Synthesis, in vitro biological investigation, and molecular dynamics simulations of thiazolopyrimidine based compounds as corticotrophin releasing factor receptor-1 antagonists

Article abstract of DOI:10.1016/j.bioorg.2021.105079

Corticotrophin releasing factor receptor-1 (CRFR1) is a potential target for treatment of depression and anxiety through modifying stress response. A series of new thiazolo[4,5-d]pyrimidine derivatives were designed, prepared and biologically evaluated as potential CRFR1 antagonists. Four compounds produced more than fifty percent inhibition in the [¹²⁵I]-Tyr⁰-sauvagine specific binding assay. Assessment of binding affinities revealed that compound (3-(2,4-dimethoxyphenyl)-7-(dipropylamino)-5-methylthiazolo[4,5-d]pyrimidin-2(3H)-one) 8c was the best candidate with highest binding affinity (K_i = 32.1 nM). Further evaluation showed the ability of compound 8c to inhibit CRF induced cAMP accumulation in a dose response manner. Docking and molecular dynamics simulations were used to investigate potential binding modes of synthesized compounds as well as the stability of 8c-CRFR1 complex. These studies suggest similar allosteric binding of 8c compared to that of the co-crystalized ligand CP-376395 in 4K5Y pdb file.

Full text of DOI:10.1016/j.bioorg.2021.105079

Bioorganic Chemistry 114 (2021) 105079
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Synthesis, in vitro biological investigation, and molecular dynamics
simulations of thiazolopyrimidine based compounds as corticotrophin
releasing factor receptor-1 antagonists
,
Hossam R. Elgiushya h, Nageh A. Abou-Taleba, George G. Holzb, Oleg G. Chepurnyb,
Ioannis Pirmettisc, Sotirios Kakabakosc, Vlasios Karageorgosd, George Liapakisd,
,
g
,
,h,*
Amgad Albohye, Khaled A.M. Abouzidf *, Sherif F. Hammada
a Pharmaceutical Chemistry Department, Faculty of Pharmacy, Helwan University, Ain Helwan 11795, Cairo, Egypt
b Department of Medicine, SUNY Upstate Medical University, Syracuse, NY, USA
c Institute of Nuclear & Radiological Sciences & Technology, Energy & Safety, National Centre for Scientific Research “Demokritos”, Athens, Greece
d Department of Pharmacology, School of Medicine, University of Crete, Voutes, 71003, Heraklion, Crete, Greece
e Department of Pharmaceutical Chemistry, Faculty of Pharmacy, The British University in Egypt (BUE), El-Sherouk City, Suez Desert Road, Cairo 11837, Egypt
f Pharmaceutical Chemistry Department, Faculty of Pharmacy, Ain Shams University, Abbassia 11566, Cairo, Egypt
g Department of Organic and Medicinal Chemistry, Faculty of Pharmacy, University of Sadat City, Sadat City, Egypt
h Basic and Applied Sciences Institute, Egypt-Japan University of Science and Technology (E-JUST), New Borg El-Arab City, 21934 Alexandria, Egypt
A R T I C L E I N F O
A B S T R A C T
Keywords:
Corticotrophin releasing factor receptor-1 (CRFR1) is a potential target for treatment of depression and anxiety
through modifying stress response. A series of new thiazolo[4,5-d]pyrimidine derivatives were designed, pre-
pared and biologically evaluated as potential CRFR1 antagonists. Four compounds produced more than fifty
percent inhibition in the [125I]-Tyr0-sauvagine specific binding assay. Assessment of binding affinities revealed
that compound (3-(2,4-dimethoxyphenyl)-7-(dipropylamino)-5-methylthiazolo[4,5-d]pyrimidin-2(3H)-one) 8c
was the best candidate with highest binding affinity (Ki = 32.1 nM). Further evaluation showed the ability of
compound 8c to inhibit CRF induced cAMP accumulation in a dose response manner. Docking and molecular
dynamics simulations were used to investigate potential binding modes of synthesized compounds as well as the
stability of 8c-CRFR1 complex. These studies suggest similar allosteric binding of 8c compared to that of the co-
crystalized ligand CP-376395 in 4K5Y pdb file.
CRFR1 antagonists
Thiazolo[4,5-d]pyrimidine
Scaffold hopping
Molecular dynamics simulation
1. Introduction
to the release of adrenocortical steroids and stress response [7–9]. CRF
receptors are type B G-protein coupled receptors (GPCR) and include
Corticotrophin releasing factor CRF (also known as corticotrophin
releasing hormone, CRH) is a peptide hormone that regulates body
response to stress and is composed of 41 amino acids [1]. It was initially
described and characterized by Vale in 1981 [2]. CRF is overexpressed
in the hypothalamic paraventricular nucleus part of the brain [3].
Elevated central CRF levels are associated with several diseases
including anxiety, depression as well as other neurodegenerative dis-
orders [4–6]. CRF is the principal mediator for stress stimulants on the
hypothalamic-pituitary-adrenocortical (HPA) axis. CRF effect takes
place via the release of adrenocorticotrophic hormone (ACTH) leading
CRFR1 and CRFR2 [10–12]. CRFR1 is expressed throughout the brain,
and found mainly on the anterior pituitary mediating the effects of CRF
on ACTH release [3,8,11,13]. Extensive studies clearly revealed that
CRFR1 is pivotal in initiating and provoking stress response and anxiety
related behaviors [5,14–20]. This crucial role in mediating stress
response at very early stages made CRFR1 a highly promising target for
the development of novel class of stress related medications. Non-
peptide small molecule antagonists of CRFR1 are investigated for
treatment of stress related disorders and other correlated neurodegen-
erative disorders [1,21,22]. Examples of non-peptide small molecule
* Corresponding authors at: Pharmaceutical Chemistry Department, Faculty of Pharmacy, Ain Shams University, Abbassia 11566, Cairo, Egypt (K.A.M. Abouzid).
Pharmaceutical Chemistry Department, Faculty of Pharmacy, Helwan University, Ain Helwan 11795, Cairo, Egypt (S.F. Hammad).
E-mail addresses: khaled.abouzid@pharma.asu.edu.eg (K.A.M. Abouzid), sherif.hammad@ejust.edu.eg (S.F. Hammad).
https://doi.org/10.1016/j.bioorg.2021.105079
Received 10 April 2021; Received in revised form 3 June 2021; Accepted 7 June 2021
Available online 10 June 2021
0045-2068/© 2021 Elsevier Inc. All rights reserved.

H.R. Elgiushy et al.
Bioorganic Chemistry 114 (2021) 105079
CRFR1 antagonists proved effective anxiolytic and antidepressant in
animal models [12] are illustrated in Fig. 1. Moreover, the crystal
structure of CRFR1 in complex with the non-peptide antagonist CP-
376395 was reported (PDB ID: 4K5Y) revealing allosteric inhibitory
effect of non-peptide CRFR1 antagonists at the transmembrane domain
(TMD) [23].
the ortho-methoxy group maintains the required orthogonality and the
two methoxy groups provide lipophilicity-balancing effect beside being
two H-bond accepting sites.
In addition, our target compounds are intended to be used as drugs
and to work at the brain level so we decided to study drugability of
synthesized compounds and their ability to cross the blood brain barrier
(BBB) [28]. Several descriptors can be used to predict drug likeness and
ability of molecules to cross BBB such as lipophilicity (expressed as logP
value), topological polar surface area (tPSA), molecular weight, number
of hydrogen bond donors and acceptors [29,30]. Herein we used Lip-
inski’s rule of five for drug likeness to predict drugability of the new
chemical entities [31]. This rule states that drug-like molecules should
have a maximum of 1 violation of the following criteria: logP value does
not exceed 5, molecular weight does not exceed 500, number of
hydrogen bond donor does not exceed 5 and number of hydrogen bond
acceptors does not exceed 10. Table SI-1 summarizes drug-likeness of
target molecules using Lipinski’s rule of five compared to reference
CRFR1 antagonists. Obtained values were calculated through Swis-
sADME server [32].
1.1. Design and rationale
Analysis of different non-peptide CRFR1 antagonists reveals four
basic pharmacophoric requirements. These include mono-, bi- or tricy-
clic central scaffold with proton accepting nitrogen atom essential for
hydrogen bonding, a methyl substitution on the carbon adjacent to the
essential nitrogen atom, an orthogonal aromatic ring and a non-bulky
mono- or dialkyl amino group [24]. The reported crystal structure of
CRFR1 bound to non-peptide antagonist CP-376395 (PDB ID: 4K5Y)
supports these pharmacophoric requirements and provided a clear
identification of the binding site. Non-peptide CRFR1 antagonists were
found to be allosteric inhibitors and their binding pocket is located be-
tween 3rd, 5th and 6th transmembranal loops. This binding mode pre-
vents conformational changes of the receptor that are required for the
binding of CRF [23]. In addition, the hydrogen bond acceptor nitrogen
was found to interact with N283.
2. Results and discussion
2.1. Chemistry
In this study we aim to design and synthesize novel CRFR1 antago-
nist. Our design is based on scaffold hopping and replacing of the central
pyridine ring with thiazolo[4,5-d]pyrimidine ring system while main-
taining the main pharmacophoric features of CP-376395 Fig. 2.
Our designed CRFR1 antagonists shown in Fig. 3 maintain all the
aforementioned essential pharmacophoric features. Selection of thiazolo
[4,5-d]pyrimidine as core scaffold was tempted by previous successful
approaches for thiazolo[4,5-d]pyrimidine based CRFR1 antagonists
[25–27]. The alkyl amino groups were selected carefully with a
maximum of four carbon chain based on previous 3D QSAR model [24].
2,4-Dimethoxyphenyl was carefully selected as the pendent ring where
Reagents and conditions: (a) CS2 (1.2 equivalents)/ k2CO3 (2
equiv.) aqueous medium-stirring 10–12 h. trichlorotriazine (0.5 equiv-
alents) stirring 0˚C, 0.5 h., 6 N NaOH, stirring, 0.5 h. (b) cyanoacetamide
(1 equivalent), elemental sulfur (1 equivalent), Triethylamine (1
equivalent),DMF 50-70˚C, (c) Acetic anhydride, reflux 140-150˚C (d)
POCl3, reflux 180˚C, 3–6 h. (e) appropriate dialkyl amine (2 equivalents),
ethanol, reflux 70˚C, 3–6 h. (f) dimethyl sulfate (2 equivalents), DMF 10
ml, reflux 150˚C, 3hrs, and then add10 ml H2O, triethyl amine few drops.
(g) POCl3, reflux 180˚C, 3–6 h. Appropriate dialkyl amine (2 equiva-
lents), ethanol, reflux 70˚C, 3–6 h. (h) appropriate dialkyl amine (2
Fig. 1. Examples of non-peptide small molecule CRFR1 antagonists.
2

