Synthesis of 2-imino and 2-hydrazono thiazolo[4,5-d]pyrimidines as corticotropin releasing factor (CRF) antagonists

Article abstract of DOI:10.1016/j.ejmech.2017.07.016

Corticotropin-releasing factor (CRF) is an important neuropeptide hormone which controls the body's overall response to stress. It plays a crucial role in regulating the behavioral, cardiovascular, immune and gastrointestinal systems. Over-activation of the CRF system has been implicated in many disorders including anxiety, depression, drug addiction, hypertension, Irritable Bowel Syndrome (IBS), peptic ulcers, inflammation and others. Thus, binding of CRF to its receptors is an attractive target to develop new medications which aim at treating ailments associated with chronic stress. Numerous small-molecule non-peptide CRF receptor antagonists were developed and many are in various stages in clinical trials. Many showed great promise in treatment of anxiety, depression, peptic ulcers, inflammation, IBS and drug addiction. In our recent previous work, the development of two series of pyrimidine and fused pyrimidine CRF antagonists were described. In continuation of our efforts in this direction, in the current manuscript, the synthesis of a third series of CRF receptor antagonists is described. The binding affinities of select compounds for the type 1 receptor of CRF (CRF₁R) were determined and compared to a standard CRF antagonist drug antalarmin. A lead compound was identified and further evaluated by measuring its effect on the inhibition of the agonist-stimulated accumulation of second messengers.

Full text of DOI:10.1016/j.ejmech.2017.07.016

Accepted Manuscript
Synthesis of 2-imino and 2-hydrazono thiazolo[4,5-d]pyrimidines as corticotropin
releasing factor (CRF) antagonists
Mohamed Teleb, Bhimanna Kuppast, Katerina Spyridaki, George Liapakis, Hesham
Fahmy
PII:
S0223-5234(17)30538-X
10.1016/j.ejmech.2017.07.016
EJMECH 9579
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 3 March 2017
Revised Date: 27 June 2017
Accepted Date: 11 July 2017
Please cite this article as: M. Teleb, B. Kuppast, K. Spyridaki, G. Liapakis, H. Fahmy, Synthesis of 2-
imino and 2-hydrazono thiazolo[4,5-d]pyrimidines as corticotropin releasing factor (CRF) antagonists,
European Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.07.016.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Synthesis of 2-imino and 2-hydrazono thiazolo[4,5-d]pyrimidines as corticotropin releasing
factor (CRF) antagonists
Mohamed Teleba, Bhimanna Kuppastb, Katerina Spyridakic, George Liapakisc, Hesham Fahmya, *
aDepartment of Pharmaceutical Sciences, College of pharmacy, South Dakota State University,
Brookings SD-57007 USA.
bDepartment of Pharmaceutical Sciences, Chicago College of pharmacy, Midwestern University,
Downers Grove IL-60151 USA.
cDepartment of Pharmacology, Faculty of Medicine, University of Crete, Voutes, Heraklion
71003, Crete, Greece
Keywords: Thiazolo[4,5-d]pyrimidines, Corticotropin releasing factor receptor antagonists,
CRF.
Abstract:
Corticotropin-releasing factor (CRF) is an important neuropeptide hormone which
controls the body’s overall response to stress. It plays a crucial role in regulating the behavioral,
cardiovascular, immune and gastrointestinal systems. Over-activation of the CRF system has
been implicated in many disorders including anxiety, depression, drug addiction, hypertension,
Irritable Bowel Syndrome (IBS), peptic ulcers, inflammation and others. Thus, binding of CRF
to its receptors is an attractive target to develop new medications which aim at treating ailments
associated with chronic stress. Numerous small-molecule non-peptide CRF receptor antagonists
were developed and many are in various stages in clinical trials. Many showed great promise in
treatment of anxiety, depression, peptic ulcers, inflammation, IBS and drug addiction. In our
recent previous work, the development of two series of pyrimidine and fused pyrimidine CRF
antagonists were described. In continuation of our efforts in this direction, in the current
manuscript, the synthesis of a third series of CRF receptor antagonists is described. The binding
affinities of select compounds for the type 1 receptor of CRF (CRF1R) were determined and
compared to a standard CRF antagonist drug antalarmin. A lead compound was identified and
further evaluated by measuring its effect on the inhibition of the agonist-stimulated accumulation
of second messengers.
_____________
Corresponding author: Tel.: +1 (605) 688-4243 (office); E-mail: Hesham.Fahmy@sdstate.edu
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1.
Introduction
Corticotropin-releasing factor (CRF) is an important neuropeptide hormone secreted from
the hypothalamus. It is a crucial component of the hypothalamus-pituitary-adrenal (HPA) axis.
