Development of Candidates for Positron Emission Tomography (PET) Imaging of Ghrelin Receptor in Disease: Design, Synthesis, and Evaluation of Fluorine-Bearing Quinazolinone Derivatives

Article abstract of DOI:10.1021/acs.jmedchem.7b01754

Molecular imaging with positron emission tomography (PET) is an attractive platform for noninvasive detection and assessment of disease. The development of a PET imaging agent targeting the ghrelin receptor (growth hormone secretagogue receptor type 1a or

Full text of DOI:10.1021/acs.jmedchem.7b01754

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Article
Development of Candidates for PET Imaging of
Ghrelin Receptor in Disease: Design, Synthesis and
Evaluation of Fluorine-bearing Quinazolinone Derivatives
Jin-Qiang Hou, Michael S. Kovacs, Savita Dhanvantari, and Leonard G. Luyt
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01754 • Publication Date (Web): 12 Jan 2018
Downloaded from http://pubs.acs.org on January 12, 2018
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Development of Candidates for PET Imaging of Ghrelin Receptor in Disease:
Design, Synthesis and Evaluation of Fluorine-bearing Quinazolinone
Derivatives
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Jinqiang Hou , Michael S. Kovacs , Savita Dhanvantari 2,3,4, Leonard G. Luyt1,2,5,*
1,2
2,3
1
2
London Regional Cancer Program, London, Canada, N6A 4L6 Lawson Health Research
3
Institute, London, Canada, N6C 2R5 Department of Medical Biophysics, University of Western
Ontario, London, Canada, N6A 3K7 Department of Pathology and Laboratory Medicine 5
Departments of Chemistry, Oncology and Medical Imaging, University of Western Ontario,
London, Canada, N6A 3K7.
4
Abstract
Molecular imaging with PET (Positron Emission Tomography) is an attractive platform for non-
invasive detection and assessment of disease. The development of a PET imaging agent targeting
the ghrelin receptor (growth hormone secretagogue receptor type 1a or GHS-R1a) has the
potential to lead to the detection and assessment of the higher than normal expression of GHS-
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R1a in diseases such as prostate, breast, and ovarian cancer. To enable the development of F
radiopharmaceuticals, we have designed and synthesized three series of quinazolinone
derivatives, resulting in the identification of two compound (5i, 17) with sub-nanomolar binding
affinity and one fluorine-bearing compound (10b) with picomolar binding affinity (20 pM),
representing the highest binding affinity for GHS-R1a reported to date. Two lead compounds (5b,
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IC50 = 20.6 nM; 5e, IC50 = 9.3 nM) were successfully F-radiolabeled with radiochemical purity
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of greater than 99%. Molecular modelling studies were performed to shed light on ligand-
receptor interactions.
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Introduction
Molecular imaging techniques, such as positron emission tomography, single photon emission
computed tomography, and magnetic resonance imaging, can allow for visualizing,
characterizing, and measuring important biological processes at the molecular and cellular levels
in vivo. Molecular imaging is rapidly being recognized as an effective method for non-invasive
detection and assessment of disease. Moreover, molecular imaging is believed to hold the
promise of personalized medicine due to its roles in choosing the right treatment for an
individual patient, guiding targeted therapies, and developing and optimizing new therapeutics.1
Among all of the imaging modalities, PET is one of the most promising imaging methods for use
in the clinic due to its high sensitivity and specificity, and has already provided a benefit to the
fields of oncology, neurology, and cardiology.2–4 In addition to patient care, PET imaging is
capable of providing important insights for medicinal chemists in drug discovery, in order to
reduce side effects arising from off-target binding of a drug candidate.5,6 However, successful
PET imaging depends on a validated radiopharmaceutical for clinically relevant targets. The
development of effective, specific, validated radiotracers targeting clinically relevant biomarkers
and their preparation methods are the most critical components to the expanding clinical
application of PET imaging.7,8
One such clinically relevant target, the growth hormone secretagogue receptor type 1a (GHS-
R1a), has potential therapeutic application towards a range of diseases due to its wide spectrum
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of biological functions.9–13 GHS-R has been identified in two isoforms thus far. GHS-R1a (also
referred to as ghrelin receptor) is one isoform and is known to be activated by its endogenous