H.R. Elgiushy et al.
Bioorganic Chemistry 114 (2021) 105079
Fig. 2. Design of the target molecules applying scaffold hopping strategy.
Fig. 3. Target CRFR1 antagonist molecules 5a-e and 8a-e.
equivalents), ethanol, reflux 70˚C, 3–6 h.
(MS) revealed the molecular ion peak at m/z = 390.54. Similarly,
Compounds 8a-e were obtained from compound 7 via gentle reflux with
appropriate amine. The formation of target compounds 8a-e was also
confirmed by different spectral analyses. Taking compound 8d as
example: 1H NMR revealed the appearance of a triplet signal at δ 0.94
ppm integrated to six protons assigned to the protons of the terminal two
(CH3) of 7-dibutyl amino, a multiplet signal at δ1.34 ppm integrated to
four protons assigned to the penultimate protons of the two (CH2) of 7-
dibutylamino, a multiplet signal obtained at δ 1.59 ppm assigned to next
four protons of the two (CH2) of 7-dibutyl amino and a multiplet signal
at δ 3.54 ppm assigned to four protons assigned to the four protons of
two (CH2-N) of 7-dibutyl amino. 13C NMR revealed the appearance of
four signals of the symmetric dibutyl at δ 14.23, 19.86, 25.01, 48.52
ppm. MS spectrum showed the appearance of molecular ion peak at m/z
= 429.88.
The adopted synthetic pathway is illustrated in scheme 1. Com-
pounds 1,2,3,6 and 7 were prepared and characterized as we previously
reported [33]. Both compounds 4 and 7 are reactive towards nucleo-
philic substitution at C7, this is attributed to the presence of imidoyl
chloride moiety (Cl-C = NR) [34]. Thus, target compounds 5a-e were
obtained via gentle reflux with two equivalents of appropriate amine.
The formation of 5a-e was confirmed via different spectral analyses. For
example, for compound 5b 1H NMR spectrum shows triplet signal at δ
1.22 ppm integrated to 6 protons with J = 7.0 Hz assigned to protons of
the terminal symmetrical two CH3 of the 7-diethylamino, and a multi-
plet signal at δ 3.60, J = 7.2 Hz integrated to four protons assigned to the
four protons of two CH2 of the 7-diethyl amino. 13C NMR showed the
appearance of five signals at the aliphatic region including a signal at δ
13.9 ppm corresponding to two (CH3) of diethyl amino and a signal at δ
42.816 ppm due to two CH2 of the 7-diethylamino. Mass spectrometry
3

H.R. Elgiushy et al.
Bioorganic Chemistry 114 (2021) 105079
Scheme.1. Synthesis of target compounds 5a-e and 8a-e.
2.2. Biological Evaluation:
Based on results above, 8c was found to bind to CRFR1 with higher
affinity than other tested compounds and with comparable binding af-
finity with that of antalarmin. Based on this finding, we tested whether
8c was able to antagonize CRF-stimulated accumulation of cAMP, which
is the secondary messenger that mediates the biological effects resulting
from stimulating CRFR1. To accomplish this, we determined its half-
To test whether the target compounds bind to CRFR1, Tyr0-sauva-
gine was radioiodinated and the ability of these compounds to inhibit
the specific binding of [125I]-Tyr0-sauvagine was investigated. The
binding affinity of [125I]-Tyr0-sauvagine was 2.2 nM (-logKi = 8.66), as
determined from homologous competitive binding experiments. The
ability of test compounds to inhibit [125I]-Tyr0-sauvagine binding was
tested by determining the specific binding of radioligand (under equi-
librium conditions) to membranes from HEK 293 cells stably expressing
the CRFR1 in the presence or absence of compounds at a single con-
centration of 500 nM. Eight final compounds (5a, 5b, 5d, 5e, 8a, 8b, 8c
and 8d) were screened for their inhibitory effect. Among tested com-
pounds, four compounds have shown less than 50% inhibition which
includes 5a, 8b, 8d and 8a Fig. 4. Compounds 5b, 5d, 5e and 8c, on the
other hand, have shown more than 50% inhibition and were further
investigated to determine their inhibitory constant (Ki) for the CRFR1.
5b, 5d, 5e and 8c were found to bind to the receptor in a dose response
manner and with binding affinities of 5-, 6-, 10-, and 2-fold lower than
that of antalarmin (Table 1, Fig. 5, Fig. 6).
maximal inhibitory concentration (or antagonistic potency, - Log IC50
)
by incubating HEK 293 cells expressing the CRFR1 with 1 nM of CRF in
the presence or absence of increasing concentrations of 8c or antalarmin
(control). As shown in Fig. 7, 8c inhibited CRF-stimulated cAMP accu-
mulation in a dose–response manner, with an antagonistic potency of
2.15 μM. Even though 8c had similar binding affinity with antalarmin, it
was 100 times less potent than antalarmin to inhibit CRF-stimulated
cAMP accumulation. Antalarmin inhibited CRF-stimulated cAMP accu-
mulation in a dose–response manner, with an antagonistic potency of 26
nM (Fig. 7). Original FRET data are also shown in the figure on the left
side.
4

H.R. Elgiushy et al.
Bioorganic Chemistry 114 (2021) 105079
Fig. 4. Screening target compounds for binding to human CRFR1. Inhibition of [125I]-Tyr0-sauvagine specific binding by 500 nM of test compounds was performed.
Table 1
Binding affinities of test compounds for CRFR1.
Analogues*
antalarmin
5b
5d
5e
8c
- LogKi S.E.
7.8 0.15
7.1
7.0
6.8
7.5
0.11
84.5
0.13
91.2
0.13
169.8
0.37
32.1
Mean Ki
(nM)
16.2
*
Kd of CP-376395 is 7.5 nM [23].
Fig. 6. Competition binding isotherms of 5e, 8c and antalarmin to CRFR1.
composed of 253 Å3 centered on co-crystalized ligand to explore the
whole area around the allosteric site. Redocking of co-crystalized ligand
predicted the correct pose with RMSD of 0.635 as calculated by
DockRMSD [35] and docking energy of ꢀ 9.1 kcal/mol. (Figure SI-1)
Docking of the four known inhibitors was able to predict a similar
pose to the crystal structure in case of the compounds with bicyclic
central core motif (Antalarmin, Pexacerfont and Verucerfont) with en-
ergies comparable or even better than the monocyclic crystalized CP-
376395 (Fig. 8). Docking of the tricyclic inhibitor, NBI35965, was not
predicted correctly relative to the co-crystalized ligand and hence its
docking energy was low (-8.0 kcal/mol). This might be due to the very
rigid structure of NBI35965.
Fig. 5. Competition binding isotherms of 5b, 5d and antalarmin to CRFR1.
2.3. Molecular modelling studies
2.3.1. Molecular Docking
Docking of the tested compounds showed that all of them were able
to adapt a similar binding mode to the co-crystalized ligand. All com-
pounds were able to maintain the hydrogen bond between the ring ni-
trogen and N283 residue. In addition, compounds 5a-e generally
showed smaller scores compared to the series 8a-e. This suggest that the
oxo-derivatives could bind better to CRFR1 when compared to the thi-
oxo derivatives which is consistent with our best hit 8c being an oxo-
Docking was used to study potential binding modes of tested com-
pounds in the allosteric binding site of CRFR1 compared to co-
crystallized ligand CP-376395. We tested our compounds along with
four known inhibitors that include antalarmin, NBI35965, Pexacerfont
and Verucerfont. We also attempted to redock the co-crystalized ligand
CP-376395 to validate the docking procedure. We used a large grid box
5

