38 mmol),
K2CO3 (0.18 g, 1.27 mmol) and Pd(PPh3)4 (37 mg, 0.03 mmol). The
reaction mixture was refluxed under N2 overnight. After cooling
down to room temperature, 1 N HCl was added slowly to neutral-
ize the mixture to pH 7–8. The mixture was extracted with dichlo-
romethane, brine and dried over Na2SO4. After removing the
solvent, the residue was purified by chromatography on silica gel
(CH2Cl2/MeOH 70:1) to yield the title compound as a pale yellow
solid (92.4 mg, 88%). Mp 296 °C. 1H NMR (300 MHz, CDCl3,
25 °C): d = 8.09–8.14 (m, 2H, ArH), 7.17 (t, J = 8.5 Hz, 2H, ArH),
4.82 (s, 2H, NH2), 3.88 (s, 4H, O(CH2)2), 3.84 (s, 4H, N(CH2)2)
ppm. 13C NMR (75 MHz, CDCl3, 25 °C): d = 172.9, 170.9, 165.2 (d,
J = 251.6 Hz), 161.9, 159.5, 129.6 (d, J = 8.8 Hz), 129.2 (d,
J = 3.3 Hz), 116.5 (d, J = 22.0 Hz), 99.8, 66.9, 46.3 ppm. HRMS: calcd
for C15H15FN5OS [M+H]+ 332.09813, found 332.09694.
5.2.12. 2-Bromo-7-morpholinothiazolo[4,5-d]pyrimidin-5-
amine (4a)
To a solution of 7-morpholinothiazolo[4,5-d]pyrimidine-2,5-
diamine 3a (1.0 g, 3.96 mmol) in acetonitrile (25 ml) were added
bromotrimethylsilane (1.57 ml, 11.9 mmol) and isoamyl nitrite
(0.67 ml, 4.76 mmol). The mixture was stirred at room tempera-
ture for two days. After removing the solvents, the crude residue
was extracted with dichloromethane, brine and dried over
Na2SO4. After evaporating the solvents, the crude residue was
purified by chromatography on silica gel (CH2Cl2/MeOH 50:1) to
yield the title compound as a pale yellow solid (0.69 g, 55%). Mp
253 °C. 1H NMR (300 MHz, CDCl3, 25 °C): d = 4.85 (s, 2H, NH2),
3.79 (s, 8H, O(CH2)2, N(CH2)2) ppm. 13C NMR (75 MHz, CDCl3,
25 °C): d = 170.8, 161.8, 158.6, 102.0, 66.7, 46.1 ppm. HRMS: calcd
for C9H11BrN5OS [M+H]+ 315.98677/317.98472, found 315.98560/
317.98353.
5.2.17. (E)-7-Morpholino-2-styrylthiazolo[4,5-d]pyrimidin-5-
amine (5b)
5.2.13. 2-Bromo-7-ethoxythiazolo[4,5-d]pyrimidin-5-amine (4b)
This compound was synthesized from 3b according to the pro-
cedure for the preparation of compound 4a. The crude residue was
purified by flash chromatography on silica (CH2Cl2/MeOH 80:1) to
yield the title compound as a pale yellow solid (48%). Mp 203 °C. 1H
NMR (300 MHz, DMSO, 25 °C): d = 5.06 (s, 2H, NH2), 4.51 (q,
J = 7.1 Hz, 2H, CH2), 1.42 (t, J = 7.1 Hz, 3H, CH3) ppm. 13C NMR
(75 MHz, CDCl3, 25 °C): d = 170.7, 163.2, 162.9, 146.2, 101.8, 62.8,
14.2 ppm. HRMS: calcd for C7H8BrN4OS [M+H]+ 274.96022/
276.95817, found 274.95958/276.95757.
This compound was synthesized according to a procedure for
the preparation of compound 5a, using (E)-styrylboronic acid.
The crude residue was purified by flash chromatography on silica
(CH2Cl2/MeOH 70:1) to yield the title compound as a pale yellow
solid (32%). Mp 255 °C. 1H NMR (300 MHz, CDCl3, 25 °C): d = 7.74
(d, J = 16.0 Hz, 1H, CH), 7.58 (d, J = 8.0 Hz, 2H, ArH), 7.39–7.43 (m,
3H, ArH), 7.26 (d, J = 16.0 Hz, 1H, CH), 4.81 (s, 2H, NH2), 3.86 (s,
4H, O(CH2)2), 3.83 (s, 4H, N(CH2)2) ppm. 13C NMR (75 MHz, CDCl3,
25 °C): d = 172.7, 170.6, 161.9, 159.4, 138.4, 135.2, 130.0, 129.1,
127.9, 124.0, 120.8, 66.9, 46.3 ppm. HRMS: calcd for C17H18N5OS
[M+H]+ 340.12321, found 340.12186.
5.2.14. 2-Bromo-7-(isopentyloxy)thiazolo[4,5-d]pyrimidin-5-
amine (4c)
This compound was synthesized from 3c, according to the pro-
cedure for the preparation of compound 4a. The crude residue was
purified by flash chromatography on silica (CH2Cl2/MeOH 50:1) to
yield the title compound as a pale yellow solid (0.19 g, 80%). Mp
181–182 °C. 1H NMR (300 MHz, CDCl3, 25 °C): d = 5.32 (s, 2H,
NH2), 4.47 (t, J = 6.7 Hz, 2H, OCH2), 1.73 (sixtet, J = 6.3 Hz, 1H,
CH), 1.69 (q, J = 6.5 Hz, 2H, CH2), 0.97 (d, J = 6.4 Hz, 6H, CH3) ppm.
13C NMR (75 MHz, CDCl3, 25 °C): d = 170.8, 164.5, 162.4, 147.0,
105.1, 66.3, 37.5, 25.3, 22.7 ppm MS: calcd for C10H14BrN4OS
[M+H]+ 317.01, 319.01, found 316.88, 318.91.
5.2.18. 7-Morpholino-2-phenylthiazolo[4,5-d]pyrimidin-5-
amine (5c)
To a mixture of Pd(PPh3)4 (15 mg, 0.01 mmol) and 2-bromo-7-
morpholinothiazolo[4,5-d]pyrimidin-5-amine 4a (40 mg, 0.13
mmol) in dioxane (1 ml) was added tributylphenylstannane
(45 ll, 0.14 mmol). The reaction mixture was refluxed overnight
under a nitrogen atmosphere and then cooled down to room tem-
perature. The solvents were removed under reduced pressure. The
crude residue was diluted with dichloromethane and washed with
water. The combined organic layers were dried over Na2SO4 and
the solvent was evaporated in vacuo. The crude residue was puri-
fied by chromatography on silica gel (CH2Cl2/MeOH 60:1), yielding
the pure title compound as a yellow solid (20 mg, 50%). Mp 278 °C.