CRF ultimately controls the release of cortisol by the adrenal cortex in response to stress, and
thus it controls the stress-coping mechanisms. It works closely with many other systems and
affects several biological processes associated with stress [1-4]. Many ailments associated with
or related to anxiety or stress are assumed to be associated with excessive activation of the CRF
system. Dysfunction of this system is also implicated in many diseases such as major depression,
anxiety, eating disorders, drug addiction, Irritable Bowel Syndrome, inflammation, peptic ulcers
and others [1-5]. The identification and characterization of a family of CRF peptides agonists
and antagonists, coupled with the cloning and molecular characterization of the two main CRF
receptor subtypes, CRF1 and CRF2, in addition to the discovery of antagonists selective to a
particular CRF receptor sub-types, altogether provided new visions to further clarify the
mechanism of how stress may affect different systems. It also signified the role of the CRF
system and its implication in several stress-related ailments [5, 6].
The CRF role and its implication in the many disorders and behavioral, cardiovascular,
gastrointestinal and immune systems suggests that new drugs that can interfere with the CRF
function or CRF binding to its intended receptors may represent a different class of
neuropsychiatric drugs for stress-related disorders such as anxiety and depressive and addictive
disorders as well as for other peripheral disorders as Irritable Bowel Syndrome, inflammation,
peptic ulcer and others [7-25]. Significant progress has been achieved in describing the structure-
activity relationships of compounds acting as a selective agonists and antagonists for CRF1 and
CRF2 receptors such as the peptides astressin and anti-sauvagine1-30 [26]. However, because of
the peptide nature of these compounds, their physical properties posed a boundary for their use in
clinical settings. This resulted in pursuing non-peptide antagonists as a better alternative [27].
Currently, only CRF1 selective non-peptide antagonists are available. Thus, the design of small
molecule, non-peptide antagonists for the CRF receptor may afford new treatment options for
many stress-related disorders. Hundreds of accounts of small molecule antagonists have
appeared in the literature and the preclinical and clinical pharmacology have been reported for
many of these agents. The main research into CRF antagonists to date has focused on non-
peptide CRF1 receptor antagonists to target health problems arising as a result of chronic stress
and thus as a potential treatment options for anxiety-related and stress-related disorders [28,29].
Several classes of small molecule non-peptide CRF receptor antagonists have been synthesized
and many are commonly used in research.
Based on all these facts, the synthesis of more non-peptide CRF1 receptor antagonists
with a diverse structure scaffolds as well as further clinical investigations may shed more light
on the how CRF is implicated in stress-related disorders and may present new potential treatment
options for stress-related disorders.
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Several major pharmaceutical companies developed small non-peptide CRF1 receptor
antagonists including Ely-Lilly [30], Neurogen [31, 32], DuPont pharmaceutical which has been
acquired by Bristol-Myers-Squibb in 2001 [33], Pfizer [34, 35], Bristol-Myers-Squibb [36-42],
Glaxo Smith Kline [43-45] and Neurocrine biosciences [46-51]. The relationship between
structure of non-peptide CRF1 antagonists and their affinity to CRF1 receptors indicates that
CRF1 antagonists are typically built of three moieties: a hydrophobic moiety up, a proton
accepting moiety in the middle, and an aromatic moiety down. Thus the basic pharmacophore is
proposed to be made of a monocyclic or bicyclic heterocyclic ring carrying a hydrophobic
dialkylamino group on one flank and an aryl ring, usually substituted at the 2-, 4- and 6-
positions, on the opposite flank (Figure 1). Having the aromatic ring orthogonal to the
heterocyclic core appears to be essential for the affinity. A methyl group on the heterocyclic core
also appears to enhance the CRF1 antagonist properties [29, 52, 53]. X-ray structure of several
non-peptide CRF1 antagonists confirms that extension of the aromatic or hetero-aromatic
hydrophobic unit (down area) and the alkyl groups on the amino functionality (up area) are out
of the plane of the core heterocyclic unit (central area) are required for optimum CRF receptor
binding affinity [54]. The most prominent CRF1 antagonists identified from these studies were
antalarmin [55-61], pexacerfont [62, 63] and verucerfont [64-66].
Heterocyclic Core: mono-
or bi-heterocyclic ring:
Hydrophobic
dialkylamino group
X = C or N
Y = C, N or S
Z = C or N
R1
X
R2
N
N
Y
Z
Methyl
group
R4
R3
R5
2, 4, 6- substituted
aromatic ring
Figure 1. The proposed pharmacophoric framework of the non-peptide CRF1 antagonists.