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ligand, ghrelin, to induce growth hormone release, while GHS-R1b is a truncated version that
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doesn’t bind ghrelin and is functionally inactive. In addition to its therapeutic potential, GHS-
R1a has been found to be highly expressed in prostate16,17, breast , and ovarian cancer and has
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been identified as a biomarker related to cardiovascular disease . Therefore, the development of
a PET imaging probe targeting GHS-R1a has the potential to detect and assess the GHS-R1a
expression in diseases such as cancer and cardiovascular disease. Our group previously reported
on fluorine and rhenium containing ghrelin analogues for potential use in the imaging of GHS-
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R1a. The Beck-Sickinger group also reported on Ga-labelled peptidomimetics as radiotracers
for PET Imaging of GHS-R1a.22
Other than peptides and peptidomimetics, a class of small molecules, based upon a
quinazolinone scaffold, was initially developed by Bayer Pharmaceuticals for the treatment of
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diabetes and obesity by targeting GHS-R1a and offers a high affinity template as a starting
point for the development of PET imaging probes. The most potent compound synthesized by
Bayer (Figure 1B) possesses a Ki of 0.9 nM. A similar class of small molecules called
azaquinazolinone derivatives was further optimized by Prosidion Ltd (UK) to achieve better
lipophilicity. As a result, bioavailability was improved but the binding affinity for GHS-R1a was
reduced as compared to quinazolinone derivatives.24 Recently, Moldovan et.al. reported the
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synthesis of fluorinated azaquinazolinone derivatives. In 2011, Potter et.al. reported on a C
radiolabeled quinazolinone derivative for PET imaging of GHS-R1a in the brain of mice. The
studies showed that the imaging properties of the radioligand was not sufficient for GHS-R1a
imaging and radiotracers with stronger binding affinity and moderate lipophilicity are required in
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order to effectively image GHS-R1a in vivo. In the present study, we report on an extensive
structure-activity relationship investigation in order to develop fluorine-bearing
azaquinazolinone derivatives with low nanomolar binding affinity and moderate cLogD.
Molecular modelling studies were employed to develop a rationale for the SAR and provide
guidance for future development of quinazolinone-based molecules for GHS-R1a. Furthermore,
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a F-radiolabeling strategy was explored for the lead molecules in order to develop PET tracers
for potential use in the molecular imaging of GHS-R1a in disease.
Results and Discussion
Design, Synthesis and Evaluation of Quinazolinone Derivatives
The structure activity relationship (SAR) investigation on quinazolinone derivatives (Figure 1 A)
previously demonstrated that, (1) the piperidine substituent is essential for the binding affinity,23
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2
(
2) a bulky group in R position is essential for binding affinity, (3) alkyl groups in R position
affects the functional profiles of the small molecules,23 (4) decreasing the size of the R3
substituent gradually reduces binding affinity,23 (5) (S)-stereoisomers are significantly more
23
favorable than the (R)-isomers. The most potent compound (5f) previously discovered from
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this class of small molecules possesses a Ki of 0.9 nM (Figure 1B). Hanrahan et al. focused on
the modification of the lipophilicity of the central core by replacing the quinazolinone type
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structure with the azaquinazolinone as exemplified in Figure 1 C. The SAR showed that adding
one or two nitrogen atoms to the quinazolinone core at any position resulted in decreased binding
1
affinity, while modification in the R position provided the opportunity to improve the binding
affinity, resulting in ligand (20) with a Ki of 10 pM (Figure 1 D). For comparative purposes, we
prepared and evaluated 20 in our lab and determined the IC50 to be 0.71 nM, as determined by
radioligand binding assay.
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D
A
B
C
R2
O
N
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N
X = N
O
N
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S
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O
N
R1
N
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R1
R3
F
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N
-
+
20 IC50 = 0.71 0.19 nM
5f
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Figure 1. Various modifications to quinazolinone derivatives.