H.R. Elgiushy et al.
Bioorganic Chemistry 114 (2021) 105079
Fig. 7. Inhibition of CRF-stimulated cAMP accumulation by 8c and antalarmin. Original FRET data are shown on the left side. EC50 of CFR is 133 pM as shown in the
top raw. 8c (middle raw) and antalarmin (bottom raw) competes with CRF and inhibit CRF-stimulated cAMP accumulation which is detect by FRET ratio.
derivative. Among the first series, compound 5d had the best docking
energy which also the best compound in this series according to the
inhibition of [125I]-Tyr0-sauvagine specific binding assay. In case of the
second series, docking predicted that compounds 8c-e have similar
binding energies. Compound 8c is indeed the most active compound but
docking was not able to differentiate between the three compounds 8c-e
(Table 2). Considering these findings we decided to further study and
compare the stability of 8c and 8d complexes as well as co-crystallized
ligands using molecular dynamics simulation.
2.3.2. Molecular dynamics
Molecular dynamics simulation was performed to investigate the
stability of ligand–protein complexes. After preparation steps, CP-
376395 crystal structure complexed with CRFR1 was subjected to 50
ns production run along with the apoprotein and 8c which is the most
potent compound. 8c and the co-crystallized ligands have shown sta-
bility in the active site of CRFR1 with ligand heavy atoms RMSD of
2.5–3.0 Å (Table 3 and Fig. 9). Both complexes maintained the hydrogen
bond with N283 during most of the run with average distance of 2.25 ±
6

H.R. Elgiushy et al.
Bioorganic Chemistry 114 (2021) 105079
Fig. 8. Docking poses of top compounds compared to the co-crystalized ligand (blue). a, Antalarmin (yellow), b, Pexacerfont (rose), c, Verucerfont (golden) and d, 8c
(orange). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
0.32 Å and 2.22 ± 0.20 Å for 8c and co-crystalized ligand, respectively.
Table 2
In addition, hydrophobic interactions between ligands (co-crystalized
Docking scores of the tested compounds in the allosteric site of
ligand and 8c) and hydrophobic amino acids in the active site (F203,
CRFR1.
L280, F284, L287 and L320) are maintained during the whole molecular
Ligand
Binding Affinity
dynamics run. The average total interaction energy between ligand and
protein, calculated by adding short-range Coulombic interaction energy
and short-range Lennard-Jones energy, for co-crystalized ligand was
found to be ꢀ 240.81 ± 0.87 kJ/mol. The total binding energy for 8c
with CRFR1 was stronger compared with the control and was found to
be ꢀ 306.71 ± 1.54 kJ/mol. Average radius of gyration of all the
complexes during the production run was around 20.0 Å. We also per-
formed MM-PBSA calculation for the target complexes with co-
crystalized ligand and 8c over the last 10 ns of the simulation. The re-
sults shows that 8c binding energy with target protein is more than that
of co-crystalized ligand with about 27 KJ/mol. The full energy compo-
nents of the MM-PBSA calculation are shown in table 4.
5a
ꢀ 8.0
ꢀ 6.9
ꢀ 8.5
ꢀ 8.8
ꢀ 8.3
ꢀ 8.3
ꢀ 8.7
ꢀ 9.0
ꢀ 9.1
ꢀ 9.0
ꢀ 9.1
ꢀ 9.4
ꢀ 8.0
ꢀ 9.2
ꢀ 9.9
5b
5c
5d
5e
8a
8b
8c
8d
8e
Co-crystalized Ligand
Antalarmin
NBI35965
Pexacerfont
Verucerfont
We were also interested to investigate the effect of elongating the
dialkyl amino substitution that leads to a large reduction in inhibitory
effect when extending the n-propyl of 8c to n-butyl in case of 8d for
example. To study that effect, 8d complex with CRFR1 was also run for
50 ns. The RMSD of ligand heavy atoms was slightly higher compared to
8c (table 3). In addition, a plot of protein RMSD compared to crystal
structure shows higher fluctuation of the 8d complex especially during
the period of 25–40 ns of the production run (Fig. 10) which might
suggest the relative instability of the complex. Also, the average number
of hydrogen bonds is less in 8d and the average distance of hydrogen
bond with N283 (2.31 ± 0.28 Å) is slightly longer. All these factors
suggest that binding of 8d is weaker compared to 8c as have been seen in
the biological assay which suggests that the N,N-di-n-propyl substitution
binds better into the CRFR1 pocket.
Table 3
Average results for the MD production run.
8c
8d
Control
Apoprotein
Average RMSD of Ligand
heavy atoms (Å)
2.52
3.01
3.05
–
Average RMSD of protein (nm)
Average RMSF of residues
(nm)
5.770
0.172
5.849
0.158
5.836
0.146
6.011
0.149
Average number of H-Bond
Length of hydrogen bond with
N283 (Å) (Between
1.82
1.63
0.87
–
2.25 ±
0.32
2.31 ±
0.28
2.22 ±
0.20
–
hydrogen and nitrogen,
–
N… ..H N)
3. Conclusion
Length of hydrogen bond with
N283 (Å)(Between heavy
atoms, N… ..N)
3.15 ±
3.20 ±
3.13 ±
–
0.23
0.23
0.16
Ten new thiazolo[4,5-d]pyrimidine derivatives were synthesized and
pharmacologically assessed for CRFR1 antagonist activity. Compound
8c was the most promising candidate and was able to cause more than
Average Radius of Gyration
(Å)
20.04
19.95
20.05
19.82
7

H.R. Elgiushy et al.
Bioorganic Chemistry 114 (2021) 105079
Fig. 9. Plot of the RMSD of the ligands heavy atoms in the allosteric site of CRFR1 of both co-crystalized ligand, compounds 8c, 8d and control.
4. Experimental
Table 4
MM-PBSA calculations for CRFR1 complexes with co-crystalized ligand (control)
4.1. Chemistry
and 8c.
Energies (kJ/mol)
4.1.1. General methods
Control
8c
Solvents, reagents and starting materials were obtained from Merck
(Darmstadt, Germany), Acros Organics (New Jersy, USA) and Sigma
Aldrich (Missori, USA). Reactions were monitored using thin-layer
chromatography (TLC) using aluminum sheets pre coated with silica
gel (Kieselgel, F254, pore size 60 Å, Merck, Darmstadt, Germany) and
observed under a UV lamp (short-wavelength, 254 nm). Silica gel (pore
size 60 Å, 230–400 mesh particle size, Merck, Darmstadt, Germany) was
used for column chromatography. Stuart melting point apparatus
(Stuart Scientific, Redhill, UK) was used for the determination of
melting points. Infra-red (IR) spectra were obtained using an FT-IR
spectrometer using KBr discs (Perkin Elmer, Waltham, Massachu-
setts). NMR spectra were obtained using Varian NMR spectrometer
(Varian Inc., Palo Alto, California) operating at 300 MHz for 1H NMR
and 75 MHz for 13C NMR at Dr. Ahmed Farag Laboratory, Faculty of
Science, Cairo University or using Bruker NMR spectrometer (Bruker
van der Waal energy
Electrostatic energy
Polar solvation energy
SASA energy
ꢀ 226.457 ± 9.611
ꢀ 24.824 ± 2.873
97.283 ± 8.519
ꢀ 276.721 ± 10.708
ꢀ 36.149 ± 5.682
133.675 ± 11.575
–23.371 ± 0.808
ꢀ 202.565 ± 13.976
ꢀ 21.915 ± 0.802
ꢀ 175.913 ± 12.113
Binding energy
50% Inhibition of [125I]-Tyr0-sauvagine specific binding. In addition, 8c
showed good binding affinity compared to antalarmin and was able to
inhibit CRF induced cAMP accumulation in a dose response manner
exhibiting an IC50 of 2.15 μM. Compound 8c was also found to have
similar binding mode compared to co-crystalized ligand CP-376395
based on docking studies and the complex of 8c with CRFR1 was
found to be stable as suggested by the molecular dynamics study.
Fig. 10. Plot of protein RMSD relative to crystal structure of 8c, 8d and co-crystalized ligand.
8