In our recent published work [67, 68], several series of substituted pyrimidines and fused
pyrimidines were synthesized. These compounds retained the main structural features of the CRF
pharmacophore, and showed structural similarities to CRF antagonists which were in clinical
trials. These series were evaluated for their binding to CRF1R in two stages. First, the ability of
the test compounds at a single concentration (500-1000 nM) to inhibit the specific binding of
[125I]-Tyr0 sauvagine, which is a strong agonist, was evaluated. Compounds showing promising
binding to CRF1R were subjected to a second pharmacological characterization by determination
of their binding affinities (IC50) for this receptor in competition experiments in comparison to the
3

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standard drug Antalarmin. Several lead compounds were identified.To continue our work in this
direction, in this manuscript the synthesis and characterization of a new serious of 2-substituted
thiazolo[4,5-d]pyrimidines is described. The binding affinities (IC50 values) for the CRF1R of
representative compounds was evaluated with promising results and compared with that of
antalarmin. These compounds were found to bind to CRF1R with better affinities than our
previous compounds [67, 68]. A lead compound was identified and its effect on the inhibition of
the agonist-stimulated accumulation of second messenger of CRF1 receptors was measured.
2.
Results and discussion
2.1. Chemistry
The general synthetic scheme of the target compounds is described in Figure 2. The
starting 4-amino-3-aryl-5-carboxamido-thiazole-2(3H)-thiones (1a & b) were synthesized
following Gewald reaction conditions by reacting the selected substituted phenyl isothiocyanate
with cyanoacetamide and sulfur base under basic catalysis [69-71]. Based on the previous SAR
studies for optimum CRF1 binding antagonist activity, the 2,4,6-trimethylphenylisothiocyanate
and 2,4,6-trichlorophenylisothiocyanate were selected so that they introduce a tri-substituted
phenyl at position 3 of the resulting thiazolo[4,5-d]pyrimidines [72, 73]. The starting thiazole-
2(3H)-thiones (1a & b) were then cyclized with acetic anhydride at reflux temperature to yield
3-aryl-5-methylthiazolo[4,5-d]pyrimidin-7(6H)-one-2(3H)-thiones (2a & b) using the reaction
conditions described by Badawey et al. [74, 75] and Fahmy et al. [76]. Compounds 2a & b
were then subjected to chlorination using phosphorous oxychloride at reflux temperature under
the reported reactions conditions [74-76] to yield 3-aryl-7-chloro-5-methylthiazolo[4,5-
d]pyrimidine-2(3H)-thiones (3a,b) in excellent yields.
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O
N
O
O
S
N
HN
S
S
+
+
S
H2N
Ac2O
H2N
S
Et3N
S
R
HN
CN
N
R
H2N
R
R
R
R
R
R
2a,b
R
1a,b
POCl3
R1
R2
N
Cl
S
N
N
S
S
N
R1R2NH
S
N
R
N
N
R
R
R
1- Me2SO4
2- MeHN2
R
4a-d
R
3a,b
1- Me2SO4
2- NH3
1- Me2SO4
2- NH2NH2
Method B
Method A
R1
N
R2
R1
N
R2
N
N
R1
N
R2
S
S
N
N
N
NH
R
NNH2
R
S
N
N
N
N
R
N
R
R
R
R
R
R
7a-d
6a-d
5a-c
R= Cl or Methyl, NR1R2= Diethylamino or (Ethyl,n-Butyl)amino
Figure 2. Synthetic scheme of compounds
Reaction of the 7-chloro derivatives (3a, b) with 2 equivalent of the selected secondary
amines in refluxing ethanol, under the established reaction conditions [74-76], afforded the 3-
aryl-7-dialkylamino-5-methylthiazolo[4,5-d]pyrimidine-2(3H)-thiones (4a & b). Diethyl amine
and [ethyl,n-butyl]amine were selected, since previous reports showed optimum CRF1 receptor
antagonist activities of derivatives with those particular amino groups at position 7 of the
thiazolo[4,5-d]pyrimidine ring [72, 73].
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The target 7-dialkyl-5-methyl-3-(substituted phenyl)-thiazolo[4,5-d]pyrimidin-2(3H)-
hydrazones (5a-c) were synthesized by the reaction of the corresponding 3-aryl-7-dialkylamino-
5-methylthiazolo[4,5-d]pyrimidine-2(3H)-thiones (4a-c) with dimethyl sulfate in acetonitrile at
reflux temperature, followed by reaction of the produced 2-methylthiazolium intermediate with
excess hydrazine following the reported reaction conditions [76]. Nucleophilic substitution of the
2-thioxo function of 3-aryl-7-dialkylamino-5-methylthiazolo[4,5-d]pyrimidine-2(3H)-thiones
with oxygen or amino functions was first described by Gewald [69, 70] and later with active
methylene-containing molecules [69]. Moreover, in the current study, this reaction was extended
to utilize ammonia as a nucleophile. Thus, the 2-imino target compounds (6a-d) were prepared
using the same reaction conditions but using ammonia (7 Normal solution in methanol). The
corresponding 2-methylimino derivatives (7a-d) were also produced, possibly due to in situ
alkylation of the 2-imino products (6a-d) by the excess dimethyl sulfate in the reaction mixture.