Our aim was to design and synthesize fluorine-containing compounds with strong binding
affinity for GHS-R1a that would be suitable for application as PET agents. Candidate molecules
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that exhibit strong binding affinity proceeded to radiolabelling using fluorine-18 ( F), a
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commonly used radioisotope with superior characteristics for PET imaging. Our initial effort
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was directed towards modifying the R and R substituents (Scheme 1) as suggested by the above
mentioned SAR information. In addition to the effort of improving the binding affinity, we have
considered the lipophilicity which is indicated by cLogP7.4. Generally speaking, less lipophilic
imaging agents result in more favorable biodistribution characteristics due to fast clearance and
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less blood protein binding. In addition, it has been shown that radiotracers with cLogD lower
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than 5 are able to cross the blood-brain barrier and thus we decided to maintain the lipophilicity
of the small molecules to cLogD < 5.
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NBoc
O
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Br
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B
r
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R
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H
HN
Br
i
ii
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NH2
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Methyl
Fluoro
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Methyl
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i
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Br
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R1
R1
N
N
N
N
N
R
R
R
R
R
5j Methyl
R
3
8
-
-
-
Methyl
Fluoro
a
e
a
4
4f M thyl
5
-
9a 9b Fluoro
0
a
10 Fl
uoro
c
1
Scheme 1. Synthesis of quinazolinone derivatives 4a-4f, 5a-5j, 9a-9b and 10a-10c. (i) a, Triethylamine,
o
dichloromethane, rt.; b, Acetic anhydride, 50 C. (ii)(R)-1-Boc-3-(aminomethyl) piperidine, toluene,
o
1
reflux. (iii) Ethylene glycol, LiOH, 150 C, microwave. (iv) a, HO- R , CuI, TMHD, Cs
2
CO , NMP,
3
2
1
20°C; b, 10% TFA, dichloromethane, rt.. (v); R -I, K
2
CO , 70°C.
3
23
Compounds 4a and 5f, reported previously, were synthesized and used as reference compounds.
Following scheme 1, a number of quinazolinone analogues were synthesized. 2-Amino-5-
bromobenzoic acid was treated with o-toluoyl chloride to form benzoxazinone 1. The addition of
BOC-protected aminomethylpiperidine led to uncyclized product 2, which was cyclized under
microwave irradiation to give compound 3. Ullmann coupling reactions were then performed
with aromatic hydroxy building block to form the intermediates, followed by BOC-deprotection
to furnish compound 4a-4f. Further N-alkylations on the piperidine nitrogen atoms using
nucleophilic substitution conditions furnished compound 5a-5j. The compounds were evaluated
using radioligand binding assay and the IC50 values are listed in Table 1.
st
Table 1. 1 series of quinazolinone derivatives with IC50 and cLogD7.4 values.
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R2
N
O
O
N
R1
N
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R1
R2
pIC50 a
cLog D7.4b
2.22
Compound
IC50 (nM)
c
F
F
4a
5a
4b
H
5.6 (0.9) 8.26 ± 0.06
F
29.5
52.0
7.63 ± 0.31
7.31 ± 0.14
4.50
N
F
H
H
H
0.90
3.40
1.24
3.53
1.24
N
F
F
F
F
5b
20.6
68.5
64.2
82.9
65.2
5.3
7.69 ± 0.07
7.17 ± 0.05
7.18 ± 0.03
7.09 ± 0.08
7.19 ± 0.08
8.30 ± 0.12
8.06 ± 0.15
N
N
4
5
c
c
F
OH
F
N
N
O
O
N
N
4
5
d
d
F
F
2.48
1.89
4.25
S
N
4
e
H
H
S
5e
9.3
N
O
N
N
4
5
f
f
587.0
6.24 ± 0.06
8.62 ± 0.43
1.62
3.32
c
F
3.7(0.9)
S
5g
1.8
18.2
0.4
8.87 ± 0.33
7.74 ± 0.03
9.46 ± 0.24
2.25
2.08
2.62
N
N
F
OH
OH
5
h
S
N
5
i
O
N
F
F
5
j
>1000
3.43
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Ghrelin (1-28)
3.3
8.53 ± 0.13
a, mean values ± SEM; , calculated using ACD Labs; ,the Ki values reported in the
b
c
literature.23
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The binding affinities of 4a and 5f, which both contain a fluorine atom, were determined to be
5
.6 nM and 3.7 nM. However, the radiolabeling of these compounds would be challenging as the
fluorine is attached to the non-activated arene in the absence of any electron-withdrawing group.