H.R. Elgiushy et al.
Bioorganic Chemistry 114 (2021) 105079
Biospin GmbH, Rheinstetten, Germany), operating at 400 MHz
for 1HNMR and 100 MHz for 13C at Center for Drug Discovery Research
and Development, Faculty of Pharmacy Ain Shams University. Chemical
shifts are expressed in δ values (ppm) relative to TMS using DMSO‑d6 or
CDCl3 as solvents. Electron impact mass spectrometry (EI-MS) spectra
were obtained at the Regional Center for Mycology and Biotechnology,
Al-Azhar University using Thermo Scientific ISQ LT mass spectrometer
(Thermo Fisher Scientific Inc., Waltham, Massachusetts). Electrospray
ionization mass (ESI-MS) spectra were collected at the Faculty of
Pharmacy, Helwan University on Thermo Scientific mass spectrometer
LC-MS (Thermo Fisher Scientific Inc., Waltham, Massachusetts).
Elemental analyses were done on a Thermo Scientific Flash 2000
elemental analyzer (Thermo Fisher Scientific Inc., Waltham, Massa-
chusetts) at Regional Center for Mycology and Biotechnology, Al-Azhar
University. Compounds 1, 2, 3, 4, 6 and 7 were prepared as reported
earlier [36].
165.16, 167.96 (10C, aromatic), 189.15 (C2, thioxo); MS (EI) m/z (%):
418.14 (M+, 18.62), 274.15 (Bp, 100); Anal. Calcd. for C20H26N4O2S2
418.15: C, 57.39; H, 6.26; N, 13.39; S, 15.32, Found: C, 57.43; H, 6.23;
N, 13.41; S, 15.31.
4.1.2.4. 7-(Dibutylamino)-3-(2,4-dimethoxyphenyl)-5-methylthiazolo
[4,5-d]pyrimidine-2(3H)-thione (5d):. Reflux time: 6 h. Buff powder,
yield 83%; m.p. 128˚C; IR (KBr)
ν
cmꢀ 1: No significant peaks; 1H NMR
(400 MHz, DMSO‑d6) δ ppm: 0.95 (t, J = 7.3 Hz, 6H, 2CH3), 1.36 (sixtet,
J = 7.2 Hz, 4H, 2CH2), 1.61 (p, J = 7.5 Hz, 4H 2CH2), 2.26 (s, 3H,CH3),
3.55 (d, J = 7.6 Hz, 4H, 2CH2), 3.70 (s, 3H,OCH3), 3.86 (s, 3H,OCH3),
6.67 (dd, J = 8.7, 2.3 Hz, 1H, aromatic), 6.78 (d, J = 2.2 Hz, 1H, aro-
matic), 7.17 (d, J = 8.6 Hz, 1H, aromatic); 13C NMR (100 MHz,
DMSO‑d6) δ ppm: 14.24 (2CH3), 19.89 (2CH2), 25.94 (CH3), 30.88
(2CH2), 48.72 (2CH2), 55.99 (OCH3), 56.41 (OCH3), 95.67, 100.13,
106.08, 117.83, 131.05, 154.92, 156.13, 160.23, 161.83, 165.13 (10C,
aromatic), 189.11 (C2, thioxo); MS (EI) m/z (%): 446.32 (M+, 5.62),
57.02 (Bp, 100); Anal. Calcd. for C22H30N4O2S2 446.63: C, 59.16; H,
6.77; N, 12.54; S, 14.36. Found: C, 59.2; H, 6.75; N, 12.57; S, 14.39.
4.1.2. Synthesis of 7-(Dialkylamino)-3-(2,4-dimethoxyphenyl)-5-
methylthiazolo[4,5-d]pyrimidine-2(3H)-thione (5a-e):
General procedure: A mixture of compound 4 (1 g, 2.83 mmol) was
reacted with the appropriate dialkyl amine (5.66 mmol) in absolute
ethanol (15 ml) with gentle heating (70˚C) for 3–6 h., the reaction
mixture was allowed to cool down then poured onto ice water and
stirred for 30 min. The obtained precipitate was filtered and recrystal-
lized from absolute ethanol or the aqueous solution was extracted with
ethyl acetate then the organic extracts were collected, dried over
anhydrous sodium sulfate and concentrated under reduced pressure.
4.1.2.5. 7-(Butyl(ethyl)amino)-3-(2,4-dimethoxyphenyl)-5-methylthiazolo
[4,5-d]pyrimidine-2(3H)-thione (5e):. Reflux time: 5 h. Yellowish brown
crystals; yield 62%; m.p. 109˚C; IR (KBr)
ν
cmꢀ 1: no significant peaks;
1H NMR (400 MHz, CDCl3) δ ppm: 1.01 (t, J = 7.3 Hz, 3H, CH3), 1.28 (t,
J = 7.0 Hz, 3H, CH3), 1.43 (h, J = 7.4 Hz, 2H, CH2), 1.69 (p, J = 7.6 Hz,
2H, CH2), 2.39 (s, 3H, CH3), 3.55 (t, J = 6. 8 Hz, 2H, CH2), 3.66 (qd, J =
7.1, 2.7 Hz, 2H, CH2), 3.77 (s, 3H, OCH3), 3.89 (s, 3H, OCH3), 6.65 –
6.69 (m, 2H, aromatic), 7.17 (d, J = 9.3 Hz, 1H, aromatic); 13C NMR
(100 MHz, DMSO‑d6) δ ppm: 14.17 (CH3), 14.23 (CH3), 19.93 (CH2),
25.95 (CH3), 31.05 (CH2), 43.73 (CH2-N), 48.22 (CH2-N), 55.98 (OCH3),
56.40 (OCH3), 95.67, 100.13, 106.05, 117.85, 131.02, 154.78, 156.15,
160.23, 161.83, 165.20 (10C, aromatic), 189.17 (C2, thioxo); MS (EI) m/
4.1.2.1. 3-(2,4-Dimethoxyphenyl)-5-methyl-7-(propylamino)thiazolo[4,5-
d]pyrimidine-2(3H)-thione (5a):. Reflux time: 3 h. Light yellow powder;
yield 85%; m.p. 129˚C; IR (KBr)
ν
cmꢀ 1: 3383 (NH); 1H NMR (400 MHz,
DMSO‑d6) δ ppm: 0.45 – 1.27 (m, 5H, C2H5), 1.58 (s, 2H, CH2), 2.27 (s,