To further confirm this finding, the same 2-methylimino derivatives (7a-d) were also prepared
from compounds 4 a-c using methylamine instead of ammonia.
2.2. Biological evaluation
Three representative compounds (Figure 3) were evaluated for theirabilities to displace
radiolabelled sauvagine binding at binding affinities concentration of 300 nM. The binding
affinities of these compounds is illustrated in Figure 4.
N
N
N
N
N
N
S
N
S
N
S
N
N
N
N
NNH2
NH
NH
6c
6d
5c
Figure 3. Structures of the representative test compounds
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Figure 4. % Inhibition of [125I]-Tyr0-sauvagine specific binding by 300 nM of
analogs 1-3 on membranes from HEK 293 cells stably expressing the human CRF1
receptor. In the absence of compounds the inhibition is 0%.
In a second assay, a specific, aquantitative assay to measure their competitive binding
compared with a standard drug antalarmin at different concentrations was carried out. In this
experiment we determined the Log IC50 values of these compounds, which under the
experimental conditions of this study are similar to their binding affinities (Log values). It
Ki
showed that the selected compounds displayed Log IC50 ± S.E values of -6.35 ± 0.158
(compound 5c), -6.05 ± 0.139 (compound 6c) and -6.77 ± 0.224 (compound 6d) compared to -
7.73 ± 0.074 value for Antalarmin (Figure 5).
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Figure 5. Competition binding isotherms of analogs 1-3 to human CRF1 receptor:
Competition of [125I]-Tyr0-sauvagine specific binding by increasing concentrations of test
compounds performed on membranes from HEK 293 cells stably expressing the human CRF1
receptor.
A third biological evaluation to screen the compound 6d with the highest binding affinity
(lowest IC50) was carried out by measuring its ability to antagonize agonist-stimulated
accumulation of cAMP, which is the second messenger that mediates the effects resulting from
stimulating CRF1 receptors,. Results of this evaluation were very promising (Figure 6). The Lead
compound (6d), at concentration of 1µΜ, was able to significantly and substantially reduce the
potency of sauvagine to cause accumulation of cAMP. In the presence of compound 6d, EC50%
of sauvagine was 1.114 nM (Log EC50% = -8.953), whereas, in its absence, EC50% was only 0.203
nM (Log EC50% = -9.692). This clearly indicates the potential of this class of compounds for
development as possible new class of medications for stress-related illnesses and that further
structural modifications may lead to compounds with superior CRF1 antagonist properties.
Figure 6. Inhibition of sauvagine-induced cAMP accumulation by compound 6d
3.
Conclusion
A novel series of thiazolo[4,5-d]pyrimidines-2-imine and 2-hydrazones were synthesized
as CRF1 receptor antagonists. These compounds were designed to have the structural features
known for optimum binding affinity for CRF1 receptors, particularly a tri-substituted phenyl at
position-3, a methyl group at position-5 and a dialkylamino group at position-7. Select
compounds were evaluated for their binding affinities for CRF1 receptors in comparison to
antalarmin, a standard CRF1 antagonist drug. A lead compound was identified and was evaluated
for its effects to antagonize sauvagine in stimulating the accumulation of cAMP. It showed an
almost 5-fold inhibition which confirmed the potential of the new series for further development
as CRF1 receptor antagonists.
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4.
Experimental
4.1. Chemistry
4.1.1 General information
All chemicals were purchased from commercial sources. Flash column chromatography
separation was performed using Acros organics silica gel 40-60 µm, 60 Å using combination of
ethyl acetate and hexanes. Preparative thin layer chromatography was performed using
UNIPLATE TM1500µm silica gel plates with UV 254 preparative layer. Whatman and sigma
TLC plates were utilized for thin layer chromatography and visualization was done using UV
fluorescence at 254 nm. Melting points were recorded on a Mel-Temp, Laboratory devices, Inc
and are uncorrected. %CHN Analyzer by combustion/ TCD and %S by O flask combustion/IC
were used for elemental analysis of final compounds and performed by Micro Analysis Inc.,
1
Wilmington DE, USA and are within 0.4%. H and 13C NMR spectra were obtained on a
BrukerAvance 400 MHz & 600MHz instrument using DMSO-d6 as solvent unless otherwise
1
stated. H NMR Spectra are reported in order; multiplicity, number of protons and signals were
characterized as s (singlet), d (doublet), dd (doublet of doublet), t (triplet), m (multiplet), br s
(broad signal), q (quartet), quin (quintet), tquin (triplet of quintet), sxt (sextet), spt (septet).
Chemical shifts are relative to TMS as an internal standard. Mass spectra were recorded on
ThermoFinnigan MAT95XL high resolution magnetic sector mass spectrometer, using
electrospray ionization method. The IR spectra were recorded on ZnSe crystal at 8 cm-1
resolution in Nicolet 380 ATR-FTIR spectrophotometer (Thermo electron Corporation, Madison,
WI).