1H NMR (300 MHz, CDCl3, 25 °C): d = 8.11 (d, J = 7.8 Hz, 2H, ArH),
7.50–7.44 (m, 3H, ArH), 4.81 (s, 2H, NH2), 3.88–3.91 (m, 4H,
O(CH2)2), 3.82–3.85 (m, 4H, N(CH2)2) ppm. 13C NMR (75 MHz,
CDCl3, 25 °C): d = 172.9, 172.3, 161.9, 159.5, 132.9, 132.0, 129.2,
127.5, 110.9, 66.9, 46.3 ppm. HRMS: calcd for C15H16N5OS [M+H]+
314.10756, found 314.10635.
5.2.15. 1-(4-(5-Amino-2-chlorothiazolo[4,5-d]pyrimidin-7-yl)
piperazin-1-yl)ethanone (4d)
A solution of NaNO2 (94 mg, 1.36 mmol) in H2SO4 (1.7 ml) was
heated at 80 °C until the solid dissolved. The solution was cooled
down to room temperature and a solution of 1-(4-(2,5-diamino-
thiazolo[4,5-d]pyrimidin-7-yl)piperazin-1-yl)ethanone 3d (0.1 g,
0.34 mmol) in acetic acid (0.5 ml) was added. After the addition
was completed, the solution was stirred at 40 °C for 30 min. A solu-
tion of CuCl (0.1 g, 1.02 mmol) in HCl (0.5 ml) was added and the
mixture was heated at 80 °C for 30 min. After cooling, water
(3 ml) was added. The mixture was neutralized with a 10 N NaOH
solution and extracted with a CHCl3/EtOH (4/1) solution, brine and
dried over Na2SO4. After removing the solvents under reduced
pressure, the crude residue was purified by flash chromatography
on silica (CH2Cl2/MeOH 20:1) affording the title compound as a
white solid (65 mg, 61%). Mp 230 °C decomposed 1H NMR
(300 MHz, DMSO, 25 °C): d = 6.46 (s, 2H, NH2), 3.71–3.80 (m, 4H,
N(CH2)2), 3.56–3.61 (m, 4H, CON(CH2)2), 2.04 (s, 3H, CH3) ppm.
13C NMR (75 MHz, DMSO, 25 °C): 169.3, 168.6, 162.1, 158.1,
157.8, 86.1, 44.9, 44.8, 44.6, 40.4, 21.2 ppm. HRMS: calcd for
5.2.19. 7-Morpholino-2-(phenylethynyl)thiazolo[4,5-d]pyrimi-
din-5-amine (5d)
To a mixture of bis(triphenylphosphine)palladium(II) acetate
(2 mg, 0.002 mmol), 2-bromo-7-morpholinothiazolo[4,5-d]pyrimi-
din-5-amine 4a (40 mg, 0.13 mmol) and triethylamine (63
l
l) in
dioxane (2 ml) was added a solution of phenylacetylene (20
ll,
0.14 mmol) in DMF (0.3 ml) over a period of 5 min. The reaction
mixture was refluxed under nitrogen for 6 h and then cooled to
room temperature. The solvents were removed under reduced
pressure. The crude residue was diluted with dichloromethane
and washed with water. The combined organic layers were dried
over MgSO4 and evaporated in vacuo. The crude residue was
C
11H14ClN6OS [M+H]+ 313.0638, found 313.0622.

M.-Y. Jang et al. / Bioorg. Med. Chem. 19 (2011) 702–714
711
purified by flash chromatography on silica gel (CH2Cl2/MeOH
60:1), yielding the pure title compound as an orange solid
(38 mg, 89%). Mp >260 °C decomposed 1H NMR (300 MHz, CDCl3,
25 °C): d = 7.62 (d, J = 7.7 Hz, 2H, ArH), 7.38–7.48 (m, 3H, ArH),
5.00 (s, 2H, NH2), 3.84 (s, 4H, O(CH2)2), 3.82 (s, 4H, N(CH2)2)
ppm. 13C NMR (75 MHz, CDCl3, 25 °C): d = 172.0, 162.3, 159.2,
153.1, 132.4, 130.4, 128.8, 120.9, 100.3, 98.3, 82.6, 66.8,
46.3 ppm. HRMS: calcd for C17H16N5OS [M+H]+ 338.10756, found
338.10645.
116.4 (d, J = 22.0 Hz), 114.2, 66.1, 37.6, 25.4, 22.8 ppm. HRMS:
calcd for C16H18FN4OS [M+H]+ 333.11854, found 333.11794.
5.2.24. 1-(4-(5-Amino-2-(4-fluorophenyl)thiazolo[4,5-d]
pyrimidin-7-yl)piperazin-1-yl)ethanone (5i)
This compound was synthesized from 4d, according to the pro-
cedure for the preparation of compound 5a. The crude residue was
purified by flash chromatography on silica (CH2Cl2/MeOH 30:1) to
yield the title compound as a yellow solid (73%). Mp 280 °C. 1H
NMR (300 MHz, DMSO, 25 °C): d = 8.09–8.14 (m, 2H, PhH), 7.42
(t, J = 8.8 Hz, 2H, PhH), 6.33 (s, 2H, NH2), 3.88 (br s, 2H, NCH2),
3.81 (br s, 2H, NCH2), 3.61 (br s, 4H, CON(CH2)2), 2.06 (s, 3H,
CH3) ppm. 13C NMR (75 MHz, DMSO, 25 °C): 172.4, 169.4, 168.6,
164.2 (d, J = 248.7 Hz), 162.2, 158.5, 129.2 (d, J = 9.0 Hz), 128.9 (d,
J = 2.9 Hz), 116.6 (d, J = 22.1 Hz), 97.5, 45.1, 44.8, 40.5, 40.3,
21.2 ppm. HRMS: calcd for C17H18FN6OS [M+H]+ 373.1247, found
373.1223.
5.2.20. 2,7-Dimorpholinothiazolo[4,5-d]pyrimidin-5-amine (5e)
A solution of 2-bromo-7-morpholinothiazolo[4,5-d]pyrimidin-
5-amine 4a (40 mg, 0.13 mmol) in morpholine (2 ml) was heated
at 80 °C under a nitrogen atmosphere overnight and then cooled
to room temperature. The volatiles were removed under reduced
pressure. The crude residue was diluted with dichloromethane
and washed with water. The combined organic layers were dried
over MgSO4 and the solvent was evaporated in vacuo. The crude
residue was purified by chromatography on silica gel (CH2Cl2/
MeOH 40:1) to yield the title compound as white solid (35 mg,
86%). Mp 251–253 °C. 1H NMR (300 MHz, CDCl3, 25 °C): d = 8.03
(s, 2H, NH2), 2.72 (s, 8H, O(CH2)2), 2.66 (s, 8H, N(CH2)2) ppm. 13C
NMR (75 MHz, CDCl3, 25 °C): d = 171.9, 171.8, 161.5, 158.5, 92.9,
66.9, 66.2, 48.2, 46.2 ppm. HRMS: calcd for C13H19N6O2S [M+H]+
323.12902, found 323.12811.