3H, CH3), 3.68 (s, 3H, OCH3), 3.84 (s, 3H, OCH3), 6.65 (s, 1H, aromatic),
6.76 (s, 1H, aromatic), 7.16 (s, 1H, aromatic), 7.82 (s, 1H, NH); 13C NMR
(100 MHz, DMSO‑d6) δ ppm: 11.39 (CH3), 22.08 (CH2), 25.57 (CH3),
42.16 (CH2), 55.50 (OCH3), 55.92 (OCH3), 96.84, 99.66, 105.53,
117.32, 130.61, 154.64, 155.69, 158.58, 161.33, 165.46 (10C, aro-
matic), 189.58 (C2, thioxo); MS (m/z, %): 376.05 (M+, 8.76), 40.15 (Bp,
100); Anal. Calcd. for C17H20N4O2S2 376.49: C, 54.23; H, 5.35; N,
14.88; S, 17.03, Found: C, 54.26; H, 5.32; N, 14.89; S, 17.02.
z
(%): 418.14 (M+, 11.63), 98.44 (Bp, 100); Anal. Calcd. for
C20H26N4O2S2 418.57: C, 57.39; H, 6.26; N, 13.39; S, 15.32, Found: C,
57.42; H, 6.24; N, 13.42; S, 15.36.
4.1.3. 3-(2,4-Dimethoxyphenyl)-7-(dialkylamino)-5-methylthiazolo[4,5-
d]pyrimidin-2(3H)-one (8a-e):
General procedure: A mixture of compound 7 (1 g, 2.96 mmol) and
the appropriate dialkyl amine (5.92 mmol) in absolute ethanol (15 ml)
using the same procedures described for 5a-e.
4.1.2.2. 7-(Diethylamino)-3-(2,4-dimethoxyphenyl)-5-methylthiazolo
[4,5-d]pyrimidine-2(3H)-thione (5b):. Reflux time: 3 h. Light buff pow-
4.1.3.1. 3-(2,4-Dimethoxyphenyl)-5-methyl-7-(propylamino)thiazolo[4,5-
d]pyrimidin-2(3H)-one (8a):. Reflux time: 3 h. Light yellow powder;
yield 85%; m.p. 124˚C; 1H NMR (400 MHz, DMSO‑d6) δ ppm: 0.76 – 0.99
(m, 5H, overlapped CH2 and CH3), 1.51 – 1.66 (m, 2H, CH2), 2.27 (s, 3H,
CH3), 3.69 (s, 3H, OCH3), 3.85 (s, 3H, OCH3), 6.66 (dd, J = 8.7, 2.5 Hz,
1H, aromatic), 6.77 (d, J = 2.5 Hz, 1H, aromatic), 7.16 (d, J = 8.6 Hz,
1H, aromatic), 7.82 (s, 1H, NH); 13C NMR (100 MHz, DMSO‑d6) δ ppm:
11.39 (CH3), 22.07 (CH2), 25.58 (CH3), 42.15 (CH2), 55.51(OCH3),
der; yield 85%; m.p. 141˚C; IR (KBr)
ν
cmꢀ 1: No significant peaks; 1
H
NMR (400 MHz, DMSO‑d6) δ ppm: δ 1.22 (t, J = 7.00 Hz, 6H, 2CH3),
2.28 (s, 3H CH3), 3.60 (h, J = 7.30 Hz, 4H, 2CH2), 3.70 (s, 3H,OCH3),
3.86 (s, 3H,OCH3), 6.67 (dd, J = 8.9, 2.5 Hz, 1H aromatic), 6.78 (d, J =
2.5 Hz, 1H aromatic), 7.17 (d, J = 8.7 Hz, 1H aromatic); 13C NMR (75
MHz, DMSO‑d6) δ ppm: 13.91 (2CH3), 25.46 (CH3), 42.82 (2CH2), 55.59
(OCH3), 56.06 (OCH3), 99.88, 105.74, 117.52, 120.98, 130.61, 154.30,
155.81, 161.45, 164.73, 164.84 (10C, aromatic), 188.87 (C2, thioxo);
MS (EI) m/z (%): 390.54 (M+, 18.62), 57.03 (Bp, 100); Anal. Calcd. for
C18H22N4O2S2 390.54: C, 55.36; H, 5.68; N, 14.35; S, 16.42, Found: C,
55.38; H, 5.65; N, 14.39; S, 16.43.
55.93 (OCH3), 89.20, 99.66, 105.54, 117.30, 130.61, 131.11, 151.88,
–
154.77, 155.68, 161.32 (10C, aromatic), 165.45 (C , C O); MS (EI) m/z
–
(%): 360.19 (M+, 9.39), 62.90 (Bp, 100); Anal. Ca2lcd. for C17H20N4O3S
360.43: C, 56.65; H, 5.59; N, 15.54; S, 8.89. Found: C, 56.67; H, 5.57; N,
15.53; S, 8.87.
4.1.2.3. 3-(2,4-Dimethoxyphenyl)-7-(dipropylamino)-5-methylthiazolo
[4,5-d]pyrimidine-2(3H)-thione (5c):. Reflux time: 3.5 h. Greenish yel-
4.1.3.2. 7-(Diethylamino)-3-(2,4-dimethoxyphenyl)-5-methylthiazolo
low crystals, yield (81%) m.p. 137˚C; IR (KBr)
ν
cmꢀ 1: No significant
[4,5-d]pyrimidin-2(3H)-one (8b):. Reflux time: 3 h. Light buff powder;
peaks; 1H NMR (400 MHz, CDCl3) δ ppm: 1.00 (t, J = 7.40 Hz, 6H,
2CH3), 1.67 – 1.78 (sixtet, 4H , 2 CH2), 2.40 (s, 3H , CH3), 3.54 (t, 4H, 2
CH2), 3.77 (s, 3H,OCH3), 3.89 (s, 3H, OCH3), 6.65 – 6.67 (m, 1H, aro-
matic), 6.68 (d, J = 2.6 Hz, 1H,aromatic), 7.16 (d, J = 9.3 Hz, 1H, ar-
omatic); 13C NMR (100 MHz, DMSO‑d6) δ ppm: 11.34 (2CH3), 22.05
(2CH2), 25.99 (CH3), 50.68 (2CH2), 55.99 (OCH3), 56.42 (OCH3),
100.14, 106.08, 117.84, 131.06, 155.00, 156.15, 160.24, 161.83,
1
–
–
yield 87%; m.p. 151˚C; IR (KBr)
ν
cmꢀ 1: 1702 (C , (C O)); H NMR (400
2
MHz, DMSO‑d6) δ ppm: 1.20 (t, J = 6.9 Hz, 6H, 2CH3), 2.24 (s, 3H, CH3),
3.59 (sixtet, J = 7.1 Hz, 4H, 2CH2), 3.72 (s, 3H, OCH3), 3.84 (s, 3H,
OCH3), 6.64 (dd, J = 8.6, 2.7 Hz, 1H, aromatic), 6.75 (d, J = 2.6 Hz, 1H,
aromatic), 7.23 (d, J = 8.6 Hz, 1H, aromatic); 13C NMR (100 MHz,
DMSO‑d6) δ ppm: 14.11 (2CH3), 25.57 (2CH2), 42.56 (CH3), 55.53
(OCH3), 55.89 (OCH3), 89.12, 99.41, 105.31, 115.38, 130.75, 154.69,
9