4.1.2 Synthesis of compounds
Compounds 1-4 were synthesized as previously reported [68, 73].
7-Dialkylamino-5-methyl-3-(substitute-phenyl)thiazolo[4,5-d]pyrimidine-2(3H)-hydrazones(5a-
c)
Dimethyl sulfate (3.0 mmol) was added to a solution of 4 (1.0 mmol) in acetonitrile (20
mL). The reaction mixture was stirred at reflux temperature for 10-12h. Additional dimethyl
sulfate (3.0 mmol) was added then the mixture was stirred at reflux temperature and monitored
by TLC till completion of the reaction (10-12h). The reaction was allowed to cool down to room
temperature. Then the reaction mixture was slowly added drop-wise toa solution of hydrazine
o
hydrate 100% (10 mmol) in methanol (10 mL) while stirring at 0-5 C to.The reaction mixture
was allowed to reach room temperature during stirring and monitored with TLC till completion
(6-10h). After completion, the reaction mixture was poured onto cold water (200 mL) and
extracted with ethyl acetate (100 mL x 3). The combined organic layers were washed with water
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and brine then dried over anhydrous sodium sulfate and evaporated under reduced pressure. The
obtained crude residue was then purified by preparative TLC using ethyl acetate / hexane (1:12).
7-Diethylamino-5-methyl-3-(2,4,6-trichlorophenyl)-2,3-thiazolo[4,5-d]pyrimidine-2(3H)-
hydrazone (5a):
Yield: semisolid (70%);1H NMR (400 MHz, CDCl3) δ 7.49 (s, 2H), 4.41 (br. s., 2H), 3.63
(q, J = 8 Hz, 4H), 2.31 (s, 3H), 1.28 (t,J = 6 Hz, 6H); 13C NMR (400 MHz, CDCl3) δ 164.67,
157.91, 155.27, 153.68, 136.25, 135.81, 130.91, 129.02, 89.26, 42.91, 25.76, 14.44; HRMS
(ES+) calculated for C16H18N6Cl3S [M+H]+ : 431.0374; found: 431.03925.
7-(Butyl,ethyl)amino-5-methyl-3-(2,4,6-trichlorophenyl)thiazolo[4,5-d]pyrimidine-2(3H)-
hydrazone (5b).
Yield: semisolid (80%);1H NMR (400 MHz, CDCl3) δ 7.48 (s, 2H), 4.42 (br. s., 2H), 3.63
(q, J = 8 Hz, 2H), 3.55 (t,J = 8 Hz, 2H), 2.30 (s, 3H), 1.69-1.64 (m, 2H), 1.43-1.38 (m, 2H), 1.26
13
(t,J = 8 Hz, 3H), 0.99 (t,J = 6 Hz, 3H); C NMR (400 MHz, CDCl3) δ 164.57, 157.91, 155.38,
153.39, 136.27, 135.78, 130.94, 129.00, 89.26, 48.15, 43.46, 31.38, 25.73, 20.11, 14.34, 14.03;
HRMS (ES+) calculated for C18H22N6Cl3S [M+H]+: 459.0687; found: 459.06924.
7-Diethylamio-3-mesityl-5-methylthiazolo[4,5-d]pyrimidin-2(3H)-hydrazone (5c).
Yield: semisolid (66%);1H NMR (400 MHz, CDCl3) δ 6.98 (d,J = 8 Hz, 2H), 4.38 (br. s.,
2H), 3.65 (q, J = 8 Hz, 4H), 2.33, 2.32 (s, 3H), 2.31, 2.30 (2s, 3H), 2.06 (s, 6H), 1.28 (t,J = 8 Hz,
6H); 13C NMR (400 MHz, CDCl3) δ 164.71, 159.01, 155.22, 138.79, 136.28, 136.18, 131.46,
129.40, 129.07, 89.19, 42.81, 25.90, 21.30, 17.99, 17.91, 14.49, 14.44; HRMS (ES+) calculated
for C19H27N6S [M+H]+ : 371.2012; found: 371.20151.
7-Dialkylamino-5-methyl-3-(substitutedphenyl)thiazolo[4,5-d]pyrimidin-2(3H)-imine (6a-d) and
7-dialkylamino-5-methyl)-3-(substituted-phenyl)thiazolo[4,5-d]pyrimidin-2(3H)-methylimine
(7a-d) [Method A]
Dimethyl sulfate (3.0 mmol) was added to a solution of 4 (1.0 mmol) in acetonitrile (20
mL). The reaction mixture was stirred at reflux temperature for 10-12 h. Additional dimethyl
sulfate (3.0 mmol) was then added and the mixture was stirred at reflux temperature and
monitored by TLC till completion of the reaction (10-12 h). The reaction mixture was allowed to
cool down to room temperature. A solution of 7N ammonia in methanol (10 mmol) was then
slowly added to the reaction mixture while stirring at 0-5 o C. The reaction mixture was allowed
to reach room temperature during stirring and monitored with TLC till completion (6-8 h). After
completion, the reaction mixture was evaporated under reduced pressure, diluted with water and
extracted with dichloromethane. The combined organic layer was washed with water, brine
solution, dried over anhydrous sodium sulfate and concentrated under vacuum. The crude residue
10

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was then purified by preparative TLC using ethyl acetate / hexane (1:12) to afford the imino and
methylimino derivatives.