5.2.25. 2-(4-Fluorophenyl)-7-(piperazin-1-yl)thiazolo[4,5-d]
pyrimidin-5-amine (6)
A
solution of 1-(4-(5-amino-2-(4-fluorophenyl)thiazolo[4,5-
d]pyrimidin-7-yl)piperazin-1-yl)ethanone 5i (0.1 g, 0.27 mmol) in
5% HCl (2 ml) was heated at 100 °C for 4 h. After cooling down to
room temperature, the mixture was neutralized with 2 N NaOH
to pH 7. The precipitate was filtered off, washed with water and
dried. The crude compound was purified by flash chromatography
on silica (CH2Cl2/MeOH 5:1) to yield the title compound as a yel-
low solid (81 mg, 91%). Mp 260 °C. 1H NMR (300 MHz, DMSO,
25 °C): d = 8.10–8.15 (m, 2H, PhH), 7.41 (t, J = 8.8 Hz, 2H, PhH),
6.26 (s, 2H, NH2), 3.76 (br s, 4H, N(CH2)2), 2.82 (br s, 4H, NH(CH2)2)
ppm. 13C NMR (75 MHz, DMSO, 25 °C): 172.4, 169.1, 164.2 (d,
J = 248.6 Hz), 162.2, 158.6, 129.2 (d, J = 9.0 Hz), 128.9 (d,
J = 3.1 Hz), 116.5 (d, J = 22.1 Hz), 97.3, 46.5, 45.4 ppm. HRMS: calcd
for C15H16FN6S [M+H]+ 331.11412, found 331.11335.
5.2.21. N-2-(2-Methoxyethyl)-7-morpholinothiazolo[4,5-d]
pyrimidine-2,5-diamine (5f)
This compound was synthesized according to a procedure for
the preparation of compound 5e, using 2-methoxyethylamine.
The crude residue was purified by flash chromatography on silica
(CH2Cl2/MeOH 50:1) to yield the title compound as a white solid
(93%). Mp 205–206 °C. 1H NMR (300 MHz, CDCl3, 25 °C): d = 5.94
(s, 1H, NH), 4.67 (s, 2H, NH2), 3.75–3.79 (m, 4H, O(CH2)2), 3.61–
3.71 (m, 6H, CH2, N(CH2)2), 3.62 (t, J = 5.0 Hz, 2H, CH2), 3.39 (s,
3H, CH3) ppm. 13C NMR (75 MHz, CDCl3, 25 °C): d = 171.8, 170.9,
161.4, 158.6, 128.9, 70.7, 66.9, 59.0, 46.2, 44.7 ppm. HRMS: calcd
for C12H19N6O2S [M+H]+ 11.12902, found 311.12837.
5.2.26. 4-(5-Amino-2-(4-fluorophenyl)thiazolo[4,5-d]pyrimidin-
7-yl)-N-m-tolylpiperazine-1-carboxamide (7a)
To a solution of 2-(4-fluorophenyl)-7-(piperazin-1-yl)thiazol-
o[4,5-d]pyrimidin-5-amine 6 (30 mg, 0.09 mmol) in DMF (1 ml)
was added m-tolyl isocyanate (13 ll, 0.10 mmol) in DMF (0.3 ml).
5.2.22. 7-Ethoxy-2-(4-fluorophenyl)thiazolo[4,5-d]pyrimidin-5-
amine (5g)
The reaction mixture was stirred for 2 h at room temperature. The
reaction was quenched with water and extracted with EtOAc.
The combined organic layers were dried over Na2SO4. After remov-
ing the solvents in vacuo, the crude residue was purified by flash
chromatography on silica (CH2Cl2/MeOH 100:1) to yield the title
compound as a white solid (40 mg, 95%). Mp 232–233 °C. 1H NMR
(300 MHz, DMSO, 25 °C): d = 8.54 (s, 1H, NH), 8.11–8.16 (m, 2H,
PhH), 7.43 (t, J = 8.7 Hz, 2H, PhH), 7.31 (s, 1H, PhH), 7.28 (d,
J = 7.5 Hz, 1H, PhH), 7.15 (t, J = 7.5 Hz, 1H, PhH), 6.77 (d, J = 7.5 Hz,
1H, PhH), 6.33 (s, 2H, NH2), 3.89 (br s, 4H, N(CH2)2), 3.63 (br s, 4H,
CON(CH2)2), 2.26 (s, 3H, CH3) ppm. 13C NMR (75 MHz, DMSO,
25 °C): 172.4, 169.4, 164.2 (d, J = 248.8 Hz), 162.2, 158.6, 154.9,
140.2, 137.3, 129.2 (d, J = 8.9 Hz), 128.9 (d, J = 2.9 Hz), 128.2, 122.6,
120.3, 116.8, 116.5 (d, J = 22.1 Hz), 97.6, 44.9, 43.3, 21.2 ppm. HRMS:
calcd for C23H23FN7OS [M+H]+ 464.16688, found 464.16510.
This compound was synthesized from 4b according to the pro-
cedure for the preparation of compound 5a. The crude residue was
purified by flash chromatography on silica (CH2Cl2/MeOH 200:1) to
yield the title compound as a white solid (76%). Mp 194 °C. 1H NMR
(300 MHz, CDCl3, 25 °C): d = 8.09–8.14 (m, 2H, ArH), 7.17 (t,
J = 8.5 Hz, 2H, ArH), 5.08 (s, 2H, NH2), 4.54 (q, J = 7.1 Hz, 2H,
OCH2), 1.45 (t, J = 7.1 Hz, 3H, CH3) ppm. 13C NMR (75 MHz, CDCl3,
25 °C): d = 173.3, 172.7, 165.4, 165.2 (d, J = 251.7 Hz), 162.3,
129.8 (d, J = 8.8 Hz), 129.5 (d, J = 3.3 Hz), 116.4 (d, J = 22.0 Hz),
63.4, 14.6 ppm. HRMS: calcd for C13H12FN4OS [M+H]+ 291.07158,
found 291.07079.
5.2.23. 2-(4-Fluorophenyl)-7-(isopentyloxy)thiazolo[4,5-d]
pyrimidin-5-amine (5h)
This compound was synthesized from 4c according to the pro-
cedure for the preparation of compound 5a. The crude residue
was purified by flash chromatography on silica (CH2Cl2/MeOH
70:1) to yield the title compound as a pale yellow solid (86%).