However, compound 4a has a secondary amine which could be used for the incorporation of a
fluoroethyl group. A two-step radiolabeling approach is commonly used for amine bearing small
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molecules. Firstly, ethylene ditosylate is treated with [ F ] to generate radioactive compound
[18
[18
F]2-fluoroethyl tosylate. In the second step, the amine bearing small molecules is reacted with
F]2-fluoroethyl tosylate via nucleophilic substitution to provide 18F-radiolabelled product
(
Scheme S1 method A). Therefore, compound 5a with a fluoroethyl group incorporated onto the
piperidine was synthesized. This compound can be readily radiolabeled using the two-step
approach, but the addition of the fluoroethyl group caused the IC50 to increase to 29.5 nM. To
reduce the cLog D7.4, compound 5b and 5c were synthesized by replacing the fluorophenyl with
fluoropyridinyl group. The IC50 of 5b was decreased to 20.6 nM while 5c was increased to 64.2
nM. A comparison of the IC50 values between 4b, 4c and 5b, 5c suggested that the incorporation
of fluoroethyl group on the piperidine has a slightly positive effect on the binding affinity. A
1
bulkier substituent than fluorophenyl in the R position is not beneficial for the binding affinity
as suggested by compound 4d, 5d, 4f. However, the benzothiazolyl group in the same position
improved the IC50 to 5.3 nM (4e). The incorporation of a fluoroethyl group on the piperidine
increased the IC50 of 5e to 9.3 nM. It has been demonstrated that a free amine on every studied
GHS-R1a ligand played an important role in binding with GHS-R1a. The positive charge at the
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amine being protonated in physiological condition is believed to establish ionic interactions with
Glu124, thus anchoring the ligands to the binding pocket of GHS-R1a.30–32 Consistently, the
introduction of the 2-fluoropropionyl group into piperidine diminished the binding affinity (5j).
This is probably due to the formation of an amide bond preventing the molecule from
protonation, thus not being able to anchor the ligand to Glu124 in the orthostatic binding pocket
GHS-R1a. Since previous studies suggest that incorporation of an isopropyl group on the
piperidine always provides a positive effect on the binding affinity, compound 5g was made and
was determined to possess an IC50 of 1.8 nM. In addition, compound 5h bearing a fluoroethyl
group has an IC50 of 18.2 while 5i has an IC50 of 0.4 nM, providing a first sub-nanomolar
binding affinity ligand.
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4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
The first round of modification resulted in two fluorine-bearing lead compounds 5b and 5e. Both
compounds then proceeded to the radiolabelling stage as described later, in the radiochemistry
section. These optimization steps also provided high-quality compounds 5g and 5i with low
nanomolar to sub-nanomolar binding affinities and optimal cLogD7.4. Unfortunately, these two
18
compounds don’t possess a fluorine atom in a position suitable for F radiolabelling. The second
round of structural modification then focused on incorporating a fluorine atom into the molecule,
while maintain the sub-nanomolar binding affinity with GHS-R1a.