7-Dialkylamino-5-methyl)-3-(substituted-phenyl)thiazolo[4,5-d]pyrimidin-2(3H)-methylimine
(7a-d) [Method B]
Dimethyl sulfate (3.0 mmol) was added to a solution of 4 (1.0 mmol) in acetonitrile (20
mL). The reaction mixture was stirred at reflux temperature for 10-12 h. Additional dimethyl
sulfate (3.0 mmol) was added then the mixture was stirred at reflux temperature and monitored
by TLC till completion of the reaction (10-12 h). The reaction was allowed to cool down to room
temperature. A solution of 2M methylamine in THF (12 eq.) was then slowly added to the
o
reaction mixture while stirring at 0-5 C. The reaction mixture was allowed to reach room
temperature during stirring and monitored with TLC till completion (4-8 h). After completion,
the reaction mixture was evaporated under reduced pressure, diluted with water and extracted
with dichloromethane. The combined organic layer was washed with water, brine solution, dried
over anhydrous sodium sulfate and concentrated under vacuum. The crude residue was then
purified by preparative TLC using ethyl acetate / hexane (1:12). Yield: semisolid (40 %).
7-Diethylamino-5-methyl-3-(2,4,6-trichlorophenyl)thiazolo[4,5-d]pyrimidin-2(3H)-imine (6a).
Yield: semisolid (40%);1H NMR (400 MHz, CDCl3) δ 7.50 (s, 2H), 3.57 (q, J = 8 Hz,
4H), 2.31 (s, 3H), 1.25 (t,J = 8 Hz, 6H); 13C NMR (400 MHz, CDCl3) δ 164.72, 157.15, 154.89,
136.38, 136.10, 130.24, 129.05, 89.53, 42.86, 25.70, 14.37; HRMS (ES+) calculated for
C16H17N5Cl3S [M+H]+ :416.0265; found: 416.02695.
7-(Butyl,ethyl)amino-5-methyl-3-(2,4,6-trichlorophenyl)thiazolo[4,5-d]pyrimidin-2(3H)-imine
(6b)
.Yield: semisolid (36%);1H NMR (400 MHz, CDCl3) δ 7.51 (s, 2H), 3.58 (q, J = 8 Hz,
2H), 3.42 (t,J = 8 Hz, 2H), 2.30 (s, 3H), 1.67-1.63 (m, 2H), 1.37-1.42 (m, 2H), 1.24 (t,J = 8 Hz,
3H), 0.99 (t,J = 8 Hz, 3H); HRMS (ES+) calculated for C18H21N5Cl3S [M+H]+ : 444.0578; found:
444.05704.
7-Diethylamino-3-mesityl-5-methylthiazolo[4,5-d]pyrimidin-2(3H)-imine (6c).
Yield: semisolid (41%);1H NMR (400 MHz, CDCl3) δ 6.93 (s, 2H), 3.51 (q, J = 8 Hz,
4H), 2.25 (s, 3H), 2.22 (s, 3H), 1.99 (s, 6H), 1.18 (t,J = 8 Hz, 6H); 13C NMR (400 MHz, CDCl3)
δ 164.66, 158.07, 155.08, 139.24, 136.90, 129.89, 129.62, 126.86, 89.34, 42.78, 25.86, 21.27,
17.80, 14.41; HRMS (ES+) calculated for C19H26N5S [M+H]+ : 356.1903; found: 356.19012.
7-(Butyl, ethyl)amino-3-mesityl-5-methylthiazolo[4,5-d]pyrimidin-2(3H)-imine (6d).
Yield: semisolid (42%);1H NMR (400 MHz, CDCl3) δ 7.01 (s, 2H), 3.60 (q, J = 8 Hz,
2H), 3.49 (t,J = 8 Hz, 2H), 2.33 (s, 3H), 2.29 (s, 3H), 2.07 (s, 6H), 1.67-1.64 (m, 2H), 1.43-1.37
11

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(m, 2H), 1.24 (t,J = 8 Hz, 3H), 0.99 (t,J = 8 Hz, 3H); 13C NMR (400 MHz, CDCl3) δ 164.56,
158.09, 155.19, 139.22, 136.91, 129.61, 89.29, 48.09, 43.32, 31.56, 25.85, 21.27, 20.06, 17.81,
14.17, 14.00; HRMS (ES+) calculated for C21H30N5S [M+H]+ : 384.2216; found: 384.22189.
