Discovery of 7- N -piperazinylthiazolo[5,4- d ]pyrimidine analogues as a novel class of immunosuppressive agents with in vivo biological activity

Article abstract of DOI:10.1021/jm101254z

Herein we describe the synthesis and in vitro and in vivo activity of thiazolo[5,4-d]pyrimidines as a novel class of immunosuppressive agents, useful for preventing graft rejection after organ transplantation. This research resulted in the discovery of a series of compounds with potent activity in the mixed lymphocyte reaction (MLR) assay, which is well-known as the in vitro model for in vivo rejection after organ transplantation. The most potent congeners displayed IC₅₀ values of less than 50 nM in this MLR assay and hence are equipotent to cyclosporin A, a clinically used immunosuppressive drug. One representative of this series was further evaluated in a preclinical animal model of organ transplantation and showed excellent in vivo efficacy. It validates these compounds as new promising immunosuppressive drugs.

Full text of DOI:10.1021/jm101254z

J. Med. Chem. 2011, 54, 655–668 655
DOI: 10.1021/jm101254z
Discovery of 7-N-Piperazinylthiazolo[5,4-d]pyrimidine Analogues as a Novel Class of
Immunosuppressive Agents with in Vivo Biological Activity
Mi-Yeon Jang,† Yuan Lin,‡ Steven De Jonghe,‡ Ling-Jie Gao,‡ Bart Vanderhoydonck,‡ Mathy Froeyen,† Jef Rozenski,†
Jean Herman,‡ Thierry Louat,‡ Kristien Van Belle,‡ Mark Waer,§ and Piet Herdewijn*,†
†Laboratory of Medicinal Chemistry, Rega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10,
3000 Leuven, Belgium, ‡Interface Valorisation Platform (IVAP), Katholieke Universiteit Leuven, Kapucijnenvoer 33, 3000 Leuven,
Belgium, and §Laboratory of Experimental Transplantation, Campus Gasthuisberg, Katholieke Universiteit Leuven, Herestraat 49,
3000 Leuven, Belgium
Received September 28, 2010
Herein we describe the synthesis and in vitro and in vivo activity of thiazolo[5,4-d]pyrimidines as a novel
class of immunosuppressive agents, useful for preventing graft rejection after organ transplantation.
This research resulted in the discovery of a series of compounds with potent activity in the mixed
lymphocyte reaction (MLR) assay, which is well-known as the in vitro model for in vivo rejection after
organ transplantation. The most potent congeners displayed IC50 values of less than 50 nM in this MLR
assay and hence are equipotent to cyclosporin A, a clinically used immunosuppressive drug. One repre-
sentative of this series was further evaluated in a preclinical animal model of organ transplantation and
showed excellent in vivo efficacy. It validates these compounds as new promising immunosuppressive
drugs.
Introduction
Solid organ transplantation has become a common medical
procedure with considerable impact on extending and im-
proving the quality of life of patients with end stage renal,
cardiac, hepatic, or pulmonary failure.1 Several immunosup-
pressive agents to prevent graft rejection after organ trans-
plantation are available such as calcineurin inhibitors (cyclo-
sporin A, tacrolimus), antimetabolites (mycophenolate mofetil,
azathioprine), mammalian target of rapamycin (mTORa)
inhibitors (sirolimus, everolimus), and corticosteroids. How-
Figure 1. Scaffold evolution.
Over the past few years, our group has been actively involved
in the search for new immunosuppressive compounds, based on
ever, continuous administration of these agents for the entire
a bicyclic heteroaromatic scaffold (Figure 1). This research
started in the late 1990s, with the discovery of 4-N-piperaziny-
life is difficult for the patient because immunosuppressive
drugs generally suppress the host’s immunocompetence and
the treatment is accompanied by serious side effects such as
opportunistic infections, nephrotoxicity, neurotoxicity, hyper-
lipidaemia, new-onset post-transplant diabetes mellitus, and
hypertension.2 Thus, a high medical need exists for safe and
lpteridines as immunosuppressive agents.3 Further scaffold
modification led to the synthesis of pyrido[3,2-d]pyrimidines
as potent immunosuppressants.4
The concept ofbioisosterismisauseful way tocomeupwith
novel scaffolds, starting from a known, biologically active com-
specific inhibitors of early T-cell activation with a novel
pound. In drug design, the purpose of exchanging one bio-
isostere for another is to enhance the desired biological or
mechanism of action.
physicochemical properties of a compound without changing
the target. Bioisosteric replacements often provide the foun-
dation for the development of new structure-activity relation-
ships, and ring equivalent bioisosteres have therefore been
used frequently indrug discovery programs.5 Asa 1,3-thiazole
*To whom correspondence should be addressed. Phone: þ32 16
a Abbreviations: AcOH, acetic acid; APBS, adaptive Poisson-
Boltzmann solver; Boc, tert-butyloxycarbonyl; BOP, benzotriazol-1-
yloxytris(dimethylamino)phosphonium hexafluorophosphate; CsA,
cyclosporin A; DBU, 1,8-diazabicyclo[5.4.0]undec-7-ene; DCC, dicyclohex-
337387. Fax: þ32 16 337340. E-mail: Piet.Herdewijn@rega.kuleuven.be.
is a well-known isostere of the pyridine ring, we envisaged
synthesis of thiazolo[5,4-d]pyrimidines as potential surrogates
of the pyrido[3,2-d]pyrimidine scaffold (Figure 1).
The thiazolo[5,4-d]pyrimidine scaffold is not very frequently
used in drug discovery programs, as the number of medicinal
chemistry projects that describes the synthesis and biological
evaluation of thiazolopyrimidines is limited. However, certain
thiazolo[5,4-d]pyrimidines have been reported as templates
ylcarbodiimide; DIPEA, diisopropylethylamine; DMF, dimethyl-
formamide; DMSO, dimethylsulfoxide; HOBt, hydroxybenzotriazole;
HSP, heat shock protein; IC50, half maximal (50%) inhibitory concen-
tration; mCPBA, m-chloroperoxybenzoic acid; MLR, mixed lympho-
cyte reaction; mTOR, mammalian target of rapamycin; NEt3, tri-
ethylamine; PCC, pyridinium chlorochromate; PNP, purine nucleoside
phosphorylase; ROCS, rapid overlay of chemical structures; rt, room
temperature; SAR, structure-activity relationship; TBTU, N,N,N0,N0-
tetramethyl-O-(benzotriazol-1-yl)uronium tetrafluoroborate; TFA, tri-
fluoroacetic acid.
r
2010 American Chemical Society
Published on Web 12/20/2010
pubs.acs.org/jmc

656 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 2
Jang et al.
Scheme 1a
a Reagents and conditions: (a) 4-fluorobenzoyl chloride, pyridine, DMF, rt; (b) carboxylic acid, DCC, DMF/CH2Cl2 (1/10), rt; (c) guanidine
hydrochloride, Na, EtOH, reflux; (d) P2S5, pyridine, reflux; (e) iodomethane, NEt3, DMSO, rt; (f) mCPBA, CH2Cl2, 0 °C to rt; (g) piperazine or
N-substituted piperazine, dioxane, 60 °C; (h) isocyanate, DMF, rt; (i) acyl chloride, pyridine, DMF, rt; (j) benzenesulfonyl chloride, pyridine, DMF, rt;
(k) benzyl chloroformate, pyridine, DMF, rt; (l) carboxylic acid, TBTU, DIPEA, DMF, rt; (m) iodomethane, 2 N NaOH, DMSO, rt.
that have been functionalized to achieve biological activities
such as inhibitors of purine nucleoside phosphorylase (PNP),6
as vanilloid receptor antagonists,7 as activators of caspases
and inducers of apoptosis,8 as antiangiogenic agents,9 as growth
factor receptor inhibitors,10 and as heat shock protein 90
(HSP-90) inhibitors.11
In this paper, we describe the synthesis of a new class of
thiazolo[5,4-d]pyrimidine analogues with in vitro as well as in
vivo immunosuppressive activity. The structure-activity rela-
tionship (SAR) studies are described in which both the sub-
stitution pattern and the scaffold are modified.
corresponding thiomethylethers 4a-dwaseffectedbytreatment
with iodomethane in the presence of triethylamine in DMSO.
Alkylation reaction on the thiazolo[5,4-d]pyrimidine 4b
was also performed, using iodomethane and a base (2 N
1
NaOH) in DMSO. Careful analysis of the H NMR spec-
trum of the isolated compound confirmed that in these
reaction conditions the benzylic methylene group is meth-
ylated, yielding compound 4e. The signal of the methyl (CH3)
protons is observed at 1.8 ppm (doublet, 3H). N-Alkyl signals
are usually observed around 3 ppm. Moreover, the deute-
rium exchange spectrum of compound 4e indicates that there
are two amino protons, further supporting the fact that
alkylation takes place at the benzylic position and not at the
exocyclic amino group or on the pyrimidine ring nitrogens.
The direct introduction of a piperazinyl group from the
thiomethyl group did not work, as only starting material
could be recovered. Therefore, the thiomethyl group was
first oxidized with m-chloroperoxybenzoic acid (mCPBA) in
dichloromethane, yielding the corresponding sulfone deri-
vatives 5a-e. The reaction of the sulfone 5a with piperazine
at 60 °C afforded the desired 2-(4-fluorophenyl)-7-(piperazin-
1-yl)thiazolo[5,4-d]pyrimidin-5-amine 6 which could be
further reacted with an isocyanate, an acyl chloride, a
carboxylic acid,14 a sulfonyl chloride, or a chloroformate,
yielding urea (compounds 7c and 7d), amides (compound 7a
using an acyl chloride and compounds 7b, 7n-q from car-
boxylic acids), sulfonamide (compound 7e), and carbamate
(compound 7f), respectively (Tables 1 and 4). Alternatively,
it is also possible to introduce commercially available N-
substituted piperazine derivatives directly onto the thiazolo-
[5,4-d]pyrimidine skeleton, yielding derivatives 7g-i (Table 1).
Chemistry
Synthesis of Thiazolo[5,4-d]pyrimidine Analogues. The
synthesis of thiazolo[5,4-d]pyrimidine derivatives starts from
commercially available diethyl aminomalonate hydrochlo-
ride or dimethyl aminomalonate hydrochloride by a known
procedure (Scheme 1).12 Dialkyl aminomalonate hydrochloride
was converted to amides by direct acylation with acyl chlo-
rides or by standard peptide coupling procedures, using car-
boxylic acids, hydroxybenzotriazole (HOBt), and dicyclo-
hexylcarbodiimide (DCC).13 In order to construct the
pyrimidine scaffold, dialkyl acylaminomalonates 1a-d were
treated with guanidine in an ethanolic sodium ethoxide solution
yielding pyrimidines 2a-d. Conversion of the lactam func-
tionalities of 5-acylaminopyrimidine-4,6-diols 2a-d into thio-
lactam groups was achieved with phosphorus pentasulfide in
pyridine, giving access to 2,5,7-substituted-thiazolo[5,4-d]pyrim-
idines 3a-d. In order to introduce a high degree of molecular
diversity at position 7, alkylation of the thio group to the