Mp 210 °C. 1H NMR (300 MHz, CDCl3, 25 °C): d = 8.10–8.15 (m,
2H, ArH), 7.17 (t, J = 8.5 Hz, 2H, ArH), 5.01 (s, 2H, NH2), 4.51 (t,
J = 6.8 Hz, 2H, OCH2), 1.81 (sixtet, J = 6.4 Hz, 1H, CH), 1.72 (q,
J = 6.5 Hz, 2H, CH2), 0.99 (d, J = 6.5 Hz, 6H, CH3) ppm. 13C NMR
(75 MHz, CDCl3, 25 °C): d = 173.2, 172.7, 165.5, 165.2 (d,
J = 251.6 Hz), 162.4, 129.8 (d, J = 8.8 Hz), 129.6 (d, J = 3.3 Hz),
5.2.27. 1-(4-(5-Amino-2-(4-fluorophenyl)thiazolo[4,5-d]pyr-
imidin-7-yl)piperazin-1-yl)-2-(4-chlorophenoxy)ethanone (7b)
To a solution of 2-(4-fluorophenyl)-7-(piperazin-1-yl)thiazol-
o[4,5-d]pyrimidin-5-amine 6 (30 mg, 0.09 mmol) and pyridine
(11 ll, 0.14 mmol) in DMF (1 ml) was added 4-chlorophenoxyace-
tyl chloride (20 mg, 0.10 mmol). The reaction mixture was stirred
for 2 h at room temperature. The reaction mixture was quenched
with water, extracted with EtOAc, brine and was dried over
Na2SO4. After removing the solvents, the crude residue was puri-
fied by flash chromatography on silica (CH2Cl2/MeOH 100:1) to

712
M.-Y. Jang et al. / Bioorg. Med. Chem. 19 (2011) 702–714
yield the title compound as a yellow solid (40 mg, 88%). Mp 267 °C.
1H NMR (300 MHz, DMSO, 25 °C): d = 8.10–8.15 (m, 2H, PhH), 7.43
(t, J = 8.6 Hz, 2H, PhH), 7.33 (d, J = 8.6 Hz, 2H, PhH), 6.97 (d,
J = 8.6 Hz, 2H, PhH), 6.34 (s, 2H, NH2), 4.92 (s, 2H, OCH2), 3.92 (br
s, 2H, NCH2), 3.87 (br s, 2H, NCH2), 3.65 (br s, 4H, CON(CH2)2)
ppm. 13C NMR (75 MHz, DMSO, 25 °C): 172.4, 169.4, 165.9, 164.2
(d, J = 248.7 Hz), 162.2, 158.5, 156.9, 129.2 (d, J = 9.0 Hz), 129.1,
128.9 (d, J = 3.0 Hz), 116.5 (d, J = 21.6 Hz), 116.4, 97.6, 65.9, 44.9,
44.7, 43.5, 40.9 ppm. HRMS: calcd for C23H21ClFN6O2S [M+H]+
499.11193, found 499.11023.
5.2.32. 2-(4-Fluorophenyl)-N7-(2-methoxyethyl)thiazolo[4,5-
d]pyrimidine-5,7-diamine (12a)
This compound was synthesized from 11a according to the pro-
cedure for the preparation of compound 5a. The crude residue was
purified by flash chromatography on silica (CH2Cl2/MeOH 40:1) to
yield the title compound as a pale yellow solid (97%). Mp 225 °C.
1H NMR (300 MHz, DMSO, 25 °C): d = 8.95 (s, 1H, NH), 7.93–7.98
(m, 2H, ArH), 7.39 (t, J = 8.7 Hz, 2H, ArH), 6.37 (s, 2H, NH2), 3.55
(br s, 4H, CH2), 3.29 (s, 3H, CH3) ppm. 13C NMR (75 MHz, DMSO,
25 °C): d = 172.3, 171.3, 162.9 (d, J = 245.9 Hz), 162.8, 154.6,
134.3 (d, J = 3.0 Hz), 129.6 (d, J = 8.7 Hz), 115.7 (d, J = 21.6 Hz),
106.7, 69.9, 57.9, 43.8 ppm. HRMS: calcd for
C14H13FN4O2S
5.2.28. 7-N-Morpholinothiazolo[4,5-d]pyrimidin-5-amine (8)
This compound was synthesized from 4a according to the pro-
cedure for the preparation of compound 11. The crude residue was
purified by flash chromatography on silica (CH2Cl2/MeOH 40:1) to
yield the title compound as a white solid (70%). 1H NMR (300 MHz,
DMSO, 25 °C): d = 9.45 (s, 1H, H-2), 6.29 (s, 2H, NH2), 3.73–3.78
(m, 4H, O(CH2)2), 3.70–3.72 (m, 4H, N(CH2)2) ppm. 13C NMR
(75 MHz, DMSO, 25 °C): d = 172.6, 162.0, 159.7, 159.1, 96.4, 65.8,
45.5 ppm.
[M+H]+ 320.07432, found 320.09738.
5.2.33. tert-Butyl-4-(5-amino-7-(4-fluorophenyl)thiazolo[4,5-
d]pyrimidin-2-yl)piperazine-1-carboxylate (12b)
This compound was synthesized from 11b according to the pro-
cedure for the preparation of compound 5a. The crude residue was
purified by flash chromatography on silica (CH2Cl2/MeOH 40:1) to
yield the title compound as a pale yellow solid (94%). Mp 211 °C.
1H NMR (300 MHz, CDCl3, 25 °C): d = 7.94–7.99 (m, 2H, ArH),
7.18 (t, J = 8.6 Hz, 2H, ArH), 5.08 (s, 2H, NH2), 3.73 (br s, 4H,
N(CH2)2), 3.59 (br s, 4H, CON(CH2)2), 1.49 (s, 9H, CH3) ppm. 13C
NMR (75 MHz, CDCl3, 25 °C): d = 173.4, 172.5, 164.1 (d,
J = 294.5 Hz), 162.6, 156.8, 154.5, 134.0 (d, J = 3.1 Hz), 130.0 (d,
J = 8.5 Hz), 116.0 (d, J = 15.9 Hz), 109.3, 80.9, 48.3, 43.1, 28.5 ppm.
5.2.29. 2,7-Dichlorothiazolo[4,5-d]pyrimidin-5-amine (10)
To
a solution of 5-amino-2-chlorothiazolo[4,5-d]pyrimidin-
7(6H)-one 928 (1.3 g, 6.42 mmol) in phosphorus oxychloride
(32 ml) was added N,N-dimethylaniline (3.2 ml). The mixture was
heated at reflux for 2 h. After cooling to room temperature, the sol-
vents were removed under reduced pressure. The crude residue
was diluted with dichloromethane and poured into ice-water.