H.R. Elgiushy et al.
Bioorganic Chemistry 114 (2021) 105079
–
156.01, 156.14, 161.24, 163.62 (10C, aromatic), 167.40 (C , C O); MS
C-18 cartridge (Waters WAT051910) and separated by sequential
elution with solutions (1 ml each) containing 5, 10, 20, 30, 35, 40, 50,
60, 80% acetonitrile in water and 0.1% TFA. Fractions of 0.5 ml were
–
2
(EI) m/z (%): 373.98 (M+, 9.13), 57.05 (Bp, 100); Anal. Calcd for
C18H22N4O3S 374.14: C, 57.74; H, 5.92; N, 14.96; S, 8.56. Found: C,
57.76; H, 5.90; N, 14.99; S, 8.59.
collected and their radioactivity was determined. Subsequently, 3.5 μL
mercaptoethanol and 10 μL 20% BSA were added to three fractions,
eluted with 35% and 40% acetonitrile, containing radioiodinated Tyr0-
sauvagine.
4.1.3.3. 3-(2,4-Dimethoxyphenyl)-7-(dipropylamino)-5-methylthiazolo
[4,5-d]pyrimidin-2(3H)-one (8c):. Reflux time: 3.5 h. Light yellow
powder; yield 82%; m.p. 133˚C; IR (KBr)
ν
cmꢀ 1: 1695 (C2
C
O); 1H
–
–
4.2.2. CRFR1 binding study
NMR (400 MHz, DMSO‑d6) δ ppm: 0.91 (d, J = 7.3 Hz, 6H, 2CH3), 1.63
(m, J = 7.5 Hz, 4H, 2CH3), 2.23 (s, 3H, CH3), 3.49 (td, J = 7.2, 2.5 Hz,
4H, 2CH2), 3.72 (s, 3H, OCH3), 3.84 (s, 3H, OCH3), 6.63 (dd, J = 8.7, 2.6
Hz, 1H, aromatic), 6.75 (d, J = 2.6 Hz, 1H, aromatic), 7.22 (d, J = 8.6
Hz, 1H, aromatic). 13C NMR (100 MHz, DMSO‑d6) δ ppm: 10.82 (CH3),
21.77 (CH2), 25.56 (CH3), 50.08 (CH2), 55.53 (OCH3), 55.89 (OCH3),
Binding studies was performed according to our reported procedure
[37] in membrane homogenates from human embryonic kidney cells
(HEK 293) stably expressing CRFR1 and using [125I]-Tyr0-sauvagine as
radioligand. Membrane homogenates were prepared according to the
method of Spyridaki et al [35]. CRFR1 -expressing HEK 293 cells, grown
in DMEM/F12 (1:1) containing 3.15 g/L glucose, 10% bovine calf serum
89.18, 99.41, 105.32, 115.36, 130.76, 155.01, 156.00, 156.16, 161.23,
and 300 μ
g/ml of the antibiotic, Geneticin at 37 ◦C and 5% CO2, were
–
–
163.49 (10C, aromatic), 167.32(C , C O); MS (EI) m/z (%): 402.17
2
(M+, 8.03), 57.63 (Bp, 100); Anal. Calcd for C20H26N4O3S, 402.17: C,
59.68; H, 6.51; N, 13.92; S, 7.96. Found: C, 59.65; H, 6.53; N, 13.90; S,
7.93.
washed with phosphate-buffered saline (PBS) (4.3 mM Na2HPO4.7 H20,
1.4 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl, 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 × g for 5 min at room temperature, and the pellets
were homogenized in 1.5 ml of buffer H (20 mM HEPES, containing 10
4.1.3.4. 7-(Dibutylamino)-3-(2,4-dimethoxyphenyl)-5-methylthiazolo
[4,5-d]pyrimidin-2(3H)-one (8d):. Reflux time: 6 h. Light buff powder;
yield 70%; m.p. 106˚C; 1H NMR (400 MHz, DMSO‑d6) δ ppm: 0.94 (t, J
= 7.4 Hz, 6H, 2CH3), 1.34 (h, J = 7.3 Hz, 4H, 2CH2), 1.59 (p, J = 7.5 Hz,
4H, 2CH2), 2.22 (s, 3H, CH3), 3.48 – 3.60 (m, 4H, 2CH2), 3.72 (s, 3H,
OCH3), 3.84 (s, 3H, OCH3), 6.63 (dd, J = 8.6, 2.6 Hz, 1H, aromatic), 6.75
mM MgCl2, 2 mM EGTA, 0.2 mg/ml bacitracin and 0.93 μg/ml aprotinin
pH 7.2 at 4 ◦C) using a Janke & Kunkel IKA Ultra Turrax T25 homoge-
nizer, at setting ~ 20, for 10–15 sec, at 4 ◦C. The homogenates were
centrifuged at 16000 × g, for 10 min, at 4 ◦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 20 ◦C). The membrane
suspensions were then diluted in buffer B and aliquots of suspensions
(d, J = 2.6 Hz, 1H, aromatic), 7.22 (d, J = 8.7 Hz, 1H, aromatic); 13
C
NMR (100 MHz, DMSO‑d6) δ ppm: 14.26 (2CH3), 19.86 (2CH2), 26.01
(CH3), 31.13 (2CH2), 48.59 (2CH2), 56.02 (OCH3), 56.38 (OCH3),
(50 μL) were added into tubes containing buffer B and 20000–30000
98.31, 99.96, 105.81, 115.82, 130.99, 131.25, 156.48, 157.96, 161.72,
cpm [125I]-Tyr0sauvagine without or with the new test compounds at the
single concentration of 500 nM (screening experiments) or with
increasing concentrations of HR compounds or antalarmin (heterolo-
gous competition binding experiments) or increasing concentrations of
Tyr0sauvagine (homologous 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 mem-
branes used were adjusted to ensure that the specific binding is always
equal to or less than 10% of the total concentration of the added radi-
oligand. Specific [125I]-Tyr0-sauvagine binding was defined as total
binding less nonspecific binding in the presence of excess of cold ligand
–
–
162.26 (10C, aromatic), 163.97 (C , C O); MS (EI) m/z (%): 429.88
2
(M+, 2.90), 186.99 (Bp, 100); Anal. Calcd for C22H30N4O3S, 430.17: C,
61.37; H, 7.02; N, 13.01; S, 7.45. Found: C, 61.39; H, 7.04; N, 12.98; S,
7.47.
4.1.3.5. 7-(Butyl(ethyl)amino)-3-(2,4-dimethoxyphenyl)-5-methylthiazolo
[4,5-d]pyrimidin-2(3H)-one (8e):. Reflux time: 5 h. Brownish yellow
–
powder; yield 65%; m.p. 125˚C; IR (KBr)
ν
cmꢀ 1: 1693 (C , C O); 1H
–
2
NMR (400 MHz, DMSO‑d6) δ ppm: 0.74 – 1.69 (m, 10H, aliphatic), 2.22
(s, 3H, CH3), 2.84 (d, 2H, CH2) 3.57 (s, 2H, CH2), 3.71 (s, 3H, OCH3),
3.83 (s, 3H, OCH3), 6.62 (d, J = 8.9 Hz, 1H, aromatic), 6.74 (s, 1H,
aromatic), 7.35 (d, J = 8.8 Hz, 1H, aromatic); 13C NMR (100 MHz,
DMSO‑d6) δ ppm: 14.15 (CH3), 14.24 (CH3), 19.92 (CH2), 25.96 (CH2),
31.03 (CH3), 43.73 (CH2), 48.21 (CH2), 55.99 (OCH3), 56.41 (OCH3),
95.66, 100.13, 106.07, 117.83, 131.04, 154.78, 156.14, 160.21, 161.83,
(1 μM antalarmin or 0.3 μM sauvagine). 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. The binding affinities for HR compounds and antalarmin (logKi
values) and for 125I-Tyr0-sauvagine (logKD values) were determined
from heterologous and homologous competition data, respectively, as
described previously using Prism 4.0 [37].
–
–
165.21 (10C, aromatic), 189.17 (C2, C O); MS (EI) m/z (%): 402.79
(M+, 4.30), 57.98 (Bp, 100); Anal. Calcd for C20H26N4O3S, 402.17: C,
59.68; H, 6.51; N, 13.92; S, 7.96. Found: C, 59.71; H, 6.48; N, 13.89; S,
7.93.
4.2. Biological evaluation
4.2.1. Iodinating Tyr0-SVG
4.2.3. FRET assays for detection of cAMP
The radioiodination of Tyr0-Sauvagine (Tyr0-SVG) was performed
FRET assays reported here used a new C4 clone of HEK293 cells
created by O.G. Cherpurny. These HEK293-C4 cells were doubly stably
transfected with the human CRFR1 and also the cAMP biosensor H188.
The methods of transfection, clonal selection, and FRET analysis in a 96-
well format were identical to those reported in greater detail by Che-
purny and colleagues for assays monitoring glucagon action at the
human glucagon receptor [38]. Briefly, suspensions of HEK293-C4 cells
were prepared in a standard extracellular saline (SES) solution con-
taining (in mM): 138 NaCl, 5.6 KCl, 2.6 CaCl2, 1.2 MgCl2, 11.1 glucose,
10 Hepes (295 mosmol, pH 7.4) supplemented with 0.1% BSA. Indi-
vidual wells of a 96-well clear-bottom assay plate (Costar 3904, Corning,
using Pre-Coated Iodination Tubes. In specific, 50 μL of Tris Iodination
Buffer (25 mM Tris•HCl, and 0.4 M NaCl, pH 7.4) were added into Pre-
Coated Iodination Tubes (Pierce, Cat. No 28601). Subsequently 20 μL
containing 1 mCi Na125I (Perkin Elmer, Cat. No NEZ033A) were added.
After 6 min incubation at room temperature, the mixture was added into
a low retention tube (Kisker, Cat. No G016) containing 7
μ
g of Tyr0-SVG
(in 1.5
μL of Tris Iodination Buffer). After 10 min incubation at room
temperature, 20
μ
L of scavenging buffer (10 mg tyrosine/ml in Tris
Iodination Buffer) was added into the mixture and the incubation
continued for 5 min at room temperature. After adding 1 ml of elution
buffer (0.1% TFA), the radiodinated species were loaded onto a Sep-Pak
NY) received 200 μL/well cell suspension so that real-time kinetic assays
10