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 2 657
Table 1. SAR of the Piperazinyl Substituent of 5-Amino-2-(4-fluoro-
phenyl)-thiazolo[5,4-d]pyrimidines
pyrimidine 10 by the reaction with phosphorus oxychlor-
ide in the presence of tetraethylammonium chloride.15
Displacement of one of the chlorines with 1 equiv of tert-
butyl piperazine-1-carboxylate furnished tert-butyl 4-(2,5-
diamino-6-chloropyrimidin-4-yl)piperazine-1-carboxylate
11. By heating with sodium sulfide nonahydrate in
DMSO, the remaining chlorine was converted into a thio
group, yielding the valuable key intermediate tert-butyl
4-(2,5-diamino-6-mercaptopyrimidin-4-yl)piperazine-1-carbo-
xylate 12.
5-Amino-7-piperazinyl-2-substituted-thiazolo[5,4-d]-
pyrimidines 14a-h were prepared from pyrimidine 12 in
two ways. It is well-known that a thiazole ring can be
mounted onto a 2-aminobenzenethiol by the reaction with
an acyl chloride16 or an aldehyde.17 Therefore, we envisaged
construction of a thiazole ring onto pyrimidine 12 by con-
densation with an appropriate acyl chloride or aldehyde.
First, pyrimidine 12 was reacted with an acyl chloride in
DMF. However, in the absence of base, the Boc group of
piperazinyl moiety was cleaved off and the free amine was
reacted with an acyl chloride, yielding 7-N-acylpiperazinyl-
thiazolo[5,4-d]pyrimidine. In the presence of base, 5- or
6-acylated pyrimidines were isolated and no ring closure
took place. Fortunately, heating of 5- or 6-acylated pyrimi-
dines in 3 M HCl in dioxane afforded thiazolo[5,4-d]pyrimidines
14d-h. Alternatively, thiazolo[5,4-d]pyrimidines 14a-c were
prepared by heating the pyrimidine 12 with an appropriate
aldehyde in DMSO. The Boc group of 13a-c was cleaved off
under acidic conditions, yielding compounds 14a-c. The
piperazine moiety of compounds 14a-h was further deriva-
tized to amides 15 by the reaction with p-chlorophenoxyacetyl
chloride (yielding compounds 15a-h, Table 3) or appro-
priate carboxylic acids (yielding compounds 15i,j, Table 4).
Synthesis of Oxazolo[5,4-d]pyrimidine 17. Oxazolo[5,4-
d]pyrimidine 17 was synthesized from 5-acylaminopyrimi-
dine-4,6-diol 2a which is described in Scheme 1 as an inter-
mediate in the synthesis of the thiazolo[5,4-d]pyrimidine
analogues (Scheme 4). By heating with phosphorus oxy-
chloride, compound 2a underwent a cyclodehydration reac-
tion yielding 7-chlorooxazolo[5,4-d]pyrimidine 16.18 Dis-
placement of the 7-chloro substituent was easily accom-
plished by heating with piperazine analogue 9, affording
compound 17.
Synthesis of Thiazolo[4,5-d]pyrimidine 23. The synthesis of
thiazolo[4,5-d]pyrimidine 23 starts from commercially avail-
able 2,6-diamino-4-chloropyrimidine. Nucleophilic displace-
ment of the chlorine by heating with N-acetylpiperazine in
water yields the pyrimidine analogue 18 (Scheme 5).3b Thio-
cyanation of compound 18 by treatment with potassium thio-
cyanate, bromine, and pyridine in DMF led to the construction
of the thiazolo[4,5-d]pyrimidine scaffold (compound 19).19 The
2-amino group was converted to chlorine via diazotation of
compound 19 by treatment with sodium nitrite and CuCl
(Sandmeyer reaction).19,20 Suzuki coupling of 20 with
4-fluorophenylboronic acid furnished compound 21. The
acetyl group of 21 was cleaved off under acidic conditions
and the piperazine moiety was further derivatized with p-
chlorophenoxyacetyl chloride, affording compound 23.
Synthesis of Purine Analogue 26. Purine based compound
26 was synthesized from commercially available 2-amino-
6-chloropurine in three steps (Scheme 6). Bromination of
2-amino-6-chloropurine by treatment with bromine in aque-
ous acetic acid afforded 2-amino-8-bromo-6-chloropurine
24.21 Suzuki reaction with 1 equiv of 4-fluorophenylboronic
a Values are the mean of two independent experiments. b Cyclosporin A.
As amide derivatized compound 7a exhibited potent
immunosuppressive activity, 2-(4-chlorophenoxy)-1-(pipe-
razin-1-yl)ethanone 9 was synthesized according to the
procedure outlined in Scheme 2. Reaction of tert-butyl
piperazine-1-carboxylate with p-chlorophenoxyacetyl
chloride followed by acidic deprotection of the Boc group
furnished piperazine analogue 9. The availability of com-
pound 9 allows a one-step displacement of the sulfone
group of compounds 5b-e, yielding derivatives 7j-m
(Table 3).
The synthetic route described in Scheme 1 allows intro-
duction of structural variety easily at position 7. However, as
we would like to make variations at position 2, we preferred
another route that makes it possible to introduce the sub-
stituent at position 2 at a later stage of the synthesis (Scheme 3).
Commercially available 2,5-diamino-4,6-dihydroxypyrimidine
hydrochloride was converted to 2,5-diamino-4,6-dichloro-

658 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 2
Table 2. SAR of Different Scaffolds
Jang et al.
a Values are the mean of two independent experiments.
acid provided compound 25 as a major product. Displace-
ment of the 6-chloro substituent was easily accomplished
by heating with piperazine analogue 9, affording com-
pound 26.
of in vivo transplant rejection. Peripheral blood lymphocytes
from two individuals are mixed together in tissue culture for
several days. Lymphocytes from incompatible individuals
will stimulate each other to proliferate significantly (measured
by tritiated thymidine uptake), whereas those from compat-
ible individuals will not. In the one-way MLR test, the
lymphocytes from one of the individuals are inactivated
(usually by treatment with mitomycin or radiation), there-
by allowing only the untreated remaining population of
cells to proliferate in response to foreign histocompati-
bility antigens.
Comparison of Different Scaffolds. The first analogue
synthesized (compound 7a) was based on a thiazolo[5,4-d]-
pyrimidine scaffold, as this is a direct isostere of a pyrido[3,2-d]-
pyrimidine structure. Furthermore, the thiazolo moiety
was replaced by an isomeric thiazole (affording thiazolo-
[4,5-d]pyrimidine 23), by an imidazole (yielding purine 26),
by an oxazole (furnishing oxazolo[5,4-d]pyrimidine 17), and
by a thiophene ring (yielding thieno[2,3-d]pyrimidine 30).
Previous SAR studies on pyrido[3,2-d]pyrimidines3 and
pteridines4 have determined the optimal substituents at-
tached to the central heterocyclic ring system. The amino,
the N-p-chlorophenoxyacetylpiperazinyl, and the p-fluoro-
phenyl groups are known to contribute to the immunosup-
pressive activity. The SAR study started by comparing the
five different scaffolds, bearing an identical substitution
pattern that allows us to study the effect of scaffold variation
on immunosuppressive activity (Table 2).
Synthesis of Thieno[2,3-d]pyrimidine 30. The synthesis of
thieno[2,3-d]pyrimidine derivative 30 has been described
before,22 however without experimental data. Therefore,
we repeated this chemistry (Scheme 7), generating spectral
data of the intermediates and the final compound. 2-(4-Fluor-
ophenyl)acetaldehyde 27 was prepared by oxidation of 2-(4-
fluorophenyl)ethanol with pyridinium chlorochromate (PCC)
in moderate yield. The condensation of aldehyde 27 with
ethyl cyanoacetate in the presence of a base and elemental
sulfur (Gewald reaction) furnished ethyl 2-amino-5-(4-fluor-
ophenyl)thiophene-3-carboxylate 28.23 Reaction of com-
pound 28 with chloroformamidine hydrochloride provided
the thieno[2,3-d]pyrimidine analogue 29 in good yield. By a
benzotriazol-1-yloxytris(dimethylamino)-phosphonium
hexafluorophosphate (BOP) promoted direct amination
reaction,24 piperazine analogue 9 was introduced in a single
step, yielding final compound 30.
Biological Evaluation and Structure-Activity Relationship
(SAR) Studies
Biological Evaluation. For evaluation of the immunosup-
pressive activity of the compounds, an allogeneic mixed
lymphocyte reaction (MLR) assay was used. The MLR assay
is a fundamental benchmark test for immunosuppressants
activity. Most of the current immunosuppressive drugs are
very potent in MLR assay, and several of them were dis-
covered by this assay. Therefore, it is appropriate to evaluate
the immunosuppressive activity of new compounds with a
MLR assay. This screening is used as a predictive in vitro test
From the data in Table 2, it can be deduced that the
thiazolo[4,5-d]pyrimidine derivative is the least active with
a MLR IC50 > 10 μM. The oxazolo[5,4-d]pyrimidine
scaffold is 6-fold less potent than the reference thiazolo-
[5,4-d]pyrimidine scaffold. The thiazolo[5,4-d]pyrimidine,

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 2 659
Table 3. SAR of 2-Substituted 5-Amino-7-N-piperazinylthiazolo[5,4-d]-
pyrimidines
Table 4. SAR of the Phenoxy Moiety of 2-Substituted 5-Amino-7-N-
piperazinylthiazolo[5,4-d]pyrimidines
a Values are the mean of two independent experiments.
Scheme 2a
a Reagents and conditions: (a) p-chlorophenoxyacetyl chloride, NEt3,
CH2Cl2, rt; (b) TFA, CH2Cl2, rt.
ligand by a target protein. In order to evaluate the similarity
(or dissimilarity) between the different scaffolds, new scaf-
folds have been overlaid with a reference scaffold. The
thiazolo[5,4-d]pyrimidine scaffold was taken as a reference.
Then a Tanimoto index based on shape and electrostatic
similarity has been calculated in order to quantify the similarity
between the scaffolds.25 A score of 1.000 means similar mole-
cules. The OpenEye software packages ROCS and EON are
well suited for this purpose.26 Only the parts of the molecules
that contain the differences were used for ROCS and EON
calculations.
ROCS (rapid overlay of chemical structures) is a fast shape
comparison application, providing a measure of molecular
shape complementarity through maximizing of shape over-
lap between molecules. The Tanimoto shape scores were
obtained from the ROCS software after superposition of the
a Values are the mean of two independent experiments.
thieno[2,3-d]pyrimidine, and purine scaffold are equally
active with IC50 values ranging from 0.49 to 0.8 μM.
The MLR test is a cell-based assay and does not give any
information about the possible molecular targets of the
immunosuppressive compounds. So 3D experimental struc-
tural information of the target protein, which could help us
to understand the differences in biological activity between
the different scaffolds, cannot be used for modeling and
docking experiments.
However, both shape and electrostatic components of the
molecules are known to play a critical role in recognition of a