The mixture was extracted with dichloromethane, washed with
NaHCO3 and brine, and dried over Na2SO4. After removing the sol-
vents under reduced pressure, the residue was purified by flash
chromatography on silica (CH2Cl2/MeOH 50:1) to yield the title
compound as a light yellow solid (0.48 g, 33%). Mp >285 °C decom-
posed. 1H NMR (300 MHz, CDCl3, 25 °C): d = 7.46 (s, 2H, NH2) ppm.
13C NMR (75 MHz, CDCl3, 25 °C): d = 168.8, 162.8, 162.5, 152.6,
113.9 ppm. HRMS: calcd for C5H3Cl2N4S [M+H]+ 220.94555/
222.94260, found 220.9448/222.94199.
HRMS: calcd for
431.16558.
C
20H24FN6O2S [M+H]+ 431.16655, found
5.2.34. 7-(4-Fluorophenyl)-2-(piperazin-1-yl)thiazolo[4,5-d]
pyrimidin-5-amine (13)
To
a
suspension of tert-butyl 4-(5-amino-7-(4-fluoro-
phenyl)thiazolo[4,5-d]pyrimidin-2-yl)piperazine-1-carboxylate
12b (0.30 g, 0.07 mmol) in dichloromethane (5 ml) was added
dropwise trifluoroacetic acid (ꢀ5 ml) until the solid was com-
pletely dissolved. The reaction mixture was stirred at room tem-
perature under a nitrogen atmosphere overnight. The solvents
were evaporated in vacuo. The residue was resuspended in water
and the solid was collected by filtration. The solid was washed with
water, dried under vacuum, yielding the title compound as a white
solid (0.175 g, 76%). Mp 280 °C. 1H NMR (300 MHz, DMSO, 25 °C):
d = 7.96–8.01 (m, 2H, PhH), 7.39 (t, J = 8.7 Hz, 2H, PhH), 6.43 (s,
2H, NH2), 3.58 (br s, 4H, N(CH2)2), 3.18 (s, 1H, NH), 2.81 (br s,
4H, HN(CH2)2) ppm. 13C NMR (75 MHz, DMSO, 25 °C): 172.3,
163.0 (d, J = 246.4 Hz), 162.9, 155.0, 134.1 (d, J = 3.0 Hz), 129.7 (d,
J = 8.7 Hz), 115.8 (d, J = 21.5 Hz), 106.7, 49.4, 45.0 ppm. HRMS:
calcd for C15H16FN6S [M+H]+ 331.11412, found 331.11309.
5.2.30. 7-Chloro-N2-(2-methoxyethyl)thiazolo[4,5-d]pyrimi-
dine-2,5-diamine (11a)
To a solution of 2,7-dichlorothiazolo[4,5-d]pyrimidin-5-amine
10 (0.10 g, 0.45 mmol) and triethylamine (69
ll, 0.50 mmol) in
dioxane (3 ml) was added 2-methoxyethylamine (39
l
l, 0.45
mmol). The reaction mixture was heated at 70 °C for 2 h. After
cooling, the volatiles were removed under reduced pressure. The
crude residue was purified by chromatography on silica gel
(CH2Cl2/MeOH 40:1) affording the title compound as a white solid
(0.11 g, 94%). Mp 236–237 °C. 1H NMR (300 MHz, DMSO, 25 °C):
d = 9.09 (s, 1H, NH), 6.71 (s, 2H, NH2), 3.52 (br s, 4H, CH2), 3.29
(s, 3H, CH3) ppm. 13C NMR (75 MHz, DMSO, 25 °C): d =171.8,
170.8, 162.3, 149.4, 107.6, 69.8, 57.9, 43.8 ppm. HRMS: calcd for
C8H9ClN4O2S [M+H]+ 260.01347, found 260.03656.
5.2.35. 4-(5-Amino-7-(4-fluorophenyl)thiazolo[4,5-d]pyrimidin-
2-yl)-N-m-tolylpiperazine-1-carboxamide (14a)
This compound was synthesized from 13 according to the pro-
cedure for the preparation of compound 7a. The crude residue was
purified by flash chromatography on silica (CH2Cl2/MeOH 50:1) to
yield the title compound as a white solid (95%). Mp 228 °C. 1H NMR
(300 MHz, DMSO, 25 °C): d = 8.62 (s, 1H, NH), 7.98–8.03 (m, 2H,
PhH), 7.41 (t, J = 8.8 Hz, 2H, PhH), 7.31 (s, 1H, PhH), 7.27 (d,
J = 7.3 Hz, 1H, PhH), 7.13 (t, J = 7.3 Hz, 1H, PhH), 6.78 (d,
J = 7.3 Hz, 1H, PhH), 6.48 (s, 2H, NH2), 3.73 (br s, 4H, N(CH2)2),
3.65 (br s, 4H, CON(CH2)2), 2.26 (s, 3H, CH3) ppm. 13C NMR
(75 MHz, DMSO, 25 °C): 172.4, 172.2, 163.1 (d, J = 246.6 Hz),
162.9, 155.2, 154.8, 140.2, 137.4, 134.0 (d, J = 2.9 Hz), 129.7 (d,
J = 8.7 Hz), 128.2, 122.6, 120.2, 116.8, 115.8 (d, J = 21.6 Hz), 106.8,
5.2.31. tert-Butyl-4-(5-amino-7-chlorothiazolo[4,5-d]pyrimi-
din-2-yl)piperazine-1-carboxylate (11b)
This compound was synthesized in a yield of 97% according to
the procedure for the preparation of compound 3a, using tert-butyl
piperazine-1-carboxylate. Mp >350 °C decomp. 1H NMR (300 MHz,
DMSO, 25 °C): d = 6.81 (s, 2H, NH2), 3.64 (br s, 4H, N(CH2)2), 3.49
(br s, 4H, CON(CH2)2), 1.43 (s, 9H, CH3) ppm. 13C NMR (75 MHz,
DMSO, 25 °C): d = 172.0, 171.7, 162.5, 153.7, 149.8, 107.8, 79.4,
47.7, 42.2, 28.0 ppm. HRMS: calcd for C14H20ClN6O2S [M+H]+
371.10570, found 371.10496.
47.7, 43.1, 21.2 ppm. HRMS: calcd for
464.16688, found 464.16510.
C
23H23FN7OS [M+H]+

M.-Y. Jang et al. / Bioorg. Med. Chem. 19 (2011) 702–714
713
5.2.36. 1-(4-(5-Amino-7-(4-fluorophenyl)thiazolo[4,5-d]pyrimi-
din-2-yl)piperazin-1-yl)-2-(4-fluorophenoxy)ethanone (14b)
This compound was synthesized from 13, according to the pro-
cedure for the preparation of compound 7b. The crude residue was
purified by flash chromatography on silica (CH2Cl2/MeOH 50:1) to
yield the title compound as a white solid (53 mg, 88%). Mp 266 °C.