H.R. Elgiushy et al.
Bioorganic Chemistry 114 (2021) 105079
of FRET could be performed using a Flexstation 3 microplate reader
(Molecular Devices, Sunnyvale, CA). Test solutions dissolved in SES
were placed in V-bottom 96-well plates (Greiner Bio-One, Monroe, NC)
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bioorg.2021.105079.
so that an automated pipetting procedure could be used to transfer 50 μL
of each test solution to each well of the assay plate containing cells. To
monitor binding of cAMP to H188, the excitation light was delivered at
435/9 nm, and the emitted light was detected at 485/15 nm
(mTurquoise2Δ FRET donor) or 535/15 nm (cp173 Venus-Venus FRET
acceptor). An increase of the 485/535 nm FRET ratio signifies an in-
crease of cAMP concentration [39]. FRET ratio values were normalized
using baseline subtraction so that a y axis value of 0 corresponds to the
initial baseline FRET ratio, whereas a value of 100 corresponds to a
100% increase (i.e. doubling) of the FRET ratio. The time course of the
ΔFRET ratio was plotted after exporting data to Origin 8.0 (OriginLab,
Northampton, MA). Origin 8.0 was also used for nonlinear regression
analysis to quantify dose–response relationships.
References
[1] G.N. Smagin, A.J. Dunn, The role of CRF receptor subtypes in stress-induced
behavioural responses, Eur. J. Pharmacol. 405 (2000) 199–206, https://doi.org/
10.1016/S0014-2999(00)00553-7.
[2] W. Vale, J. Spiess, C. Rivier, J. Rivier, Characterization of a 41-residue ovine
hypothalamic peptide that stimulates secretion of corticotropin and β-endorphin,
Obstet. Gynecol. Surv. 37 (1982) 334, https://doi.org/10.1097/00006254-
198205000-00013.
[3] K. Van Pett, V. Viau, J.C. Bittencourt, R.K.W. Chan, H.Y. Li, C. Arias, G.S. Prins,
M. Perrin, W. Vale, P.E. Sawchenko, Distribution of mRNAs encoding CRF
receptors in brain and pituitary of rat and mouse, J. Comp. Neurol. 428 (2000)
191–212, https://doi.org/10.1002/1096-9861(20001211)428:2<191::AID-
CNE1>3.0.CO;2-U.
[4] F. Holsboer, M. Ising, Central CRH system in depression and anxiety - Evidence
from clinical studies with CRH1 receptor antagonists, Eur. J. Pharmacol. 583
(2008) 350–357, https://doi.org/10.1016/j.ejphar.2007.12.032.
[5] A.V. Kaluev, D.J. Nutt, On the role of GABA in anxiety and depression, Eksp. i Klin.
Farmakol. 67 (2004) 71–76. https://doi.org/10.30906/0869-2092-2004-6
7-4-71-76.
4.3. Molecular modelling studies
4.3.1. Molecular Docking
[6] G. Aguilera, M. Flores, P. Carvallo, J.P. Harwood, M. Millan, K.J. Catt, M. Flores,
P. Carvallo, J.P. Harwood, M. Millan, K.J. Catt, Receptors for Corticotropin-
Releasing Factor, in: Corticotropin-Releasing Factor Basic Clin. Stud. a Neuropept,
1st ed.,, CRC Press, 2018, pp. 153–174, https://doi.org/10.1201/9781351070935-
11.
The proposed binding mode of tested compounds with Corticotro-
phin releasing factor receptor (CRFR1) was studied using Autodock Vina
[40] Compounds were built and prepared as reported earlier [41]
Crystal structure of CRFR1 (PDB ID:4K5Y) was used after adding hy-
drogens, removing waters, and adding Gasteiger charges. Prepared and
co-crystalized ligands were docked in a grid box in the allosteric site
(25*25*25 Å3, centered on co-crystalized ligand) using exhaustiveness
of 16. For each ligand, the top 9 binding poses were ranked according to
their binding affinities and the predicted binding interactions were
analyzed. The pose with the best binding affinity and binding mood
similar to co-crystalized ligand was reported. 3D images were prepared
using PyMOL software.
[7] C. Rivier, M. Smith, W. Vale, Regulation of adrenocorticotropic hormone (ACTH)
secretion by corticotropin-releasing factor (CRF), in: Corticotropin-Releasing
Factor Basic Clin. Stud. a Neuropept, 1st editio,, CRC Press, 2018, pp. 175–189,
https://doi.org/10.1201/9781351070935.
[8] J.A. Majzoub, Corticotropin-releasing hormone physiology, Eur. J. Endocrinol.
Suppl. 155 (2006) S71–S76, https://doi.org/10.1530/eje.1.02247.
[9] C. Chen, Recent Advances in Small Molecule Antagonists of the Corticotropin-
Releasing Factor Type-1 Receptor-Focus on Pharmacology and Pharmacokinetics,
Curr. Med. Chem. 13 (2006) 1261–1282, https://doi.org/10.2174/
092986706776873014.
´
[10] E.M. Fekete, E.P. Zorrilla, Physiology, pharmacology, and therapeutic relevance of
urocortins in mammals: Ancient CRF paralogs, Front. Neuroendocrinol. 28 (2007)
1–27, https://doi.org/10.1016/j.yfrne.2006.09.002.
4.3.2. Molecular dynamics
[11] T.L. Bale, W.W. Vale, CRF and CRF Receptors : Role in Stress Responsivity and
Other Behaviors, Annu. Rev. Pharmacol. Toxicol. 44 (2004) 525–557, https://doi.
org/10.1146/annurev.pharmtox.44.101802.121410.
Docking poses were used as initial coordinates for molecular dy-
namics simulation. Missing loop (221–223) of CRFR1 was build using
SWISS-MODEL [42] and was filled with YST sequence. Preparation of
protein and ligands and used parameters were done according to
methods reported earlier [43]. In summary, CHARMM36 all-atom force
field [44] was used for protein parameterization while ligands param-
eters were obtained from SwissParam [45] Complexes were built, boxed
in dodecahedron box, solvated with TIP3P water [46] and neutralized
with Na+ or Cl- ions as needed. All molecular dynamics simulations were
done using GROMACS 2020.3 [47] Initially, complexes were minimized
using steepest descent algorithm and maximum force was set to less than
1000 kJ molꢀ 1 nmꢀ 1 then systems were equilibrated using 100 ps of
NVT followed by NPT ensembles. V-rescale thermostat [48] was used to
control temperature at 300 oK while Parrinello-Rahman barostat [49]
was used to control pressure. Particle mesh Ewald (PME) method [50]
was used for calculation of long-range electrostatics. Timestep of 2 fs
was used for all simulations and Van der Waals cut-off distance (rvdw) of
1.2 nm was used. MM-PBSA calculation was done using g_mmpbsa tool
[51].
[12] E.P. Zorrilla, G.F. Koob, Progress in corticotropin-releasing factor-1 antagonist
development, Drug Discov. Today. 15 (2010) 371–383, https://doi.org/10.1016/j.
drudis.2010.02.011.
[13] M. Arai, I.Q. Assil, A.B. Abou-Samra, Characterization of three corticotropin-
releasing factor receptors in catfish: A novel third receptor is predominantly
expressed in pituitary and urophysis, Endocrinology. 142 (2001) 446–454, https://
doi.org/10.1210/endo.142.1.7879.
[14] P. Timpl, R. Spanagel, I. Sillaber, A. Kresse, J.M.H.M. Reul, G.K. Stalla, V. Blanquet,
T. Steckler, F. Holsboer, W. Wurst, Impaired stress response and reduced anxiety in
mice lacking a functional corticotropin-releasing hormone receptor 1, Nat. Genet.
19 (1998) 162–166, https://doi.org/10.1038/520.
[15] G.W. Smith, J.M. Aubry, F. Dellu, A. Contarino, L.M. Bilezikjian, L.H. Gold,
R. Chen, Y. Marchuk, C. Hauser, C.A. Bentley, P.E. Sawchenko, G.F. Koob, W. Vale,
K.F. Lee, Corticotropin releasing factor receptor 1-deficient mice display decreased
anxiety, impaired stress response, and aberrant neuroendocrine development,
Neuron. 20 (1998) 1093–1102.
[16] M.P. Stenzel-Poore, S.C. Heinrichs, S. Rivest, G.F. Koob, W.W. Vale,
Overproduction of corticotropin-releasing factor in transgenic mice: a genetic
model of anxiogenic behavior, J. Neurosci. 14 (1994) 2579–2584.
[17] S.C. Heinrichs, J. Lapsansky, T.W. Lovenberg, E.B. De Souza, D.T. Chalmers,
Corticotropin-releasing factor CRF1, but not CRF2, receptors mediate anxiogenic-
like behavior, Regul. Pept. 71 (1997) 15–21.
[18] G. Liebsch, R. Landgraf, R. Gerstberger, J.C. Probst, C.T. Wotjak, M. Engelmann,
F. Holsboer, A. Montkowski, Chronic infusion of a CRH1 receptor antisense
oligodeoxynucleotide into the central nucleus of the amygdala reduced anxiety-
related behavior in socially defeated rats, Regul. Pept. 59 (1995) 229–239.
Declaration of Competing Interest
¨
[19] G. Liebsch, R. Landgraf, M. Engelmann, P. Lorscher, F. Holsboer, Differential
behavioural effects of chronic infusion of CRH 1 and CRH 2 receptor antisense
oligonucleotides into the rat brain, J. Psychiatr. Res. 33 (1999) 153–163.
[20] T. Skutella, J.C. Probst, U. Renner, F. Holsboer, C. Behl, Corticotropin-releasing
hormone receptor (type I) antisense targeting reduces anxiety, Neuroscience. 85
(1998) 795–805.
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
[21] D.A. Gutman Michael J. Owens, and Charles B. Nemeroff, Corticotropin-releasing
factor antagonists as novel psychotherapeutics, Drugs Future. 25 (2000) 923–931.
file:///C:/Users/Hosam/AppData/Local/Mendeley Ltd./Mendeley Desktop/
Downloaded/Unknown - Unknown - crf antagonists as novel psycothearapeutics.
pdf.pdf.
Acknowledgements
This work was partially funded by NIH grant funding (NIH R01
DK069575) received by Professor G. Holz.
11