660 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 2
Jang et al.
Scheme 3a
a Reagents and conditions: (a) tetraethylammonium chloride, POCl3, reflux; (b) tert-butyl piperazine-1-carboxylate, DIPEA, dioxane, reflux;
(c) sodium sulfide nonahydrate, DMSO, 50 °C; (d) aldehyde, DMSO, 150 °C; (e) TFA, CH2Cl2, rt; (f) acyl chloride, pyridine, DMF, rt, then 3 M HCl in
dioxane, dioxane, 60 °C; (g) acyl chloride, pyridine, DMF, rt; (h) carboxylic acid, TBTU, DIPEA, DMF, rt.
Scheme 4a
the thieno[2,3-d]pyrimidine and thiazolo[5,4-d]pyrimidine
scaffolds. The SAR of thieno[2,3-d]pyrimidines has been
reported by our group in a separate paper.22 Because of the
innovative character of thiazolo[5,4-d]pyrimidine chemistry,
we further explored the immunosuppressive properties of this
scaffold.
SAR of the Thiazolo[5,4-d]pyrimidine Analogues. The
chemistry of the thiazolopyrimidine scaffold is centered
around the 5-amino-7-N-piperazinylthiazolo[5,4-d]pyrimidine
pattern. The SAR started with the biological evaluation
of the unsubstituted piperazinyl group (compound 6),
which shows already reasonable MLR activity (IC50
=
4.3 μM) and functions as an excellent starting point for
further optimization. In order to improve the in vitro
potency, a series of substituents was introduced (Table 1). The
introduction of aryl groups (compounds 7h and 7i) or
alkyl group (compound 7g) led to analogues that did not
show any immunosuppressive activity at 10 μM, the high-
est tested concentration. The piperazinyl group also offers
the opportunity to be derivatized as amides, urea, carbamates,
and sulfonamides. The sulfonamide derivative (compound 7e)
did not show any activity at 10 μM, and the carbamate derivative
(compound 7f) showed similar activity as unsubstituted
compound 6. On the other hand, potent activity was asso-
ciated with urea and amide. The choice for the m- and
p-tolylamide (compounds 7d and 7c) and the p-chloro-
phenoxyacetyl (compound 7a) side chain is based on the
fact that these substituents are known from previous SAR
studies on other heterocycles to impart potent immuno-
suppressive activity. The introduction of a geminal dimethyl
substituent in R-postion of the carbonyl moiety of com-
pound 7a led to compound 7b, which does not show any
immunosuppressive activity at 10 μM.
a Reagents and conditions: (a) POCl3, DIPEA, 90 °C; (b) 2-(4-chlo-
rophenoxy)-1-(piperazin-1-yl)ethanone 9, dioxane, 80 °C.
different scaffolds on the thiazolo[5,4-d]pyrimidine scaffold,
which was used as a reference (Figure 2 and Table 2).
EON compares electrostatic potential distribution overlap
between molecules.27 The Adaptive Poisson-Boltzmann Solver
software package (APBS) was used to calculate the electro-
staticpotential around the different scaffolds.28 The resulting
potential maps were mapped onto the solvent accessible surface
(calculated by MSMS)29 of the molecules and visualized by
Chimera.30 Pictures showing the electrostatic potentials
mapped on the surface of the five molecular scaffolds are
given in Figure 3. The electrostatic Poisson-Boltzmann scores
(electrostatic Tanimoto score) were calculated by OpenEye’s
EON software and are also mentioned in Table 2. On the
basis of the calculations, it can be concluded that the shape of
the molecules seems to be more important for the activity
than the electrostatic potential map in the neighborhood of
the five-membered ring.
The SAR at position 2 has been investigated intensively
(Table 3). Different linkers were introduced between the
aromatic p-fluorophenyl ring and the heterocyclic scaffold.
No linker (compound 7a) and a methylene (compound 7j), a
propyl (compound 15a), and a butyl (compound 15b) linker
all show very comparable MLR activity with IC50 ranging
Having the biological data in hand, a selection of scaffolds
for further optimization has to be done. Priority was given to

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 2 661
Scheme 5a
a Reagents and conditions: (a) 1-acetylpiperazine, water, reflux; (b) KSCN, pyridine, Br2, DMF, 65-5 °C; (c) NaNO2, conc H2SO4, 80 °C, then conc
HCl, CuCl, 40 °C; (d) 4-fluorophenylboronic acid, K2CO3, Pd(PPh3)4, dioxane/water (3/1), 100 °C; (e) 5% HCl, 100 °C; (f) 4-chlorophenoxyacetyl
chloride, pyridine, DMF, rt.
Scheme 6a
a Reagents and conditions: (a) Br2, AcOH/H2O, 60 °C; (b) 4-fluorophenylboronic acid, K2CO3, Pd(PPh3)4, dioxane/water (3/1), 100 °C; (c) 2-(4-
chlorophenoxy)-1-(piperazin-1-yl)ethanone 9, dioxane, 80 °C.
Scheme 7a
Figure 2. Predicted three-dimensional shape of compounds 7a, 17,
23, 26, and 30. Color code of carbon atoms is as follows: for com-
a Reagents and conditions: (a) PCC, CH2Cl2, rt; (b) ethyl cyanoace-
pound 7a, red; for compound 17, green; for compound 23, khaki; for
tate, S, NEt3, DMF, 50 °C to rt; (c) chloroformamidine hydrochloride,
compound 26, magenta; for compound 30, cyan.
dimethylsulfone, 130 °C; (d) BOP, DBU, 2-(4-chlorophenoxy)-1-(piperazin-
1-yl)ethanone 9, CH3CN, rt to 60 °C.
These two compounds all exhibited IC50 values around 300 nM.
from 0.49 to 0.92 μM. On the other hand, an ethyl
Even an aliphatic alkyl chain (compound 15c) retained
immunosuppressive activity with a MLR IC50 of 0.69 μM. It
indicated that quite some structural variation is tolerated at
that position. Furthermore, a number of heterocyclic aro-
matics were introduced, as exemplified by the synthesis of the
three pyridyl analogues 15f-h. The 2-pyridyl analogue 15h is
equipotent with the original lead compound 7a, whereas the
3- and 4-pyridyl analogues show a 10-fold increase in MLR
activity, yielding IC50 values of 50 and 40 nM, respectively.
This is as potent as cyclosporin A (MLR IC50 = 54 nM), a
(compound 7l) or an ethoxy linker (compound 7m) led to a
10-fold decrease in MLR activity. Branching of the benzylic
carbon of compound 7j led to the preparation of compound
7k. This compound (which was tested as a racemate) shows a
6-fold drop in immunosuppressive activity when compared
with its unsubstituted analogue 7j.
In order to further establish the SAR at position 2,
a number of additional analogues was made in which the
p-fluorophenyl group was replaced by a p-methylphenyl
(compound 15d) and a p-chlorophenyl (compound 15e).

662 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 2
Jang et al.
Figure 3. Electrostatic potentials mapped on the surface of the compounds 7a, 17, 23, 26, and 30: -0.5, red; 0, white; 0.5, blue.
Table 5. Effect of Compound 15g in Suppression of Rejection of C57-
to-Balb/c Mouse Cardiac Allografts
thiazolo[5,4-d]pyrimidine scaffold. By selection of the right
decoration pattern of the scaffold, potent in vitro immuno-
suppressive activity could be obtained. One representative of
this series (compound15g) was selected todemonstrate in vivo
biological activity in a mouse model of cardiac allograft trans-
plantation. From these in vitro and in vivo studies, we can
conclude that novel immunosuppressive agents have been
discovered that are equally active to cyclosporin A, a marketed
immunosuppressive drug that is routinely used in transplant
patients. Therefore, these findings are an excellent starting
point for the development of a new generation of immuno-
suppressive drugs. Further pharmacokinetic and pharma-
codynamic studies are underway to evaluate the importance
of those findings.
compd
dosea ((mg/kg)/day)
surviving days
6, 7, 8, 8, 9, 9
p b
vehicle
CsA
15g
40
40
>30, >30, >30, >30
17, >30, >30, >30
<0.001
<0.03
a By daily gavage. b Student t test; vs vehicle group.
clinically used immunosuppressive drug, which was included
as positive control.
In order to probe the optimal substitution pattern on the
phenoxy ring, a number of additional analogues were made
(Table 4). The p-chloro and p-bromo analogues display
comparable immunosuppressive activity, whereas the unsub-
stituted analogue (compound 7n) and especially the p-fluoro
(compound 7o) and the p-methoxy (compound 7q) conge-
ners demonstrate profound immunosuppressive activity
with MLR IC50 values of 70 and 40 nM, respectively.
The combination of both optimized structural features
(the 3-pyridinyl ring at position 2 and a 4-fluoro/methoxy
phenoxy acetyl side chain on the piperazinyl moiety) led to
the synthesis of compounds 15i and 15j. Both analogues
demonstrate very potent immunosuppressive activity in the
MLR assay (IC50 for both compounds is 12 nM).
In Vivo Evaluation of Compound 15g. The procedures
described above led to the identification of compounds with
strong immunosuppressive activity in the MLR assay. This,
however, does not guarantee in vivo efficacy of the com-
pounds. Therefore, we selected compound 15g, as represen-
tative compound, for in vivo evaluation of its immuno-
suppressive activity.
Graft Survival. The in vivo efficacy of compound 15g was
studied in a mouse model of cardiac allograft transplanta-
tion. Drug vehicle (n = 6) or compound 15g (n = 4) was
given by oral gavage daily, from the day of transplantation,
until day 30 after transplantation. Cyclosporin A, the major
immunosuppressive drug used in organ transplantation, was
used as a reference and was also administered by daily gavage
(n = 4). The results of this experiment are shown in Table 5.
Animals treated with vehicle alone rejected their allograft
within 6-9 days after transplantation. CsA at the given dose
achieved survival of all four grafts as long as the treatment
was continued. Oral administration of compound 15g (at a
dose of 40 mg/kg) resulted in continuous graft survival in 3
out of 4 grafts. No signs of toxicity were observed by treatment
of mice with compound 15g at a dose of 40 (mg/kg)/day for
30 days.
Experimental Section
Chemistry. For all reactions, analytical grade solvents were
used. All moisture-sensitive reactions were carried out in oven-
dried glassware (135 °C). 1H and 13C NMR spectra were recorded
with a Bruker Advance 300 instrument (1H NMR, 300 MHz;
13C NMR, 75 MHz), using tetramethylsilane as internal stan-
dard for 1H NMR spectra and DMSO-d6 (39.5 ppm) or CDCl3
(77.2 ppm) for 13C NMR spectra. Abbreviations used are the
following: s = singlet, d = doublet, t = triplet, q = quartet, m =
multiplet, br s = broad signal. Coupling constants are expressed
in hertz. Mass spectra are obtained with a Finnigan LCQ
advantage Max (ion trap) mass spectrophotometer from Thermo
Finnigan, San Jose, CA, U.S. Exact mass measurements are
performed on a quadrupole time-of-flight mass spectrometer
(Q-tof-2, Micromass, Manchester, U.K.) equipped with a stan-
dard electrospray-ionization (ESI) interface. Samples were in-
fused in i-PrOH/H2O (1:1) at 3 μL/min. Melting points are
determined on a Barnstead IA 9200 and are uncorrected. Pre-
coated aluminum sheets (Fluka Silica gel/TLC-cards, 254 nm)
were used for TLC. Column chromatography was performed on
˚
ICN silica gel 63-200, 60 A. All final compounds possess a purity
of at least 95%, as determined by a RP-HPLC analysis.
Diethyl 2-(4-Fluorobenzamido)malonate (1a). To a solution of
diethyl aminomalonate hydrochloride (5.0 g, 23.6 mmol) and
pyridine (7.64 mL, 94.5 mmol) in DMF (60 mL) was added
p-fluorobenzoyl chloride (4.19 mL, 35.4 mmol). The reaction
mixture was stirred at room temperature for 15 h. After removal
of the solvents, the residue was redissolved in dichloromethane,
washed with water, brine, and dried over Na2SO4. After removal
of the solvent, the crude residue was purified by flash chroma-
tography on silica (CH2Cl2/MeOH 30:1) to yield the title
1
compound as a white solid (4.9 g, 70%). Mp 108-109 °C. H
NMR (300 MHz, DMSO, 25 °C): δ 9.37 (d, J= 7.5 Hz, 1H,
NH), 7.96-8.01 (m, 2H, ArH), 7.33 (t, J= 8.8 Hz, 2H, ArH),
5.30 (d, J= 7.5 Hz, 1H, CH), 4.12-4.28 (m, 4H, CH2), 1.22
(t, J= 7.1 Hz, 6H, CH3) ppm. 13C NMR (75 MHz, DMSO,
25 °C): δ166.5, 165.4, 164.2 (d, J= 247.9 Hz), 130.4 (d, J= 9.2 Hz),
129.4 (d, J= 2.8 Hz), 115.4 (d, J= 21.8 Hz), 61.7, 56.5, 13.9 ppm.
HRMS: calcd for C14H17FNO5 298.1091, found 298.1092.
General Method for Preparation of Dialkyl 2-Acylaminoma-
lonate (1b-d). To a solution of carboxylic acid (9.3 mmol) and
1-hydroxybenzotriazole (32.2 mmol) in dichloromethane (140 mL)
was added DCC (32.2 mmol). The reaction mixture was stirred
at room temperature for 2 h. The resulting solution was cooled
to 0 °C. Then a solution of diethyl aminomalonate hydrochloride
These data indicate that compound 15g can suppress a
robust in vivo allogeneic response.
Conclusion
On the basis of previously published research and using the
concept of isosterism, a novel series of immunosuppressive
compounds have been designed and synthesized. The SAR
studieshavebeen guided by a cell-based MLRassay and led to
the synthesis of immunosuppressive compounds based on a