1H NMR (300 MHz, DMSO, 25 °C): d = 7.97–8.02 (m, 2H, PhH), 7.41
(t, J = 8.8 Hz, 2H, PhH), 7.33 (d, J = 8.8 Hz, 2H, PhH), 6.98 (d,
J = 8.8 Hz, 2H, PhH), 6.49 (s, 2H, NH2), 4.93 (s, 2H, NCH2), 3.78 (br
s, 4H, NCH2), 3.66 (br s, 4H, CON(CH2)2) ppm. 13C NMR (75 MHz,
DMSO, 25 °C): 172.4, 172.1, 165.9, 163.1 (d, J = 246.7 Hz), 163.0,
156.9, 155.3, 134.0 (d, J = 2.9 Hz), 129.7 (d, J = 8.7 Hz), 129.1,
124.5, 116.4, 115.8 (d, J = 21.6 Hz), 106.8, 65.9, 47.6, 43.3 ppm.
100 mM imidazole, pH 8.0). Peak fractions containing recombinant
SecA were pooled and dialysed against buffer D (50 mM Tris–HCl,
50 mM NaCl, 10% glycerol, 2 mM EDTA, pH 8.0). Protein samples
were concentrated using Vivaspin centrifugal devices (Sartorius,
Göttingen, Germany) with a MWCO of 3 kDa and dialysed against
buffer E (50 mM Tris–HCl, 50 mM NaCl, 50% glycerol, 2 mM EDTA,
pH 8.0). Enzyme preparations were aliquoted and stored at ꢁ80 °C
until use.
5.3.2. E. coli preprotein AlkProPhoA(Cys-)
The preprotein AlkProPhoA(Cys-) from E. coli was overexpressed
in E. coli BL21.19 and purified as described.39,40 Briefly, cell pellets
were suspended in buffer F (50 mM Tris–HCl, 0.5 M NaCl, 5%
HRMS: calcd for
499.11023.
C
23H21ClFN6O2S [M+H]+ 499.11193, found
glycerol, pH 8.0) containing2.5 mM PMSF and 50 lg/ml DNase. After
lysis by passing the cells three times through a French pressure cell
(69 MPa), ProPhoA(Cys-) was isolated as inclusion bodies and
purified by Ni-NTA affinity chromatography under denaturing
conditions. Peak fractions containing the recombinant preprotein
(in 50 mM Tris–HCl, 50 mM NaCl, 5% glycerol, 100 mM imidazole,
6 M urea, pH 8.0) were pooled and dialysed against 50 mM Tris–HCl,
50 mM KCl, 5% glycerol, 6 M urea, 1 mM EDTA, pH 8.0. Enzyme
preparations were aliquoted and stored at ꢁ80 °C until use.
5.2.37. tert-Butyl 4-(5-acetamidothiazolo[4,5-d]pyrimidin-2-
yl)piperazine-1-carboxylate (15)
A solution of tert-butyl 4-(5-amino-7-chlorothiazolo[4,5-d]pyr-
imidin-2-yl)piperazine-1-carboxylate 11b (40 mg, 0.11 mmol) and
10% Pd/C (3 mg) in THF/MeOH (2:1, 1 ml) was stirred at room tem-
perature under H2 for 4 h. The mixture is filtered over Celite and
solvent are evaporated in vacuo. The crude product was dissolved
in acetic anhydride and heated at 80 °C for 3 h. After removing the
solvent, the crude residue was purified by chromatography on sil-
ica gel (CH2Cl2/MeOH 50:1) to yield the title compound as a white
solid (13 mg, 32%). 1H NMR (300 MHz, CDCl3, 25 °C): d = 8.67 (s,
1H, H-7), 8.62 (s, 1H), 3.76 (br s, 4H, N(CH2)2), 3.61 (br s, 4H,
CON(CH2)2), 2.55 (s, 3H, COCH3), 1.49 (s, 9H, O(CH3)3) ppm. 13C
NMR (75 MHz, CDCl3, 25 °C): d = 173.8, 170.5, 156.1, 154.3, 148.5,
117.6, 80.8, 48.4, 43.0, 28.3, 25.2 ppm.
5.3.3. Isolation of inner membrane vesicles
Inner membrane vesicles (IMVs) were prepared from E. coli
strain BL31(DE3) using pET610 to overexpress SecYEG.41 Cells pel-
lets were suspended in 30 ml 50 mM Tris–HCl, 20% glycerol, pH 8.0
containing 2.5 mM PMSF and 50 lg/ml DNase (buffer F). Cells were
lysed by three passages through a French pressure cell (69 MPa),
diluted with an equal volume of buffer F and centrifuged (4000g,
10 min, 4 °C) to remove cell debris. Membranes were collected
from the supernatant by ultracentrifugation (35,000g, 90 min,
4 °C). The resulting pellet was resuspended in buffer F and loaded
onto a five-step sucrose gradient consisting of 1.1, 1.3, 1.5, 1.7 and
1.9 M sucrose layers (in 50 mM Tris–HCl, pH 8.0). After ultracentri-
fugation (24,000g, 16 h, 4 °C), the brown fraction containing the
IMVs was collected, diluted in 300 ml buffer F and recollected by
ultracentrifugation (35,000g, 90 min, 4 °C). Purified IMVs were sus-
pended in 5 ml 50 mM Tris–HCl, 50 mM KCl, 5 mM MgCl2, pH 8.0
(buffer G). To inactivate endogenous SecA, IMVs were treated with
6 M urea (30 min, 4 °C). Urea-stripped IMVs were sedimented
(35,000g, 35 min, 4 °C) through a sucrose cushion (0.2 M sucrose,
50 mM Tris–HCl pH 8.0, 50 mM KCl), washed with buffer G, and
after a second centrifugation step resuspended into 5 ml buffer
G. The suspension was homogenized with a homogenizer and the
membrane suspension was passed through a lipid extruder for bet-
ter homogenization. Protein concentrations were estimated by the
Bradford assay, using bovine serum albumin as the standard. SecA-
stripped IMVs were stored in small aliquots at ꢁ80 °C.
5.2.38. 7-Ethoxy-N2-(2-methoxyethyl)thiazolo[4,5-d]pyrimi-
dine-2,5-diamine (16)
5.2.38.1. Method A. This compound was synthesized from 11a
according to the procedure for the preparation of compound 1b.
The crude residue was purified by flash chromatography on silica
(CH2Cl2/MeOH 40:1) to yield the title compound as a white solid
(70%).
5.2.38.2. Method B. This compound was synthesized from 4b
according to the procedure for the preparation of compound 11a.