H.R. Elgiushy et al.
Bioorganic Chemistry 114 (2021) 105079
[22] S. Okuyama, S. Chaki, N. Kawashima, Y. Suzuki, S. Ogawa, A. Nakazato,
T. Kumagai, T. Okubo, K. Tomisawa, Receptor binding, behavioral, and
electrophysiological profiles of nonpeptide corticotropin-releasing factor subtype 1
receptor antagonists CRA1000 and CRA1001, J. Pharmacol. Exp. Ther. 289 (1999)
926–935.
Structural-functional analysis of the third transmembrane domain of the
corticotropin-releasing factor type 1 receptor: role in activation and allosteric
antagonism, J. Biol. Chem. 289 (2014) 18966–18977, https://doi.org/10.1074/
jbc.M113.544460.
[38] B. Milliken, O. Chepurny, R. Doyle, G. Holz, FRET Reporter Assays for cAMP and
Calcium in a 96-well Format Using Genetically Encoded Biosensors Expressed in
Living Cells, Bio-Protocol. 10 (2020) 3514–3531. https://doi.org/10.21769/biopro
toc.3641.
´
[23] K. Hollenstein, J. Kean, A. Bortolato, R.K.Y.Y. Cheng, A.S. Dore, A. Jazayeri, R.
M. Cooke, M. Weir, F.H. Marshall, K. Hollenstein, J. Kean, A. Bortolato, R.K.Y.
Y. Cheng, A.S. Dore, M. Weir, F.H. Marshall, Structure of class B GPCR
corticotropin-releasing factor receptor 1, Nature. 499 (2013) 438–443, https://doi.
org/10.1038/nature12357.
[39] J. Klarenbeek, J. Goedhart, A. van Batenburg, D. Groenewald, K. Jalink, Fourth-
Generation Epac-Based FRET Sensors for cAMP Feature Exceptional Brightness,
Photostability and Dynamic Range: Characterization of Dedicated Sensors for
FLIM, for Ratiometry and with High Affinity, PLoS One. 10 (2015)
e0122513–e0122513. https://doi.org/10.1371/JOURNAL.PONE.0122513.
[40] O. Trott, A.J. Olson, AutoDock Vina: Improving the speed and accuracy of docking
with a new scoring function, efficient optimization, and multithreading, J. Comput.
Chem. 31 (2010) 455–461, https://doi.org/https://doi.org/10.1002/jcc.21334.
[41] S.S. Ebada, N.A. Al-Jawabri, F.S. Youssef, A. Albohy, S.M. Aldalaien, A.M. Disi,
P. Proksch, In vivo antiulcer activity, phytochemical exploration, and molecular
modelling of the polyphenolic-rich fraction of Crepis sancta extract,
Inflammopharmacology. 28 (2020) 321–331, https://doi.org/10.1007/s10787-
019-00637-x.
[24] Y. Ye, Q. Liao, J. Wei, Q. Gao, 3D-QSAR study of corticotropin-releasing factor 1
antagonists and pharmacophore-based drug design, Neurochem. Int. 56 (2010)
107–117, https://doi.org/10.1016/j.neuint.2009.09.008.
[25] J.P. Beck, M.A. Curry, R.J. Chorvat, L.W. Fitzgerald, P.J. Gilligan, R. Zaczek, G.
L. Trainor, Thiazolo[4,5-d]pyrimidine thiones and -ones as corticotropin-releasing
hormone (CRH-R1) receptor antagonists, Bioorganic Med. Chem. Lett. 9 (1999)
1185–1188, https://doi.org/10.1016/S0960-894X(99)00159-6.
[26] B. Kuppast, K. Spyridaki, G. Liapakis, H. Fahmy, Synthesis of substituted
pyrimidines as corticotropin releasing factor (CRF) receptor ligands, Eur. J. Med.
Chem. 78 (2014) 1–9, https://doi.org/10.1016/j.ejmech.2014.03.040.
[27] 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, Eur. J. Med. Chem. 138 (2017) 900–908, https://doi.org/10.1016/j.
ejmech.2017.07.016.
[42] A. Waterhouse, M. Bertoni, S. Bienert, G. Studer, G. Tauriello, R. Gumienny, F.
T. Heer, T.A.P. De Beer, C. Rempfer, L. Bordoli, R. Lepore, T. Schwede, SWISS-
MODEL: Homology modelling of protein structures and complexes, Nucleic Acids
Res. 46 (2018) W296–W303, https://doi.org/10.1093/nar/gky427.
[43] S.S. Ebada, N.A. Al-Jawabri, F.S. Youssef, D.H. El-Kashef, T.O. Knedel, A. Albohy,
M. Korinek, T.L. Hwang, B.H. Chen, G.H. Lin, C.Y. Lin, S.M. Aldalaien, A.M. Disi,
C. Janiak, P. Proksch, Anti-inflammatory, antiallergic and COVID-19 protease
inhibitory activities of phytochemicals from the Jordanian hawksbeard:
Identification, structure-Activity relationships, molecular modeling and impact on
its folk medicinal uses, RSC Adv. 10 (2020) 38128–38141, https://doi.org/
10.1039/d0ra04876c.
[28] C.L. Graff, G.M. Pollack, Drug transport at the blood-brain barrier and the choroid
plexus, Curr. Drug Metab. 5 (2004) 95–108. http://www.ncbi.nlm.nih.gov/pub
med/14965253.
[29] V.A. Levin, Relationship of octanol/water partition coefficient and molecular
weight to rat brain capillary permeability, J. Med. Chem. 23 (1980) 682–684,
https://doi.org/10.1021/jm00180a022.
[30] H. Pajouhesh, G.R. Lenz, Medicinal chemical properties of successful central
nervous system drugs, NeuroRx. 2 (2005) 541–553, https://doi.org/10.1602/
neurorx.2.4.541.
[44] J. Huang, A.D. Mackerell, CHARMM36 all-atom additive protein force field:
Validation based on comparison to NMR data, J. Comput. Chem. 34 (2013)
2135–2145, https://doi.org/10.1002/jcc.23354.
[31] C.A. Lipinski, F. Lombardo, B.W. Dominy, P.J. Feeney, Experimental and
computational approaches to estimate solubility and permeability in drug
discovery and development settings, Adv. Drug Deliv. Rev. 46 (2001) 3–26. http
://www.ncbi.nlm.nih.gov/pubmed/11259830.
[45] V. Zoete, M.A. Cuendet, A. Grosdidier, O. Michielin, SwissParam: A fast force field
generation tool for small organic molecules, J. Comput. Chem. 32 (2011)
2359–2368, https://doi.org/10.1002/jcc.21816.
[32] A. Daina, O. Michielin, V. Zoete, SwissADME: a free web tool to evaluate
pharmacokinetics, drug-likeness and medicinal chemistry friendliness of small
molecules, Sci. Rep. 7 (2017) 42717, https://doi.org/10.1038/srep42717.
[33] S.H. Mohamed, H.R. Elgiushy, H. Taha, S.F. Hammad, N.A. Abou-Taleb, K.A.
M. Abouzid, H. Al-Sawaf, Z. Hassan, An investigative study of antitumor properties
of a novel thiazolo[4,5-d]pyrimidine small molecule revealing superior antitumor
activity with CDK1 selectivity and potent pro-apoptotic properties, Bioorganic
Med. Chem. 28 (2020), 115633, https://doi.org/10.1016/j.bmc.2020.115633.
[34] H. Ulrich, Cyclic Imidoyl Halides, in: Chem. Imidoyl Halides, 1st ed.,, Springer, US,
New York, NY, 1968, pp. 193–210, https://doi.org/10.1007/978-1-4684-8947-7_
8.
[46] P. Mark, L. Nilsson, Structure and dynamics of the TIP3P, SPC, and SPC/E water
models at 298 K, J. Phys. Chem. A. 105 (2001) 9954–9960, https://doi.org/
10.1021/jp003020w.
´
[47] M.J. Abraham, T. Murtola, R. Schulz, S. Pall, J.C. Smith, B. Hess, E. Lindah,
Gromacs: High performance molecular simulations through multi-level parallelism
from laptops to supercomputers, SoftwareX. 1–2 (2015) 19–25, https://doi.org/
10.1016/j.softx.2015.06.001.
[48] G. Bussi, D. Donadio, M. Parrinello, Canonical sampling through velocity rescaling,
J. Chem. Phys. 126 (2007), https://doi.org/10.1063/1.2408420.
[49] M. Parrinello, A. Rahman, Polymorphic transitions in single crystals: A new
molecular dynamics method, J. Appl. Phys. 52 (1981) 7182–7190, https://doi.org/
10.1063/1.328693.
[35] E.W. Bell, Y. Zhang, DockRMSD: An open-source tool for atom mapping and RMSD
calculation of symmetric molecules through graph isomorphism, J. Cheminform.
11 (2019), https://doi.org/10.1186/s13321-019-0362-7.
[50] T. Darden, D. York, L. Pedersen, Particle mesh Ewald: An N⋅log(N) method for
Ewald sums in large systems, J. Chem. Phys. 98 (1993) 10089–10092, https://doi.
org/10.1063/1.464397.
[36] Y.Y. Liao, J.C. Deng, Y.P. Ke, X.L. Zhong, L. Xu, R.Y. Tang, W. Zheng,
Isothiocyanation of amines using the Langlois reagent, Chem. Commun. 53 (2017)
6073–6076, https://doi.org/10.1039/c7cc02373a.
[51] R. Kumari, R. Kumar, A. Lynn, G-mmpbsa -A GROMACS tool for high-throughput
MM-PBSA calculations, J. Chem. Inf. Model. 54 (2014) 1951–1962, https://doi.
org/10.1021/ci500020m.
[37] K. Spyridaki, M.-T.T. Matsoukas, A. Cordomi, K. Gkountelias, M. Papadokostaki,
T. Mavromoustakos, D.E. Logothetis, A.N. Margioris, L. Pardo, G. Liapakis,
12