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 2 663
(29.3 mmol) and pyridine (29.3 mmol) in DMF (10 mL) was
added. The temperature was allowed to rise to ambient tem-
perature. After 1 h, the reaction mixture was concentrated under
reduced pressure, and the residue was partitioned between ethyl
acetate and a 5% NaHCO3 solution. The organic layer was
washed with water, brine and was dried over Na2SO4. After
removal of the solvent, the crude residue was purified by flash
chromatography on silica (CH2Cl2/MeOH 50:1) to yield the title
compound.
Diethyl 2-(2-(4-Fluorophenyl)acetamido)malonate (1b). White
solid (yield 93%). Mp 91-92 °C. 1H NMR (300 MHz, CDCl3,
25 °C): δ 7.26-7.29 (m, 2H, ArH), 7.05 (t, J = 8.7 Hz, 2H, ArH),
6.44 (d, J = 6.9 Hz, 1H, NH), 5.12 (d, J = 6.9 Hz, 1H, CH),
4.16-4.32 (m, 4H, CH2), 3.61 (s, 2H, CH2Ph), 1.27 (t, J = 7.1
Hz, 6H, CH3) ppm. 13C NMR (75 MHz, CDCl3, 25 °C): δ 170.6,
166.3, 162.2 (d, J = 244.2 Hz), 131.0 (d, J = 7.7 Hz), 130.1
(d, J = 3.4 Hz), 115.8 (d, J = 21.3 Hz), 62.7, 56.6, 42.1, 14.0
ppm. HRMS: calcd for C15H19FNO5 [M þ H]þ 312.1247, found
312.1247.
General Method for Preparation of 5-Acylamino-2-amino-4,6-
dihydroxypyrimidine (2a-d). Guanidine hydrochloride (14.1 mmol)
and dialkyl 2-aminoacylmalonate (10.1 mmol) were added to a
solution of sodium (20.2 mmol) in ethanol (50 mL). The reaction
mixture was refluxed for 3 h. The mixture was cooled to room
temperature. The solid product was filtered off and washed with
ethanol. The product was dissolved in the minimum amount
of water and acidified to pH 4-5 with 5 M HCl. The precipitate was
collected, washed with water, and dried to give the title compound.
N-(2-Amino-4,6-dihydroxypyrimidin-5-yl)-4-fluorobenzamide
(2a). White solid (yield 53%). Mp 298 °C. 1H NMR (300 MHz,
DMSO, 25 °C): δ 10.62 (s, 2H, OH), 8.81 (s, 1H, NH), 7.96-8.01
(m, 2H, ArH), 7.28 (t, J = 8.8 Hz, 2H, ArH), 6.63 (s, 2H, NH2)
ppm. 13C NMR (75 MHz, DMSO, 25 °C): δ 164.6, 163.7 (d, J =
245.8 Hz), 162.3, 152.4, 131.3, 130.2 (d, J = 9.0 Hz), 114.9 (d,
J = 21.5 Hz), 91.1 ppm. HRMS: calcd for C11H10FN4O3
[M þ H]þ 265.073 69, found 265.071 48
2-(2-(4-Fluorophenoxy)ethyl)-7-(methylthio)thiazolo[5,4-d]-
pyrimidin-5-amine (4d). A solution of N-(2-amino-4,6-dihydrox-
ypyrimidin-5-yl)-3-(4-fluorophenoxy)propanamide 2d (2.5 g,
8.11 mmol) and phosphorus pentasulfide (3.60 g, 16.2 mmol)
in dry pyridine (40 mL) was refluxed for 6 h. After the mixture
was cooled to room temperature, the precipitate was filtered off,
washed with EtOAc, and dried. The crude 5-amino-2-(2-(4-
fluorophenoxy)ethyl)thiazolo[5,4-d]pyrimidine-7-thiol (2.0 g,
6.20 mmol) was redissolved in DMSO (30 mL). Triethylamine
(0.85 mL, 6.12 mmol) and iodomethane (0.31 mL, 4.90 mmol)
were added. The reaction mixture was stirred for 12 h at room
temperature under a nitrogen atmosphere. The mixture was poured
into water and extracted with EtOAc. The combined organic
layers were dried over Na2SO4, and the solvents were removed
under reduced pressure. The crude residue was purified by chroma-
tography on silica gel (CH2Cl2/MeOH 100:1), yielding the title
compound as a light yellow solid (0.45 g, 15% over two steps).
Mp 115 °C. 1H NMR (300 MHz, CDCl3, 25 °C): δ 6.96 (t, J =
9.1 Hz, 2H, ArH), 6.34-6.88 (m, 2H, ArH), 5.19 (s, 2H, NH2),
4.31 (t, J = 6.2, 2H, CH2), 3.48 (t, J = 6.2, 2H, CH2) ppm. 13
C
NMR (75 MHz, CDCl3, 25 °C): δ 164.7, 164.3, 161.9, 159.2,
157.6 (d, J = 237.1 Hz), 135.4, 116.0 (d, J = 23.0 Hz), 115.9 (d,
J = 7.8 Hz), 66.8, 34.7, 12.1 ppm. HRMS: calcd for C14H14-
FN4OS2 [M þ H]þ 337.0593, found 337.058 27.
2-(1-(4-Fluorophenyl)ethyl)-7-(methylthio)thiazolo[5,4-d]-
pyrimidin-5-amine (4e). To a solution of 2-(4-fluorobenzyl)-
7-(methylthio)thiazolo[5,4-d]pyrimidin-5-amine 4b (0.4 g,
1.31 mmol) and 2 N NaOH (0.65 mL, 1.31 mmol) in DMSO
(7 mL) was added iodomethane (81 μL, 1.31 mmol). The
reaction mixture was stirred at room temperature for 2 h. The
mixture was poured into water and extracted with ethyl acetate,
brine and dried over Na2SO4. After removal of the solvents
under reduced pressure, the residue was purified by chromatog-
raphy on silica gel (hexane/EtOAc 5:1), yielding the title com-
pound as a white solid (0.29 g, 69%). Mp 134-135 °C. 1H NMR
(300 MHz, CDCl3, 25 °C): δ 7.31(br s, 2H, ArH), 7.02 (br s, 2H,
ArH), 5.16 (s, 2H, NH2), 4.47 (br s, 1H, CH), 2.58 (s, 3H, CH3),
1.76 (d, J = 6.6 Hz, 3H, CHCH3) ppm. 13C NMR (75 MHz,
CDCl3, 25 °C): δ 169.9, 164.8, 164.4, 162.2 (d, J = 244.5 Hz),
159.1, 138.5 (d, J = 3.1 Hz), 135.6, 129.3 (d, J = 7.9 Hz), 115.8
(d, J = 21.3 Hz), 44.4, 21.3, 12.2 ppm. HRMS: calcd for
C14H14FN4S2 [M þ H]þ 321.064 39, found 321.063 77.
General Method for Preparation of 5-Amino-7-thio-2-substi-
tuted-thiazolo[5,4-d]pyrimidine (3a-c). A solution of 5-acylamino-
2-amino-4,6-dihydroxypyrimidine (4.92 mmol) and phosphorus
pentasulfide (9.84 mmol) in dry pyridine (25 mL) was refluxed for
6 h. The solvents were evaporated in vacuo. The crude residue was
purified by flash chromatography on silica (CH2Cl2/MeOH 30:1),
yielding the title compound.
5-Amino-2-(4-fluorophenyl)thiazolo[5,4-d]pyrimidine-7-thiol (3a).
Yellow solid (yield 93%). Mp 314 °C. 1H NMR (300 MHz, DMSO,
25 °C): δ 12.51 (s, 1H, SH), 7.96-8.01 (m, 2H, ArH), 7.38 (t, J =
8.8 Hz, 2H, ArH), 7.10 (s, 2H, NH2) ppm. 13C NMR (75 MHz,
DMSO, 25 °C): δ 176.3 163.5, 163.4 (d, J = 247.5 Hz), 157.2, 140.7,
129.4 (d, J = 2.9 Hz), 128.7 (d, J = 8.9 Hz), 116.4 (d, J = 22.1 Hz)
ppm. HRMS: calcd for C11H8FN4S2 [M þ H]þ 279.0174, found
279.0165
General Method for Preparation of 5-Amino-7-methylthio-2-
substituted-thiazolo[5,4-d]pyrimidine (4a-c). To a solution of
5-amino-7-thio-2-substituted-thiazolo[5,4-d]pyrimidine (4.31 mmol)
and triethylamine (10.8 mmol) in DMSO (25 mL) was added
iodomethane (8.62 mmol). The reaction mixture was stirred for
12 h under N2 at 25 °C. The mixture was poured onto water and
extracted with EtOAc. The organic extracts were dried over
Na2SO4, and the solvent was removed under reduced pressure.
The crude residue was purified by flash chromatography on
silica (CH2Cl2/MeOH 80:1), yielding the title compound.
2-(4-Fluorophenyl)-7-(methylthio)thiazolo[5,4-d]pyrimidin-5-
amine (4a). Light yellow solid (yield 60%). Mp 217-218 °C. 1H
NMR (300 MHz, DMSO, 25 °C): δ 7.98-8.03 (m, 2H, ArH),
7.39 (t, J = 8.8 Hz, 2H, ArH), 7.09 (s, 2H, NH2), 2.59 (s, 3H,
CH3) ppm. 13C NMR (75 MHz, DMSO, 25 °C): δ 164.5, 163.6
(d, J = 247.7 Hz), 163.3, 159.8, 157.5, 134.7, 129.3 (d, J =
2.8 Hz), 128.9 (d, J = 8.9 Hz), 116.4 (d, J = 22.2 Hz), 11.3 ppm.
HRMS: calcd for C12H10FN4S2 [M þ H]þ 293.0331, found
293.0328
General Method for Preparation of 5-Amino-7-methylsulfonyl-
2-substituted-thiazolo[5,4-d]pyrimidine (5a-e). To a solution of
5-amino-7-thio-2-substituted-thiazolo[5,4-d]pyrimidine (1.03 mmol)
in dichloromethane (5 mL) was added mCPBA (70%, 2.57
mmol) at 0 °C. The reaction mixture was stirred for 3 h, whereby
the reaction temperature was gradually increased from 0 °C to
room temperature. The reaction mixture was diluted with CHCl3
and was washed with a saturated NaHCO3 solution, brine and
was dried over Na2SO4. After removal of the solvent under
reduced pressure, the residue was purified by flash chromato-
graphy on silica (CH2Cl2/MeOH 50:1), affording the title
compound.
2-(4-Fluorophenyl)-7-(methylsulfonyl)thiazolo[5,4-d]pyrimidin-
1
5-amine (5a). White solid (yield 93%). Mp 231 °C. H NMR
(300 MHz, DMSO, 25 °C): δ 8.11-8.16 (m, 2H, ArH), 7.12
(s, 2H, NH2), 7.44 (t, J = 8.8 Hz, 2H, ArH), 3.58 (s, 3H, CH3)
ppm. 13C NMR (75 MHz, DMSO, 25 °C): δ 171.5, 164.2 (d, J =
249.2 Hz), 160.9, 159.7, 156.9, 131.5, 129.9 (d, J = 9.1 Hz), 128.8
(d, J = 2.9 Hz), 116.6 (d, J = 22.3 Hz), 41.4 ppm. HRMS: calcd
for C12H10FN4O2S2 [M þ H]þ 325.0229, found 325.0222
General Method for Introduction of Amine at Position 7 (6,
7g-m). To a solution of 5-amino-7-methylsulfanyl-2-substi-
tuted-thiazolo[5,4-d]pyrimidine (1.23 mmol) and triethylamine
(1.85 mmol) in dioxane (6 mL) was added amine (1.85 mmol).
The reaction mixture was heated at 60 °C for 5 h. After the
mixture was cooled, the volatiles were removed under reduced
pressure. The crude residue was purified by flash chromatography