7-Diethylamino-5-methyl-3-(2,4,6-trichlorophenyl)thiazolo[4,5-d]pyrimidin-2(3H)-methylimine
(7a).
Yield: (30 %); MP: 172; 1H NMR (400 MHz, CDCl3) δ 7.47 (s, 2H), 3.62 (q, J = 8 Hz,
4H), 3.06 (s, 3H), 2.31 (s, 3H), 1.26 (t,J = 6 Hz, 6H);13C NMR (400 MHz, CDCl3) δ 164.54,
157.53, 155.02, 151.93, 136.35, 135.59, 131.42, 128.95, 89.03, 42.91, 40.38, 25.80, 14.46;
HRMS (ES+) calculated for C17H19N5Cl3S [M+H]+ : 430.0421; found: 430.04169
7-(Butyl,ethyl)amino-5-methyl-3-(2,4,6-trichlorophenyl)thiazolo[4,5-d]pyrimidin-2(3H)-
methylimine (7b)
Yield: (31%); MP: 136;1H NMR (400 MHz, CDCl3) δ 7.47 (s, 2H), 3.63 (q, J = 8 Hz,
2H), 3.55 (t,J = 8 Hz, 2H), 3.06 (s, 3H), 2.30 (s, 3H), 1.70-1.65 (m, 2H), 1.43-1.38 (m, 2H), 1.26
13
(t,J = 8 Hz, 3H), 0.99 (t,J = 8 Hz, 3H); C NMR (400 MHz, CDCl3) δ 164.45, 157.53, 155.12,
151.85, 136.32, 135.55, 131.44, 128.95, 88.99, 48.13, 43.43, 40.41, 31.39, 25.78, 20.09, 14.31,
14.01; HRMS (ES+) calculated for C19H23N5Cl3S [M+H]+ : 458.0734; found: 458.07184.
7-Diethylamino-3-mesityl-5-methylthiazolo[4,5-d]pyrimidin-2(3H)-methylimine (7c).
Yield: (31%); MP: 132;1H NMR (400 MHz, CDCl3) δ 6.89 (s, 2H), 3.57 (q, J = 8 Hz,
4H), 2.96 (s, 3H), 2.22 (s, 6H), 1.96 (s, 6H), 1.20 (t,J = 8 Hz, 6H); 13C NMR (400 MHz, CDCl3)
δ 164.58, 158.68, 154.97, 138.50, 136.32, 131.91, 129.32, 128.47, 126.92, 88.93, 42.80, 40.56,
25.95, 21.32, 17.96, 14.49; HRMS (ES+) calculated for C20H28N5S [M+H]+ : 370.2060; found:
370.20651
7-(Butyl,ethyl)amino-3-mesityl-5-methylthiazolo[4,5-d]pyrimidin-2(3H)-methylimine (7d).
Yield: semisolid (30%);1H NMR (400 MHz, CDCl3) δ 6.97 (s, 2H), 3.65 (q, J = 8 Hz,
2H), 3.56 (t,J = 8 Hz, 2H), 3.04 (s, 3H), 2.30 (s, 3H), 2.29 (s, 3H), 2.04 (s, 6H), 1.71-1.65 (m,
2H), 1.44-1.38 (m, 2H), 1.26 (t,J = 8 Hz, 3H), 1.00 (t,J = 8 Hz, 3H); 13C NMR (400 MHz,
CDCl3) δ 164.46, 158.69, 155.06, 153.22, 138.45, 136.34, 129.30, 88.90, 48.05, 40.63, 31.44,
25.92, 21.31, 20.12, 17.96, 14.34, 14.02; HRMS (ES+) calculated for C22H32N5S [M+H]+ :
398.2373; found: 398.23746.
4.2. Biological Evaluation
4.2.1. CRF1 Receptor binding study
Binding studies was performed according to our reported procedure [67,68] in membrane
homogenates from human embryonic kidney cells (HEK 293) stably expressing CRF1 receptors
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and using [125I]-Tyr0-sauvagine as radioligand. Membrane homogenates were prepared according
to the method of Gkountelias [77]. CRF1-expressing HEK 293 cells, grown in DMEM/F12 (1:1)
containing 3.15 g/L glucose, 10% bovine calf serum and 300 µg/ml of the antibiotic, Geneticin at
37o C and 5% CO2, were washed with phosphate-buffered saline (PBS) (4.3 mM Na2HPO4.7
H20, 1.4 mM KH2PO4, 137 mMNaCl, 2.7 mMKCl, pH 7.2-7.3 at R.T). Then the cells were
briefly treated with PBS containing 2 mM EDTA (PBS/EDTA), and then dissociated in
PBS/EDTA. Cells suspensions were centrifuged at 1000 x g for 5 min at room temperature, and
the pellets were homogenized in 1.5 ml of buffer H (20 mM HEPES, containing 10 mM MgCl2,
2 mM EGTA, 0.2 mg/ml bacitracin and 0.93 µg/ml aprotinin pH 7.2 at 4o C) using a Janke&
Kunkel IKA Ultra Turrax T25 homogenizer, at setting ~20, for 10-15 sec, at 4o C. The
homogenates were centrifuged at 16000 x g, for 10 min, at 4o C. The membrane pellets were re-
suspended by homogenization, as described above, in 1 ml buffer B (buffer H containing 0.1%
BSA, pH 7.2 at 20o C). The membrane suspensions were then diluted in buffer B and aliquots of
suspensions (50 µl) were added into tubes containing buffer B and 20-25 pM [125I]-
Tyr0sauvagine without or with the new test compounds at the single concentration of 300 nM or
with increasing concentrations of the test compounds (competition binding experiments) in a
final volume of 0.2 ml. The mixtures were incubated at 20-21° C for 120 min and then were
filtered through Whatman 934AH filters, presoaked for 1 h in 0.3% polyethylene imine at 4° C.