The crude residue was purified by flash chromatography on silica
(CH2Cl2/MeOH 40:1) to yield the title compound as a white solid
(82%). Mp 205 °C. 1H NMR (300 MHz, CDCl3, 25 °C): d = 6.32 (s,
1H, NH), 4.86 (s, 2H, NH2), 4.43 (q, J = 7.1, 2H, CH2CH3), 3.61–3.67
(m, 4H, CH2CH2), 3.38 (s, 3H, OCH3), 1.38 (t, J = 7.1 Hz, 3H,
OCH2CH3) ppm. 13C NMR (75 MHz, CDCl3, 25 °C): d = 172.4, 172.2,
163.9, 161.9, 95.1, 70.6, 62.5, 59.0, 44.9, 14.8 ppm. HRMS: calcd
for C10H16N5O2S [M+H]+ 270.10247, found 270.10168.
5.4. SecA ATPase activity measurements
5.3. Protein overexpression and purification
The ATPase activity of SecA was quantified in 96-well microtiter
5.3.1. E. coli and S. aureus SecA
(50 ll reaction volume) plates using the malachite green colori-
E. coli and S. aureus SecA were overexpressed as histidine-
tagged fusion proteins in E. coli BL21.19 cells. Cell pellets were sus-
pended in buffer A (50 mM Tris–HCl, 1 M NaCl, 10% glycerol, 5 mM
metric method for the detection of free inorganic phosphate
(Pi).38 E. coli SecA intrinsic ATPase activity measurements were per-
formed at 37 °C in 50 mM Tris–HCl, 50 mM KCl, 5 mM MgCl2,
imidazole, pH 8.0) containing 2.5 mM PMSF and 50
l
g/ml DNase.
0.4 mg/ml BSA, 0.5 lM (50 lg/ml) ecSecA, 100 lM ATP, pH 8.0. S.
After lysis by passing the cells three times through a French pres-
aureus SecA intrinsic ATPase activity measurements were per-
sure cell (69 MPa), cell debris was removed by centrifugation. The
formed at 28 °C in 50 mM HEPES, 50 mM KCl, 2 mM MgCl2,
cleared extract was applied to
a
nickel-nitrilotriacetic acid
0.4 mg/ml BSA, 4 lM (400 lg/ml) saSecA, 100 lM ATP, pH 7.5.
(Ni-NTA) column pre-equilibrated with buffer A. After washing
the column with 10 column volumes buffer A and 5 column vol-
umes buffer B (50 mM Tris–HCl, 50 mM NaCl, 10% glycerol, 5 mM
imidazole, pH 8.0), proteins were eluted by applying 10 column
volumes of buffer C (50 mM Tris–HCl, 50 mM NaCl, 10% glycerol,
E. coli SecA translocation ATPase activity measurements were per-
formed at 37 °C in 50 mM Tris–HCl, 50 mM KCl, 5 mM MgCl2,
0.4 mg/ml BSA, 0.2
recombinant ecSecYEG (12
(40 g/ml), 100 M ATP, pH 8.0.
lM (20
l
g/ml) ecSecA, E. coli IMVs containing
lg/ml protein), E. coli ProPhoA(Cys-)
l
l

714
M.-Y. Jang et al. / Bioorg. Med. Chem. 19 (2011) 702–714
Bradford, P.; Dushin, R. G.; Rothstein, D.; Projan, S. J. Antimicrob. Agents
Chemother. 2000, 44, 1418.
Initial rates of ATP hydrolysis were measured in the presence of
200 M compound or DMSO (2% final DMSO concentration). For
background correction, all reactions were performed with negative
controls with no SecA in the reaction mixture. After incubation (2 h
for intrinsic ATPase, 30 min for translocation ATPase activity mea-
surements) at the appropriate temperature, ATPase reactions were
l
14. Papanikolau, Y.; Papadovasilaki, M.; Ravelli, R. B.; McCarthy, A. A.; Cusack, S.;
Economou, A.; Petratos, K. J. Mol. Biol. 2007, 366, 1545.
15. Li, M.; Huang, Y.; Tai, P. C.; Wang, B. Biochem. Biophys. Res. Commun. 2008, 368,
839.
16. Chen, W.; Huang, Y.; Gundala, S. R.; Yang, H.; Li, M.; Tai, P. C.; Wang, B. Bioorg.
Med. Chem. 2010, 18, 1617.
17. Payne, D. J.; Gwynn, M. N.; Holmes, D. J.; Pompliano, D. L. Nat. Rev. Drug Disc.
2007, 6, 29.
18. Lewis, A. F.; Revankar, G. R.; Fennanwald, S. M.; Huffman, J. H.; Rando, R. F. J.
Heterocycl. Chem. 1995, 32, 547.
stopped by adding 150 ll Malachite Green Reagent (Enzo
LifeSciences, Plymouth, USA) to the reaction mixture. Microtiter
plates were incubated for 20 min at room temperature and read
at 660 nm using a Tecan Infinite M200 microplate reader. After
background correction, rates of SecA ATP hydrolysis in the pres-
ence of compound were expressed relative to rate of ATP hydroly-
sis in the absence of compound.
For the determination of the Ki value of compound 14b for inhi-
bition of the ecSecA translocation ATPase activity, initial rates of
ATP hydrolysis were measured as described above, in the presence
19. Carson, D. WO/2000/069861.
20. (a) Beck, J. P.; Curry, M. A.; Chorvat, R. J.; Fitzgerald, L. W.; Gilligan, P. J.; Zaczek,
R.; Trainor, G. L. Bioorg. Med. Chem. Lett. 1999, 9, 1185; (b) Beck, J. P. WO/1999/
051608.
21. Fahmy, H. T. Y.; Rostom, S. A. F.; Saudi, M. N.; Zjawiony, J. K.; Robins, D. J. Arch.
Pharm. Pharm. Med. Chem. 2003, 336, 216.
22. Bekhit, A. A.; Fahmy, H. T. Y.; Rostom, S. A. F.; Baraka, A. M. Eur. J. Med. Chem.
2003, 38, 27.
23. (a) Baxter, A.; Cooper, A.; Kinchin, E.; Moakes, K.; Unitt, J.; Wallace, A. Bioorg.
Med. Chem. Lett. 2006, 16, 960; (b) Walters, I.; Austin, C.; Austin, R.; Bonnert, R.;
Cage, P.; Christie, M.; Ebden, M.; Gardiner, S.; Grahmes, C.; Hill, S.; Hunt, F.;
Jewell, R.; Lewis, S.; Martin, I.; Nicholls, D.; Robinson, D. Bioorg. Med. Chem. Lett.
2007, 17, 2731.
of different inhibitor (25, 50, 100 and 200 lM) and ATP concentra-
tions (0–2 mM). Apparent Km and Vmax values were estimated by
fitting the data by nonlinear regression analysis to the Michaelis–
Menten equation. The Ki value of compound 14b for inhibition of
the ecSecA translocation ATPase activity was determined by linear
regression using Dixon plot analysis (1/v vs [I]).