664 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 2
Jang et al.
on silica (CH2Cl2/MeOH 15:1 for comound 6 and CH2Cl2/
MeOH 50:1 for the others), furnishing the title compound.
2-(4-Fluorophenyl)-7-(piperazin-1-yl)thiazolo[5,4-d]pyrimidin-
5-amine (6). Light yellow solid (yield 67%). Mp 205 °C. 1H
NMR (300 MHz, DMSO, 25 °C): δ 9.09 (t, J = 9.2 Hz, 1H,
NH), 7.98-8.03 (m, 2H, ArH), 7.37 (t, J = 8.8 Hz, 2H, ArH),
6.58 (s, 2H, NH2), 4.45 (br s, 4H, NCH2), 3.26 (br s, 4H,
NHCH2) ppm. 13C NMR (75 MHz, DMSO, 25 °C): δ 167.9,
163.1 (d, J = 246.5 Hz), 159.9, 154.5, 151.5, 129.8 (d, J =
3.0 Hz), 128.3 (d, J = 8.6 Hz), 116.3 (d, J = 22.0 Hz), 46.5,
45.7 ppm. HRMS: calcd for C15H16FN6S [M þ H]þ 331.1141,
found 331.1129
General Method of Coupling of 5-Amino-7-(piperazin-1yl)-2-
substituted-thiazolo[5,4-d]pyrimidine with Acyl Chloride (7a, 15a-h).
To a solution of 5-amino-7-(piperazin-1-yl)-2-substituted-thiazolo-
[5,4-d]pyrimidine (0.12 mmol) and pyridine (0.18 mmol) in DMF
(1 mL) was added 4-chlorophenoxyacetyl chloride (0.13 mmol). The
reaction mixture was stirred for 2 h at room temperature. The
reaction mixture was quenched with water, extracted with EtOAc,
brine, and dried over Na2SO4. After removal of the solvents, the
crude residue was purified by flash chromatography on silica
(CH2Cl2/MeOH 50:1) to yield the title compound.
1-(4-(5-Amino-2-(4-fluorophenyl)thiazolo[5,4-d]pyrimidin-7-yl)-
piperazin-1-yl)-2-(4-chlorophenoxy)ethanone (7a). White solid
(yield 53%). Mp 240 °C. 1H NMR (300 MHz, CDCl3, 25 °C):
δ 7.86-7.91 (m, 2H, ArH), 7.26 (d, J = 9.0 Hz, 2H, ArH), 7.15
(t, J = 8.4 Hz, 2H, ArH), 6.93 (d, J = 9.0 Hz, 2H, ArH), 4.81 (s,
2H, NH2), 4.75 (s, 2H, CH2), 4.32 (br s, 4H, N(CH2)2), 3.75
(quint, J = 5.0 Hz, 4H, CON(CH2)2) ppm. 13C NMR (75 MHz,
CDCl3, 25 °C): δ 168.3, 166.6, 164.2 (d, J = 249.9 Hz), 159.4,
156.6, 155.4, 155.2, 130.1 (d, J = 3.1 Hz), 129.8, 129.0, 128.7
(d, J = 8.6 Hz), 127.0, 116.3 (d, J = 22.0 Hz), 116.1, 68.2, 45.7,
42.5 ppm. HRMS: calcd for C23H21ClFN6O2S [M þ H]þ
499.1119, found 499.1130.
General Method For Coupling of 5-Amino-7-(piperazin-1-yl)-
2-substituted-thiazolo[5,4-d]pyrimidine with Carboxylic Acids
(7b,n-q and 15i,j). To a solution of 5-amino-7-(piperazin-1-yl)-
2-substituted-thiazolo[5,4-d]pyrimidine (0.09 mmol) and car-
boxylic acid (0.14 mmol) in DMF (2 mL) was added TBTU
(N,N,N0,N0-tetramethyl-O-(benzotriazol-1-yl)uronium tetrafluoro-
borate) (0.14 mmol), followed by DIPEA (0.14 mmol). The
reaction mixture was stirred at room temperature for 3 h. The
mixture was diluted with water and extracted with dichloro-
methane. The combined organic layers were washed with brine
and dried over Na2SO4. After removal of the solvents under
reduced pressure, the crude residue was purified by flash chro-
matography on silica (CH2Cl2/MeOH 50:1), yielding the title
compound.
1-(4-(5-Amino-2-(4-fluorophenyl)thiazolo[5,4-d]pyrimidin-7-yl)-
piperazin-1-yl)-2-(4-chlorophenoxy)-2-methylpropan-1-one (7b).
White solid (yield 59%). 1H NMR (300 MHz, DMSO, 25 °C):
δ 7.94-7.98 (m, 2H, ArH), 7.32-7.38 (m, 4H, ArH), 6.87 (d,
2H, ArH), 6.46 (s, 2H, NH2), 4.14 (br s, 2H, NCH2), 3.93 (br s,
4H, N(CH2)2), 3.68 (br s, 2H, NCH2), 1.56 (s, 6H, CH3, CH3)
ppm. 13C NMR (75 MHz, DMSO, 25 °C): δ 170.2, 167.8, 163.1
(d, J = 246.6 Hz), 159.8, 154.4, 153.8, 152.0, 129.7 (d, J =
3.2 Hz), 129.3, 128.5 (d, J = 8.6 Hz), 125.3, 124.7, 118.8, 116.2
(d, J = 21.9 Hz), 80.8, 45.2, 42.6, 25.6 ppm. HRMS: calcd for
C25H25ClFN6O2S [M þ H]þ 527.1432, found 527.1445.
4-(5-Amino-2-(4-fluorophenyl)thiazolo[5,4-d]pyrimidin-7-yl)-
N-p-tolylpiperazine-1-carboxamide (7c). To a solution of 2-(4-
fluorophenyl)-7-(piperazin-1-yl)thiazolo[5,4-d]pyrimidin-5-amine 6
(50 mg, 0.15 mmol) in DMF (1 mL) was added p-tolyl isocyanate
(21 μL, 0.17 mmol) in DMF (0.3 mL). The reaction mixture was
stirred for 2 h at room temperature. The reaction mixture was
quenched with water, extracted with EtOAc, brine, and dried
over Na2SO4. After removal of the solvent, the crude residue
was purified by flash chromatography on silica (CH2Cl2/MeOH
100:1) to yield the title compound as a white solid (31 mg, 44%).
Mp 233-235 °C.1H NMR (300 MHz, CDCl3, 25 °C): δ 7.86-7.91
(m, 2H, ArH), 7.26 (d, J = 1.6 Hz, 2H, tolyl H), 7.10-7.23
(m, 4H, ArH, tolyl H), 6.34 (s, 1H, NH), 4.81 (s, 2H, NH2), 4.42
(br s, 4H, NCH2), 3.28 (t, J = 5.3 Hz, 4H, CONCH2), 2.31
(s, 3H, CH3) ppm. 13C NMR (75 MHz, CDCl3, 25 °C): δ 168.3,
164.1 (d, J = 249.8 Hz), 159.5, 155.4, 155.3, 155.2, 136.3, 133.3,
130.2 (d, J = 3.3 Hz), 129.7, 128.6 (d, J = 8.6 Hz), 126.8, 120.5,
116.3 (d, J = 21.9 Hz), 45.6, 44.1, 20.9 ppm. HRMS: calcd for
C23H23FN7OS [M þ H]þ 464.1669, found 464.1671.
2-(4-Fluorophenyl)-7-(4-(phenylsulfonyl)piperazin-1-yl)thiazolo-
[5,4-d]pyrimidin-5-amine (7e). To a solution of 2-(4-fluoro-
phenyl)-7-(piperazin-1-yl)thiazolo[5,4-d]pyrimidin-5-amine 6
(50 mg, 0.15 mmol) and pyridine (18 μL, 0.23 mmol) in DMF
(1mL) wasadded benzenesulfonyl chloride (21 μL, 0.17 mmol).
The reaction mixture was stirred for 3 h at room temperature.
The reaction mixture was quenched with water, extracted with
EtOAc, brine, and dried over Na2SO4. After removal of the
solvent, the crude residue was purified by flash chromatogra-
phy on silica (CH2Cl2/MeOH 80:1) to yield the title compound
as a white solid (32 mg, 45%). Mp 280 °C. 1H NMR (300 MHz,
DMSO, 25 °C): δ 7.94-7.98 (m, 2H, ArH), 7.64-7.79 (m, 5H,
ArH), 7.35 (t, J = 8.6 Hz, 2H, ArH), 6.48 (s, 2H, NH2), 4.35
(br s, 4H, NCH2), 3.06 (br s, 4H, CONCH2) ppm. 13C NMR
(75 MHz, DMSO, 25 °C): δ 167.9, 163.2 (d, J = 246.8 Hz),
159.9, 154.3, 152.2, 134.7, 133.4, 129.5 (d, J = 3.0 Hz), 129.5,
128.5 (d, J = 8.6 Hz), 127.6, 124.7, 116.2 (d, J = 21.9 Hz), 45.9,
44.3 ppm. HRMS: calcd for C21H20FN6O2S2 [M þ H]þ
471.107 32, found 471.106 94.
Benzyl 4-(5-Amino-2-(4-fluorophenyl)thiazolo[5,4-d]pyrimidin-
7-yl)piperazine-1-carboxylate (7f). To a solution of 2-(4-fluoro-
phenyl)-7-(piperazin-1-yl)thiazolo[5,4-d]pyrimidin-5-amine
6 (50 mg, 0.15 mmol) and pyridine (18 μL, 0.23 mmol) in
DMF (1 mL) was added benzyl chloroformate (24 μL,
0.17 mmol). The reaction mixture was stirred for 3 h at room
temperature. The reaction mixture was quenched with water,
extracted with EtOAc, brine, and dried over Na2SO4. After
removal of the solvent, the crude residue was purified by flash
chromatography on silica (CH2Cl2/MeOH 100:1) to yield the
title compound as a white solid (54 mg, 77%). Mp 196 °C. 1H
NMR (300 MHz, CDCl3, 25 °C): δ 7.85-7.89 (m, 2H, ArH),
7.40-7.35 (m, 5H, ArH), 7.13 (t, J = 8.6 Hz, 2H, ArH), 5.19
(s, 2H, CH2), 4.82 (s, 2H, NH2), 4.34 (br s, 4H, NCH2), 3.66
(br s, 4H, CONCH2) ppm. 13C NMR (75 MHz, CDCl3,
25 °C): δ 168.3, 164.1 (d, J = 249.8 Hz), 159.5, 155.5,
155.3, 155.02, 155.00, 136.7, 130.1 (d, J = 3.3 Hz), 128.7,
128.6 (d, J = 8.3 Hz), 128.4, 128.3, 128.2, 126.7, 116.2 (d, J =
22.0 Hz), 45.8, 44.1 ppm. HRMS: calcd for C23H22FN6O2S
[M þ H]þ 465.150 90, found 465.150 05.
tert-Butyl 4-(2-(4-Chlorophenoxy)acetyl)piperazine-1-carbox-
ylate (8). To a solution of tert-butyl piperazine-1-carboxylate
(0.30 g, 0.16 mmol) and triethylamine (0.34 mL, 2.42 mmol) in
dichloromethane (8 mL) was added p-chlorophenoxyacetyl
chloride (0.36 mg, 1.77 mmol). The reaction mixture was stirred
at room temperature overnight. The mixture was diluted with
dichloromethane and washed with water, brine and dried over
Na2SO4. After removal of the solvents, the crude residue was
purified by chromatography on silica gel (CH2Cl2/MeOH 80:1)
to yield the title compound as a white solid (0.57 g, 100%). Mp
89 °C. 1H NMR (300 MHz, CDCl3, 25 °C): δ 7.25 (d, J = 8.9 Hz,
2H, ArH), 6.88 (d, J = 8.9 Hz, 2H, ArH), 4.68 (s, 2H, CH2), 3.57
(br s, 4H, CON(CH2)2), 3.41 (br s, 4H, CON(CH2)2), 1.46 (s,
3H, CH3) ppm. 13C NMR (75 MHz, CDCl3, 25 °C): δ 166.5,
156.5, 154.6, 129.7, 126.9, 116.0, 80.6, 68.1, 45.4, 43.8, 42.1, 28.5
ppm. HRMS: calcd for C17H24ClN2O4 [M þ H]þ 355.142 46,
found 355.141 67.
2-(4-Chlorophenoxy)-1-(piperazin-1-yl)ethanone (9). A sus-
pension of tert-butyl 4-(2-(4-chlorophenoxy)acetyl)piperazine-
1-carboxylate 8 (0.58 g, 0.16 mmol) in dichloromethane (8 mL)
was treated dropwise at room temperature with TFA until the
solid completely dissolved. The reaction mixture was stirred under
nitrogen at room temperature overnight. The volatiles were