The filters were washed 3 times with 0.5 ml of ice-cold PBS, pH 7.1 containing 0.01% Triton X-
100 and assessed for radioactivity in a gamma counter. The amount of membranes used were
adjusted to insure that the specific binding is always equal to or less than 10% of the total
concentration of the added radio-ligand. Specific [125I]-Tyr0-sauvagine binding was defined as
total binding less nonspecific binding in the presence of 1000 nMantalarmin. Data for
competition binding was analyzed by nonlinear regression analysis, using Prism 4.0 (GraphPad
Software, San Diego, CA). IC50 values were obtained by fitting the data from competition studies
to a one-site competition model.
4.2.2. cAMP Accumulation Assays:
The cAMP Accumulation Assays was carried out according to the reported procedure
[78]. HEK293 cells stably expressing the CRF1R were plated in 96-well cell culture plates
(pretreated with 0.1 mg/ml poly-L-lysine). After overnight incubation (37°C, 5% CO2) of cells
(95–100% confluence) the medium was removed, and 100 µl of assay buffer (25 mM HEPES,
pH 7.4, 2 mM choline, 288 mM sucrose, 0.9 mM CaCl2, 0.5 mM MgCl2, and 1 mM 3-isobutyl-
1-methylxanthine) were added. After 1 h of incubation at 37°C, more assay buffer without (basal
levels) or with increasing concentrations of CRF in the presence or absence of 1µM non-peptide
CRF antagonist was added to a total volume of 200 µl, and the incubation was continued for 30
min at 37 °C. At the end of the incubation, the assay buffer was removed. The cells were placed
on ice and lysed with 3% trichloroacetic acid. Lysates were incubated on ice for 30–60 min and
stored at -20 °C. After 1–5 days, frozen lysates were thawed and centrifuged at 1800xg for 10
min at 4 °C, and the supernatants were neutralized with 2 N NaOH. Quantification of cAMP in
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the neutralized supernatants was performed using a competitive binding assay. In brief,
supernatants were transferred to polypropylene mini-tubes (20 µl/tube) containing buffer A (100
mMTris-HCl, pH 7.4, 100 mMNaCl, and 5 mM EDTA) with 1–1.5 nM [2,8-3H]cAMP.
Subsequently, cAMP-binding protein (~100 mg of crude bovine adrenal cortex extract in 500 ml
of buffer A) was added to each tube. After incubation on ice for 3 h, the mixtures were filtered
through Whatman 934AH glass fiber filters using buffer C (120 mMNaCl and 10mM TrisHCl,
pH 7.4, at 4°C) as washing buffer. The amount of cAMP in each sample (one-tenth of a well)
was determined by comparison with a standard curve of known concentrations of unlabeled
cAMP (1–100 pmol/tube). The logEC50 values were obtained by fitting the data to a one-site
sigmoidal model using nonlinear regression analysis
4.2.3. Statistical Analysis
Prism 4.0 was used for all statistical analysis (GraphPad Software, San Diego, CA).
Competition binding were analyzed by nonlinear regression analysis, and IC50 values were
obtained by fitting the data from competition studies to a one-site competition model by
nonlinear regression.
Acknowledgments
This research project was funded the Department of Pharmaceutical Sciences, College of
Pharmacy, South Dakota State University and Department of Pharmacology, Faculty of
Medicine, University of Crete, Greece.
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Highlights:
 2-iminothiazolo[4,5-d]pyrimdines as CRF receptor antagonists.
 2-hydrazonothiazolo[4,5-d]pyrimdines as CRF receptor antagonists.
 Inhibition of binding of radiolabelled sauvagive to CRF 1 receptors by thiazolo[4,5-
d]pyrimdines.
 Inhibition of suavagine-induced accumulation of cAMP by thiazolo[4,5-d]pyrimdines.