24. Binnun, E. US/2007/0185139.
25. Evans, B. E.; Rittle, K. E.; Bock, M. G.; Dipardo, R. M.; Freidinger, R. M.; Whitter,
W. L.; Lundell, G. F.; Veber, D. F.; Anderson, P. S.; Chang, R. S. L.; Lotti, V. J.;
Cerino, D. J.; Chen, T. B.; Kling, P. J.; Kunkel, K. A.; Springer, J. P.; Hirshfield, J. J.
Med. Chem. 1988, 31, 2235.
26. Mesguiche, V.; Parsons, R. J.; Arris, C. E.; Bentley, J.; Boyle, F. T.; Curtin, N. J.;
Davies, T. G.; Endicott, J. A.; Gibson, A. E.; Golding, B. T.; Griffin, R. J.; Jewsbury,
P.; Johnson, L. N.; Newell, D. R.; Noble, M. E. M.; Wang, L. Z.; Hardcastle, I. R.
Bioorg. Med. Chem. Lett. 2003, 13, 217.
27. (a) Waer, M. J.; Herdewijn, P. A. M. M.; Pfleiderer, W.; Marchand, A. D.; De
Jonghe, S. WO/2005/021003.; (b) Sen, D.; Bhowmik, S.; Sengupta, P. J. Indian
Chem. Soc. 1986, 63, 420.
Acknowledgments
We are grateful to A. Economou for providing the E. coli SecA,
SecYEG and AlkProPhoA(Cys-) expression plasmids and to
M. Koukaki for biochemicals, protocols and advice.
28. Willis, P. A. US 6790,850, 2004.
29. Schomaker, J. M.; Delia, T. J. J. Org. Chem. 2001, 66, 7125.
30. Espinet, P.; Echavarren, A. M. Angew. Chem., Int. Ed. 2004, 43, 4704.
31. (a) Sonogashira, K.; Tohda, Y.; Hagihara, N. Tetrahedron Lett. 1975, 16,
4467; (b) Lester, R. M.; Julia, M. D.; Razwan, A.; Ian, J. S. Tetrahedron Lett.
2002, 43, 8853.
32. (a) Kataoka, N.; Shelby, Q.; Stambuli, J. P.; Hartwig, J. F. J. Org. Chem. 2002, 67,
5553; (b) Wagaw, S.; Buchwald, S. L. J. Org. Chem. 1996, 61, 7240.
33. Baker, J. A.; Chattfield, P. V. J. Chem. Soc. (C) 1970, 2478.
34. Nagahara, K.; Anderson, J. D.; Kini, G. D.; Dally, N. K.; Larson, S. B.; Smee, D. F.;
Jin, A.; Sharma, B. S.; Jolley, W. B.; Robins, R. K.; Cottam, H. B. J. Med. Chem.
1990, 33, 407.
35. Tikad, A.; Routier, S.; Akssira, M.; Leger, J. M.; Jarry, C.; Guillaumet, G. Org. Lett.
2007, 9, 4673.
36. Lavecchia, G.; Berteina-Raboin, S.; Guillaumet, G. Tetrahedron Lett. 2005, 46,
5851.
37. (a) Gerster, J. F.; Hinshaw, B. C.; Robins, R. K.; Townsend, L. B. J. Org. Chem. 1968,
33, 1070; (b) Ratsep, P. C.; Robins, R. K.; Vaghefi, M. M. Nucleosides Nucleotides
1990, 9, 197; (c) Kelly, J. L.; McLean, E. W.; Linn, J. A.; Krochmal, M. P.; Ferris, R.
M.; Howard, J. L. J. Med. Chem. 1990, 33, 196.
References and notes
1. Klevens, R. M.; Morrison, M. A.; Nadle, J.; Petit, S.; Gershman, K.; Ray, S.;
Harrison, L. H.; Lynfield, R.; Dumyati, G.; Townes, J. M.; Craig, A. S.; Zell, E. R.;
Fosheim, G. E.; McDougal, L. K.; Carey, R. B.; Fridkin, S. K. JAMA 2007, 298, 1763.
2. Devasahayam, G.; Scheld, W. M.; Hoffman, P. S. Expert Opin. Invest. Drugs 2010,
19, 215.
3. Payne, D. J. Science 2008, 321, 1644.
4. Economou, A. Expert Opin. Ther. Targets 2001, 5, 141.
5. Baron, C.; Coombes, B. Infect. Disord. Drug Targets 2007, 7, 19.
6. Papanikou, E.; Karamanou, S.; Economou, A. Nat. Rev. Microbiol. 2007, 5, 839.
7. Cunningham, K.; Lill, R.; Crooke, E.; Rice, M.; Moore, K.; Wickner, W.; Oliver, D.
EMBO J. 1989, 8, 955.
8. Lill, R.; Cunningham, K.; Brundage, L. A.; Ito, K.; Oliver, D.; Wickner, W. EMBO J.
1989, 8, 961.
9. Stephens, C.; Shapiro, L. Chem. Biol. 1997, 4, 637.
10. Oliver, D. B.; Cabelli, R. J.; Dolan, K. M.; Jarosik, G. P. Proc. Natl. Acad. Sci. U.S.A.
1990, 87, 8227.
11. Sugie, Y.; Inagaki, S.; Kato, Y.; Nishida, H.; Pang, C. H.; Saito, T.; Sakemi, S.; Dib-
Hajj, F.; Mueller, J. P.; Sutcliffe, J.; Kojima, Y. J. Antibiot. 2002, 55, 25.
12. Parish, C. A.; de la Cruz, M.; Smith, S. K.; Zink, D.; Baxter, J.; Tucker-Samaras, S.;
Collado, J.; Platas, G.; Bills, G.; Diez, M. T.; Vicente, F.; Pelaez, F.; Wilson, K. J.
Nat. Prod. 2009, 72, 59.
38. Van Veldhoven, P. P.; Mannaerts, G. P. Anal. Biochem. 1987, 161, 45.
39. Gouridis, G.; Karamanou, S.; Gelis, I.; Kalodimos, C. G.; Economou, A. Nature
2009, 462, 363.
40. Gouridis, G.; Karamanou, S.; Koukaki, M.; Economou, A. Methods Mol. Biol.
2010, 619, 157.
13. Alksne, L. E.; Burgio, P.; Hu, W.; Feld, B.; Singh, M. P.; Tuckman, M.;
Petersen, P. J.; Labthavikul, P.; McGlynn, M.; Barbieri, L.; McDonald, L.;
41. Kaufmann, A.; Manting, E. H.; Veenendaal, A. K.; Driessen, A. J.; van der Does, C.
Biochemistry 1999, 38, 9115.