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 2 665
evaporated to dryness, diluted with water, and the solid was
collected by filtration. The solid was washed with water and
dried to yield the title compound as a white solid (0.30 g, 72%).
Mp 239-240 °C. H NMR (300 MHz, DMSO, 25 °C): δ 9.02
(s, 1H, NH), 7.32 (d, J = 8.8 Hz, 2H, ArH), 6.96 (d, J = 8.8 Hz,
2H, ArH), 4.90 (s, 2H, CH2), 3.65 (br s, 4H, CON(CH2)), 3.18
(br s, 2H, NCH2), 3.10 (br s, 2H, NCH2) ppm. 13C NMR
(75 MHz, DMSO, 25 °C): δ 165.9, 156.9, 129.1, 124.6, 116.5,
65.7, 42.6, 41.2, 38.1 ppm. HRMS: calcd for C12H16ClN2O2
[M þ H]þ 255.090 03, found 255.089 13.
NMR (300 MHz, CDCl3, 25 °C): δ 7.83 (d, J = 7.7 Hz, 2H, ArH),
7.42 (d, J = 7.7 Hz, 2H, ArH), 4.85 (s, 2H, NH2), 4.32 (br s, 4H,
N(CH2)2), 3.59 (br s, 4H, N(CH2)2), 1.52 (s, 9H, t-Bu) ppm. 13
C
1
NMR (75 MHz, CDCl3, 25 °C): δ 168.2, 159.5, 155.3, 154.9, 154.6,
136.2, 132.3, 129.3, 127.8, 126.8, 80.2, 45.8, 43.9, 28.6 ppm. HRMS:
calcd for C20H24ClN6O2S [M þ H]þ 447.1370, found 447.1369.
General Method of Deprotection of Boc Group (14a,c,d). To a
solution of tert-butyl 4-(5-amino-2-substituted-thiazolo[5,4-d]-
pyrimidin-7-yl)piperazine-1-carboxylate (0.51 mmol) in dichloro-
methane (5 mL) was added TFA (0.38 mL). The reaction
mixture was stirred at room temperature overnight. The volatile
was evaporated under reduced pressure, and the residue was
diluted with water and neutralized with 1 N NaOH. The precipitates
were collected by filtration, washed with water, and dried over
P2O5, yielding the title compound.
4,6-Dichloropyrimidine-2,5-diamine (10). A suspension of 2,5-
diamino-4,6-dihydroxypyrimidine hydrochloride (5.0 g, 28 mmol)
and tetraethylammonium chloride (27.8 g, 0.167 mmol) in
phosphorus oxychloride (80 mL) was heated at 105 °C for 20 h.
After the mixture was cooled, excess phosphorus oxychloride
was distilled off under vacuum. The reaction mixture was
poured into ice-water and the pH adjusted to 4, and the mixture
was stirred for 1 h at 50 °C. The pH was adjusted to 7, cooled to
35 °C, and the product was extracted with ethyl acetate, washed
with a saturated NaHCO3 solution, brine, and dried over Na2SO4.
After removal of the solvents under reduced pressure, the residue
was purified by flash chromatography on silica (CH2Cl2/MeOH
50:1), affording the title compound (3.21 g, 64%). 1H NMR
(300 MHz, DMSO, 25 °C): δ 6.49 (s, 2H, NH2), 4.71 (s, 2H,
NH2) ppm. 13C NMR (75 MHz, DMSO, 25 °C): δ 154.6, 145.7,
126.3 ppm. HRMS: calcd for C4H5Cl2N4 [M þ H]þ 178.9891/
1780.9862, found 178.9910/180.9884.
2-(4-Chlorophenyl)-7-(piperazin-1-yl)thiazolo[5,4-d]pyrimidin-
5-amine (14a). White solid (yield 88%). H NMR (300 MHz,
1
DMSO, 25 °C): δ 7.47 (d, J = 7.9 Hz, 2H, ArH), 7.57 (d, J =
7.9 Hz, 2H, ArH), 6.62 (s, 2H, NH2), 4.45 (br s, 4H, N(CH2)2),
3.29 (br s, 4H, N(CH2)2) ppm. 13C NMR (75 MHz, DMSO, 25 °C):
δ 168.1, 159.9, 154.4, 152.2, 134.8, 131.8, 129.2, 127.8, 124.8,
42.6, 42.1 ppm. MS: 346.94 [M þ H]þ
General Method for Condensation of Pyrimidine with Acyl
Chloride (14b,e-h). To a solution of tert-butyl 4-(2,5-diamino-6-
mercaptopyrimidin-4-yl)piperazine-1-carboxylate 12 (0.31 mmol)
and pyridine (0.46 mmol) in DMF (3 mL) was added acyl chloride
(0.34 mmol). The reaction mixture was stirred for 2 h. The
mixture was extracted with ethyl acetate, washed with water,
brine, and dried over sodium sulfate. After removal of the solvent
under reduced pressure, the crude mixture was diluted with
dioxane (5 mL) and 3 M HCl in dioxane (1 mL) was added and
the mixture was heated at 60 °C for 5 h. After cooling, the
mixture was concentrated under reduced pressure and the residue
was diluted with water and neutralized with 1 N NaOH. The
precipitates were collected by filtration, washed with water, and
dried over P2O5, yielding the title compound.
tert-Butyl 4-(2,5-Diamino-2-substituted-6-chloropyrimidin-4-
yl)piperazine-1-carboxylate (11). To a solution of 2,5-diamino-
4,6-dichloropyrimidine (2.0 g g, 11.2 mmol) and DIPEA (2.9 mL,
16.8 mmol) in dioxane (40 mL) was added tert-butyl piperazine-
1-carboxylate (3.12 g, 16.8 mmol). The reaction mixture was
heated at 100 °C for overnight. After the mixture was cooled, the
volatile was removed under reduced pressure. The crude residue
was diluted with CHCl3 and was washed with a saturated
NaHCO3 solution, brine and dried over Na2SO4. After removal
of the solvents under reduced pressure, the residue was purified
by flash chromatography on silica (CH2Cl2/MeOH 50:1), af-
fording the title compound. 1H NMR (300 MHz, DMSO, 25 °C): δ
5.81 (s, 2H, NH2), 3.85 (s, 2H, NH2), 3.43 (br s, 4H, N(CH2)2),
3.25 (br s, 4H, N(CH2)2), 1.41 (s, 9H, t-Bu) ppm. 13C NMR
(75 MHz, CDCl3, 25 °C): δ 158.4, 155.3, 154.9, 146.0, 119.6,
80.2, 47.2, 43.5, 28.6 ppm. HRMS: calcd for C13H22ClN6O2
[M þ H]þ 329.1493, found 329.1516.
7-(Piperazin-1-yl)-2-(pyridin-4-yl)thiazolo[5,4-d]pyrimidin-5-
amine (14b). Yellow solid (yield 48%). H NMR (300 MHz,
1
DMSO, 25 °C): δ 8.69 (d, J = 4.6 Hz, 2H, pyridyl-H), 7.87 (d,
J = 4.6 Hz, 2H, pyridyl-H), 6.70 (s, 2H, NH2), 4.46 (br s, 4H,
N(CH2)2), 3.19 (br s, 4H, N(CH2)2) ppm.
7-Chloro-2-(4-fluorophenyl)oxazolo[5,4-d]pyrimidin-5-amine
(16). To a solution of N-(2-amino-4,6-dihydroxypyrimidin-5-yl)-
4-fluorobenzamide 2 (0.30 g, 1.14 mmol) in POCl3 (6 mL) was
added diisopropylethylamine (0.39 mL, 2.27 mmol). The reac-
tion mixture was stirred under N2 at 90 °C for 3.5 h. The reaction
mixture was allowed to cool to room temperature, and the volatiles
were evaporated to dryness. The residue was diluted with water,
and the aqueous phase was extracted with diethyl ether. The
combined organic layers were washed with a saturated NaHCO3
solution and brine, dried over Na2SO4, and concentrated under
reduced pressure. The residue was purified by chromatography
on silica gel (CH2Cl2/MeOH 100:1) to yield the title compound
tert-Butyl 4-(2,5-Diamino-6-mercaptopyrimidin-4-yl)piper-
azine-1-carboxylate (12). To a solution of tert-butyl 4-(2,5-
diamino-6-chloropyrimidin-4-yl)piperazine-1-carboxylate (2.0 g,
6.1 mmol) in DMSO (15 mL) was added sodium sulfide non-
ahydrate (2.9 g, 12.1 mmol). The reaction mixture was heated at
50 °C for overnight., After the mixture was cooled, water (15 mL)
was added and the solution was evaporated under reduced
pressure. The crude residue was diluted with water (20 mL)
and neutralized with HCl. The precipitates were collected by
filtration, washed with water, and dried over P2O5, yielding the
1
as a white solid (0.085 g, 28%). H NMR (300 MHz, DMSO,
1
25 °C): δ 8.13-8.18 (m, 2H, ArH), 7.48 (s, 2H, NH2), 7.45 (t, J =
8.9 Hz, 2H, ArH) ppm. 13C NMR (75 MHz, DMSO, 25 °C):
δ 167.1, 164.3 (d, J = 249.4 Hz), 160.7, 157.5, 149.2, 129.7 (d,
J = 9.7 Hz), 122.3 (d, J = 2.6 Hz), 120.7, 116.6 (d, J = 22.3 Hz)
ppm. HRMS: calcd for C11H7ClFN4O [M þ H]þ 265.029 24,
found 265.028 51.
1-(4-(5-Amino-2-(4-fluorophenyl)oxazolo[5,4-d]pyrimidin-7-yl)-
piperazin-1-yl)-2-(4-chlorophenoxy)ethanone (17). To a solution
of 7-chloro-2-(4-fluorophenyl)oxazolo[5,4-d]pyrimidin-5-amine
16 (30 mg, 0.11 mmol) and triethylamine (24 μL, 0.17 mmol) in
dioxane (1 mL) was added 2-(4-chlorophenoxy)-1-(piperazin-1-yl)-
ethanone 9 (43 mg, 0.17 mmol). The reaction mixture was heated
at 70 °C for 3 h. After the mixture was cooled, the volatiles were
removed under reduced pressure. The crude residue was purified
by chromatography on silica gel (CH2Cl2/MeOH 70:1) to yield
the title compound as a white solid (40 mg, 73%). Mp 244 °C.
title compound. H NMR (300 MHz, DMSO, 25 °C): δ 11.60
(s, 1H, SH), 6.23 (s, 2H, NH2), 3.99 (s, 2H, NH2), 3.39 (br s, 8H,
N(CH2)2, N(CH2)2), 1.41 (s, 9H, t-Bu) ppm. 13C NMR (75 MHz,
DMSO, 25 °C): δ 164.5, 153.9, 151.1, 148.6, 119.6, 78.9, 45.7,
43.1, 28.0 ppm. MS: 326.65 [M þ H]þ
General Method for Condensation of Pyrimidine with Alde-
hydes (13a,c,d). A solution of tert-butyl 4-(2,5-diamino-6-
mercaptopyrimidin-4-yl)piperazine-1-carboxylate 12 (6.1 mmol)
in DMSO (10 mL) was added aldehyde (2.36 mmol). The reaction
mixture was heated at 150 °C for 1 h. After the mixture was cooled,
the mixture was diluted with ethyl acetate and washed with water,
brine and dried over Na2SO4. After removal of the solvent, the
crude residue was purified by flash chromatography on silica
(CH2Cl2/MeOH 100:1) to yield the title compound.
tert-Butyl 4-(5-Amino-2-(4-chlorophenyl)thiazolo[5,4-d]pyrim-
idin-7-yl)piperazine-1-carboxylate (13a). White solid (yield 55%). 1H

666 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 2
Jang et al.
1H NMR (300 MHz, CDCl3, 25 °C): δ 8.03-8.08 (m, 2H, ArH),
7.40 (t, J = 8.8 Hz, 2H, ArH), 7.33 (d, J = 8.8 Hz, 2H, ArH),
6.98 (d, J = 8.8 Hz, 2H, ArH), 6.54 (s, 2H, NH2), 4.93 (s, 2H,
CH2), 4.19 (br s, 2H, NCH2), 4.09 (br s, 2H, N(CH2)2), 3.62 (br s,
4H, CON(CH2)2) ppm. 13C NMR (75 MHz, CDCl3, 25 °C):
δ 167.4, 165.9, 163.5 (d, J = 247.4 Hz), 160.5, 156.9, 153.7,
152.5, 129.1, 128.5 (d, J = 8.9 Hz), 124.5, 123.1 (d, J = 2.9 Hz),
116.4, 116.3 (d, J = 22.1 Hz), 108.6, 65.9, 43.9, 41.1 ppm.
HRMS: calcd for C23H21ClFN6O3 [M þ H]þ 483.134 77, found
483.133 67.
with dichloromethane, brine and dried over Na2SO4. After
removal of the solvent, the residue was purified by chromatog-
raphy on silica gel (CH2Cl2/MeOH 30:1) to yield the title
compound as a pale yellow solid (0.13 g, 73%). Mp 280 °C.
1H NMR (300 MHz, DMSO, 25 °C): δ 8.09-8.14 (m, 2H, ArH),
7.42 (t, J = 8.8 Hz, 2H, ArH), 6.33 (s, 2H, NH2), 3.88 (br s, 2H,
CH2), 3.81 (br s, 2H, CH2), 3.61 (br s, 4H, CH2), 2.06 (s, 3H,
CH3) ppm. 13C NMR (75 MHz, DMSO, 25 °C): δ 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 C20H24FN6O2S
[M þ H]þ 431.166 55, found 431.165 58.
1-(4-(2,6-Diaminopyrimidin-4-yl)piperazin-1-yl)ethanone (18). 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 solution was cooled
and made alkaline with 10 M NaOH to give a white precipitate. The
solution was filtered, washed with cold water, and dried over P2O5
in a vacuum desiccator to yield the title compound as a white solid
2-(4-Fluorophenyl)-7-(piperazin-1-yl)thiazolo[4,5-d]pyrimidin-
5-amine (22). The mixture of 1-(4-(5-amino-2-(4-fluorophenyl)-
thiazolo[4,5-d]pyrimidin-7-yl)piperazin-1-yl)ethanone 21 (0.1 g,
0.27 mmol) in 5% HCl (2 mL) was heated at 100 °C for 4 h. After
cooling, the mixture was neutralized with 2 N NaOH to pH 7.
The precipitate was filtered and 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
1
(3.3 g, 67%). Mp 274 °C. H NMR (300 MHz, DMSO, 25 °C):
δ 5.74 (s, 2H, NH2), 5.51 (s, 2H, NH2), 5.04 (s, 1H, CH), 3.33-3.46
(m, 8H, CH2), 2.02 (s, 3H, CH3) ppm. 13C NMR (75 MHz, DMSO,
25 °C): δ 168.3, 165.2, 163.5, 162.7, 74.1, 45.2, 43.7, 43.3, 40.5, 21.2
ppm. HRMS: calcd for C6H11N4O [M þ H]þ 155.093 29, found
155.092 60.
1
yellow solid (81 mg, 91%). Mp 260 °C. H NMR (300 MHz,
DMSO, 25 °C): δ 8.10-8.15 (m, 2H, ArH), 7.41 (t, J = 8.8 Hz,
2H, ArH), 6.26 (s, 2H, NH2), 3.76 (br s, 4H, CH2), 2.82 (br s, 4H,
CH2) ppm. 13C NMR (75 MHz, DMSO, 25 °C): δ 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 C20H24FN6O2S [M þ H]þ 431.166 55,
found 431.165 58.
1-(4-(2,5-Diaminothiazolo[4,5-d]pyrimidin-7-yl)piperazin-1-yl)-
ethanone (19). A solution of 1-(4-(2,6-diaminopyrimidin-4-yl)-
piperazin-1-yl)ethanone 18 (3.3 g, 26.2 mmol) and potassium
thiocyanate (15.2 g, 0.16 mol) in DMF (120 mL) was heated at
65 °C. Pyridine (4.2 mL, 52.3 mmol) was added and the solution
cooled to 5 °C. Bromine (1.34 mL, 26.2 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 reduced pressure and the
resulting solid was diluted with water, filtered, and dried over
P2O5, yielding the title compound as a pale yellow solid (2.5 g,
52%). Mp 263 °C. 1H NMR (300 MHz, DMSO, 25 °C): δ 7.85
(s, 2H, NH2), 5.90 (s, 2H, NH2), 3.53-3.64 (m, 8H, CH2), 2.03
(s, 3H, CH3) ppm. 13C NMR (75 MHz, DMSO, 25 °C): δ 172.2,
170.2, 168.5, 161.6, 157.5, 90.9, 45.3, 45.2, 44.9, 40.6, 21.2 ppm.
HRMS: calcd for C6H11N4O [M þ H]þ 155.093 29, found
155.092 60.
1-(4-(5-Amino-2-chlorothiazolo[4,5-d]pyrimidin-7-yl)piper-
azin-1-yl)ethanone (20). 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 to room temperature, and a solution of
1-(4-(2,5-diaminothiazolo[4,5-d]pyrimidin-7-yl)piperazin-1-yl)eth-
anone 19 (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 solution 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 the
mixture was cooled, 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 removal of
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 (dec).
1H NMR (300 MHz, DMSO, 25 °C): δ 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):
δ 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 C11H14ClN6OS [M þ H]þ 313.0638, found
313.0622.
1-(4-(5-Amino-2-(4-fluorophenyl)thiazolo[4,5-d]pyrimidin-7-yl)-
piperazin-1-yl)-2-(4-chlorophenoxy)ethanone (23). This com-
pound was synthesized from compound 22 according to a
procedure for the preparation of compound 7a. The crude residue
was purified by flash chromatography on silica (CH2Cl2/MeOH
40:1) to yield the title compound as a yellow solid (45 mg, 99%).
Mp 267 °C. 1H NMR (300 MHz, DMSO, 25 °C): δ 8.10-8.15
(m, 2H, ArH), 7.43 (t, J = 8.6 Hz, 2H, ArH), 7.33 (d, J = 8.6 Hz,
2H, ArH), 6.97 (d, J = 8.6 Hz, 2H, ArH), 6.34 (s, 2H, NH2), 4.92
(s, 2H, OCH2), 3.92 (br s, 2H, CH2), 3.87 (br s, 2H, CH2), 3.65
(br s, 4H, CH2) ppm. 13C NMR (75 MHz, DMSO, 25 °C):
δ 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 þ H]þ 499.111 93,
found 499.110 23.
8-Bromo-6-chloro-9H-purin-2-amine (24). To a solution of
2-amino-6-chloropurine (1.00 g, 5.90 mmol) in acetic acid (25 mL)
was added bromine (1.21 mL, 23.6 mmol) slowly. And water
(5 mL) was added to the mixture. The temperature was main-
tained between 50 and 60 °C for 36 h, and the volatiles were
evaporated under reduced pressure. The crude solid was redis-
solved in MeOH, preadsorbed on silica gel, and purified by
chromatography on silica gel (CH2Cl2/MeOH 30:1) to yield
8-bromo-6-chloro-9H-purin-2-amine as a yellow solid (0.69 g,
47%). 1H NMR (300 MHz, DMSO, 25 °C): δ 13.67 (s, 1H, NH),
6.88 (s, 2H, NH2) ppm. 13C NMR (75 MHz, DMSO, 25 °C):
δ 159.8, 156.5, 147.2, 126.8, 124.0 ppm. HRMS: calcd for
C5H4BrClN5 [M þ H]þ 247.933 86, found 247.933 26.
6-Chloro-8-(4-fluorophenyl)-9H-purin-2-amine (25). To a solu-
tion of 8-bromo-6-chloro-9H-purin-2-amine 24 (0.30 g, 1.21 mmol)
in dioxane/water (3:1, 6 mL) was added 4-fluorophenylboronic
acid (0.17 g, 1.21 mmol), K2CO3 (0.67 g, 4.83 mmol), and
Pd(PPh3)4 (0.14 g, 0.12 mmol). The reaction mixture was refluxed
under N2 for 2 h. After the mixture was cooled to room tempera-
ture, 1 N HCl was added slowly to neutralize the mixture to pH
7-8. The solvents were removed under reduced pressure. The
residue was diluted with water (10 mL) and then filtered. The crude
solid was redissolved in MeOH, preadsorbed on silica gel, and
purified by chromatography on silica gel (CH2Cl2/MeOH 70:1),
yielding 6-chloro-8-(4-fluorophenyl)-9H-purin-2-amine as a pale
yellow solid (0.10 g, 31%). 1H NMR (300 MHz, DMSO, 25 °C): δ
1-(4-(5-Amino-2-(4-fluorophenyl)thiazolo[4,5-d]pyrimidin-7-yl)-
piperazin-1-yl)ethanone (21). To a solution of 1-(4-(5-amino-2-
chlorothiazolo[4,5-d]pyrimidin-7-yl)piperazin-1-yl)ethanone 20
(0.10 g, 0.32 mmol) in dioxane/water (3:1, 4 mL) were added
4-fluorophenylboronic acid (45 mg, 0.32 mmol), K2CO3 (0.18 g,
1.28 mmol), and Pd(PPh3)4 (37 mg, 0.03 mmol). The reaction
mixture was refluxed under N2 overnight. After the mixture was
cooled to room temperature, 1 N HCl was added slowly to
neutralize the mixture to pH 7-8. The mixture was extracted

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 2 667
13.31 (s, 1H, NH), 8.14-8.19 (m, 2H, ArH), 7.39 (t, J = 8.6 Hz,
2H, ArH), 6.84 (s, 2H, NH2) ppm. 13C NMR (75 MHz, DMSO,
25 °C): δ 163.3 (d, J = 246.7 Hz), 159.7, 156.4, 149.2, 148.4, 8.8 (d,
J = 8.6 Hz), 125.8 (d, J = 2.7 Hz), 116.1 (d, J = 21.9 Hz) ppm.
HRMS: calcd for C11H8ClFN5 [M þ H]þ 264.045 23, found
264.044 61.
7.51 (s, 1H, CH), 7.23 (t, J = 8.8 Hz, ArH), 6.73 (s, 2H, NH2)
ppm. 13C NMR (75 MHz, DMSO, 25 °C): δ 167.8, 161.4 (d, J =
243.4 Hz), 158.0, 153.5, 130.9, 130.2 (d, J = 3.0 Hz), 127.0
(d, J = 8.1 Hz), 117.3, 116.9, 115.9 (d, J = 21.6 Hz) ppm.
HRMS: calcd for C12H9FN3OS [M þ H]þ 262.045 04, found
262.044 13.
1-(4-(2-Amino-8-(4-fluorophenyl)-9H-purin-6-yl)piperazin-1-yl)-
2-(4-chlorophenoxy)ethanone (26). To a solution of 6-chloro-
8-(4-fluorophenyl)-9H-purin-2-amine 25 (35 mg, 0.13 mmol)
and triethylamine (37 μL, 0.27 mmol) in dioxane (1 mL) was
added 2-(4-chlorophenoxy)-1-(piperazin-1-yl)ethanone 9 (51 mg,
0.20 mmol). The reaction mixture was heated at 70 °C for 3 h.
After the mixture was cooled, the volatiles were removed under
reduced pressure. The crude residue was purified by chroma-
tography on silica gel (CH2Cl2/MeOH 20: 1) to yield 1-(4-(2-
amino-8-(4-fluorophenyl)-9H-purin-6-yl)piperazin-1-yl)-2-(4-
chlorophenoxy)ethanone as a white solid (50 mg, 78%). 1H
NMR (300 MHz, DMSO, 25 °C): δ 12.76 (s, 1H, NH), 8.05-
8.09 (m, 2H, ArH), 7.31-7.36 (m, 4H, ArH), 6.98 (d, J = 7.9 Hz,
2H, ArH), 4.32 (s, 2H, NH2), 4.92 (s, 2H, CH2), 4.32 (br s, 2H,
1-(4-(2-Amino-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl)-
piperazin-1-yl)-2-(4-chlorophenoxy)ethanone (30). To a solution
of 2-amino-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4(3H)-one 29
(40 mg, 0.15 mmol) and BOP (88 mg, 0.20 mmol) in CH3CN
(1 mL) was added DBU (34 μL, 0.23 mmol). After the mixture
was stirred for 10 min at room temperature, 2-(4-chloro-
phenoxy)-1-(piperazin-1-yl)ethanone (59 mg, 0.23 mmol) was
added. The mixture was stirred at room temperature overnight
and then heated at 60 °C for 4 h. The solvents were removed
under reduced pressure, and the crude residue was purified by
flash chromatography on silica gel (CH2Cl2/MeOH 60:1) to
yield the title compound as a white solid (42 mg, 55%). Mp 128 °C.
1H NMR (300 MHz, DMSO, 25 °C): δ 7.52-7.57 (m, 2H, ArH),
7.27 (s, 1H, CH), 7.22 (d, J = 8.9 Hz, 2H, ArH), 7.10 (t, J =
8.7 Hz, ArH), 6.91 (d, J = 8.9 Hz, 2H, ArH), 4.78 (s, 2H, NH2),
4.74 (s, 2H, CH2), 3.86 (br s, 4H, NCH2), 3.78 (br s, 4H, NCH2)
ppm. 13C NMR (75 MHz, DMSO, 25 °C): δ 171.1, 165.9, 161.5
(d, J = 243.5 Hz), 159.7, 158.6, 156.9, 130.4 (d, J = 3.3 Hz), 129.0,
128.6, 127.3 (d, J = 7.9 Hz), 124.5, 117.4, 116.4, 115.8 (d, J =
21.5 Hz), 109.9, 65.9, 45.8, 45.6, 43.5, 40.9 ppm. HRMS: calcd for
C24H22ClFN5O2S [M þ H]þ 498.116 68, found 498.115 11.
MLR Assay. Peripheral blood mononuclear cells (PBMCs)
were isolated from heparinized blood of healthy donors by
density-gradient centrifugation over Lymphoprep. PBMCs were
resuspended at a concentration of 1.6 ꢀ 106 cells/mL in complete
medium (RPMI-1640 containing 10% heat-inactivated fetal calf
serum (FCS) and antibiotics). RPMI1788 cells were treated with
30 μg/mL mitomycin C for 20 min at 37 °C, washed four times
with medium, and finally suspended in complete RPMI
medium to a density of 0.6 ꢀ 106 cells/mL. An amount of
75 μL of each cell suspension was mixed with 50 μL of diluted
compound. The mixed cells were cultured at 37 °C for 5 days
in 5% CO2. DNA synthesis was assayed by the addition of
1 μCi (methyl-3H)thymidine per well during the last 18 h in
culture. Thereafter, the cells were harvested on glass filter
paper and the counts per minute (cpm) determined in a liquid
scintillation counter.
In Vivo Evaluation. Animals and Surgery. Inbred C57BL/6
H-2b and Balb/c H-2d female mice, 8-10 weeks old, 20-25 g,
were used as donor and recipient, respectively. Heterotopic heart
transplantation was performed by implanting the donor heart
on the neck of recipients using conventional microsurgery tech-
niques as described previously.31 Grafts were implanted in the
recipient neck, and graft beating was checked daily by inspection
and palpation. Cessation of beating indicated graft rejection,
which was confirmed by histological examination. Housing
and all experimental animal procedures were approved by the
Institutional Animal Care and Research Advisory Committee
of the KU Leuven, Belgium.
Animals were randomly divided into three groups: (i) vehicle
group, with vehicle (30% 2-hydroxypropyl-β-cyclodetrin) only
by daily gavage, n = 6; (ii) reference drug group, with CsA of
40 mg/kg by daily gavage, n = 4; (iii) compound 15g group, with
40 mg/kg by daily gavage, n = 4.
NCH2), 4.14 (br s, 2H, NCH2), 3.60 (s, 4H, CONCH2) ppm. 13
C
NMR (75 MHz, DMSO, 25 °C): δ 172.6, 165.8, 162.5 (d, J =
246.0 Hz), 159.5, 156.9, 155.6, 153.2, 129.1, 127.6 (d, J = 8.6 Hz),
126.7 (d, J = 3.0 Hz), 116.4, 115.8 (d, J = 21.8 Hz), 114.4, 65.9,
44.2, 41.2 ppm. HRMS: calcd for C23H22ClFN7O2 [M þ H]þ
482.150 75, found 482.149 55.
2-(4-Fluorophenyl)acetaldehyde (27). To a stirred suspension
of pyridinium chlorochromate (6.9 g, 21.4 mmol) in CH2Cl2
(100 mL) was added a solution of 2-(4-fluorophenyl)ethanol
(3.0 g, 21.4 mmol) in CH2Cl2 (10 mL). The resulting suspension
was stirred for 2 h at room temperature and was then diluted
with ether. The resulting suspension was filtered through a pad
of Celite and washed with ether. The solvents were removed
under reduced pressure to yield the crude title compound as a
green oil (2.6 g, 86%), which was used as such for further reaction.
1H NMR (300 MHz, CDCl3, 25 °C): δ 9.75 (s, 1H, CH),
7.19-7.22 (m, 2H, ArH), 7.06 (t, J = 8.5 Hz, ArH), 3.68 (s, 2H,
CH2) ppm.
Ethyl 2-Amino-5-(4-fluorophenyl)thiophene-3-carboxylate (28).
Triethylamine (0.98 mL, 7.01 mmol) was added to a stirred sus-
pension of ethyl cyanoacetate (2.79 mL, 13.7 mmol) and sulfur
(0.44 g, 13.7 mmol) in DMF (70 mL). A solution of 2-(4-fluor-
ophenyl)acetaldehyde 27 (1.9 g, 13.7 mmol) in DMF (5 mL) was
added dropwise over a period of 50 min, while the temperature was
maintained at 50 °C. The solution was cooled to room temperature
and stirred overnight. The mixture was poured into water, and the
aqueous phase was extracted with diethyl ether. The organic layer
was separated and washed with water, brine and dried over Na2SO4.
The solvents were evaporated and the crude residue was purified by
flash chromatography on silica gel (EtOAc/hexane 1:15) to yield the
title compound as a white solid (1.3 g, 38%). 1H NMR (300 MHz,
CDCl3, 25 °C): δ 7.33-7.39 (m, 2H, ArH), 7.14 (s, 1H, CH), 6.99
(t, J = 8.6 Hz, ArH), 6.06 (s, 2H, NH2), 4.29 (q, J = 7.1, 2H, CH2),
1.36 (t, J = 7.1, 3H, CH3) ppm. 13C NMR (75 MHz, CDCl3,
25 °C): δ165.5, 162.3, 161.8 (d, J= 244.6 Hz), 130.4 (d, J=3.3Hz),
126.4 (d, J = 7.4 Hz), 123.8, 121.3, 115.8 (d, J = 21.7 Hz), 107.9,
60.0, 14.6 ppm. HRMS: calcd for C13H13FNO2S [M þ H]þ
266.065 10, found 266.064 25.
2-Amino-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4(3H)-one (29).
A mixture of ethyl 2-amino-5-(4-fluorophenyl)thiophene-
3-carboxylate 28 (0.3 g, 1.13 mmol), chloroformamidine hydro-
chloride (0.33 g, 2.83 mmol), and dimethylsulfone (0.53 g,
5.65 mmol) was heated at 120-130 °C for 30 min. After the
mixture was cooled to room temperature, water (10 mL) was
added and ammonium hydroxide was used to neutralize the sus-
pension. The solid was filtered off, washed with water, and
dried. The crude residue was purified by flash chromatography
on silica gel (CH2Cl2/MeOH 10:1) to yield the title compound as
Treatment started from the day of transplantation until day
30 after transplantation. Long-term surviving grafts after with-
drawing treatment remained under monitoring until day 60 after
transplantation.
Supporting Information Available: Experimental and spectro-
scopic data of all compounds as well as HPLC purity determi-
nation of all tested compounds. This material is available free of
charge via the Internet at http://pubs.acs.org.
1
a white solid (0.28 g, 95%). Mp 376 °C. H NMR (300 MHz,
DMSO, 25 °C): δ 11.05 (s, 1H, NH), 7.66-7.71 (m, 2H, ArH),

668 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 2
Jang et al.
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