Given the fact that the size of a methyl group is similar to that of fluorine, we decided to replace
the methyl group with a fluorine. Following the synthesis route as depicted in Scheme 1,
compounds 9a-b, 10a-c were made. Surprisingly, as compared to the methyl analog (4b, IC50
=
52.0 nM), the fluorine replacement dramatically reduced the binding affinity when there is
1
fluoropyridine (9a, IC50 = 935.2 nM) group in the R position (Table 2), but the effect was
1
minimal when there is benzothiazolyl group in the R position (9b, IC50 = 16.0 nM), as compared
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
to the corresponding methyl analog (4e, IC50 = 5.3 nM). Upon incorporation of an isopropyl
2
group in the R position, the binding affinity was significantly enhanced for compounds 10a
(IC50 = 82.3 nM) and 10b (IC50 = 0.02 nM). The fluorine bearing compound 10b, with picomolar
binding affinity and a cLogD7.4 suitable for brain penetration (2.39), appears to be the best
candidate for developing into a radiotracer. We thus synthesized the respective radiolabeling
precursor, with a nitro group available for substitution, as described in Scheme S2. However, the
radiolabeling was unsuccessful (Figure S1), probably due to the quinazolinone scaffold being a
weak electron withdrawing group and the existence of steric hindrance. To enable radiolabeling,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
compound 10c was made with fluoroethyl group in the R position, but the IC50 increased to 8.1
nM. These modifications provided one fluorine bearing compound (10b) with picomolar binding
affinity. To the best of our knowledge, this is the highest affinity ligand for GHS-R1a reported to
date.
nd
Table 2. The 2 series of quinazolinone derivatives with IC50 and cLogD7.4 values.
R2
N
O
O
N
F
R1
N
pIC50 a
cLog D7.4b
Compds.
R1
R2
IC50 (nM)
N
F
9
a
H
935.2
0.53
S
9
b
H
16.0
70.5
0.02
7.86 ± 0.23
7.16 ± 0.07
1.57
1.53
2.39
N
N
F
1
0a
S
N
1
0b
10.92 ± 0.32
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S
N
F
1
0c
8.1
8.15 ± 0.23
3.99
a, mean values ± SEM; , calculated using ACD Labs
b
Nucleophilic aromatic substitution (as described in Scheme S1, Method B) has been commonly
used for 18F-radiolabeling of aromatic systems, but it requires sufficient activation of the
aromatic ring, which can be achieved by electron withdrawing group such as carbonyl group in
the ortho or para position to the leaving group.6,33 The nitro group has been reported to be an
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
8
efficient leaving group for F nucleophilic aromatic substitution and a number of clinically used
1
8
F-radiopharmaceuticals (e.g., F-altanserin,34 18F-fluorodopamine ) have been synthesized by
18
35
aromatic nucleophilic substitution using nitro as a leaving group. Compound 4a and 5f have
similar binding affinity to that of natural ghrelin and a cLogD7.4 suitable for brain penetration as
listed in Table 1. The compound possesses fluorine, which can potentially be substituted by
18
radioactive F. To make the radiolabeling of the compounds feasible, the 4-fluorophenoxy was
replaced with fluorobenzoyl group. We hypothesized that the carbonyl would serve as electron
1
8
withdrawing group, thus facilitating the nucleophilic aromatic substitution with F anion. The
1
8
precursor with a nitro group in replacement of fluorine can be made readily for F radiolabelling.
Based on these considerations, compounds 13 and 14 were made following the synthesis route as
described in Scheme 2. Starting from compound 3, the aryl stannane 11 was made by treating 3
with bis(tributyltin), which was followed by a Stille coupling reaction to give compound 12.
Boc-deprotection was then performed to furnish compound 13, which was followed by
alkylation to provide compound 14. As expected, compound 14 has an IC50 of 3.8 nM, which is
equivalent to that of compound 5f (Table 3). Compound 13 also showed very strong binding
affinity (4.0 nM). Both compounds have a cLogD7.4 suitable for brain penetration as listed in
Table 3. We next sought to develop the radiolabeling precursor for compound 14. The precursor
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
with the nitro group in replacement of fluorine was synthesized (Scheme S3), but the
radiolabeling proceeded in extremely low yield (Figure S2). Given the similarity of a methylene
group to an ether linker, the methylene analog 17 was also synthesized (Scheme 2). Surprisingly,
the IC50 of 17 was determined to be 0.32 nM.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
NBoc
NBoc
NBoc
O
O
N
O
N
R1
i
ii
Br
Sn
N
N
N
N
12 A
15 B
1
1
3
R1
NH
N
N
O
O
v
O
N
iii
i
R1
R1
A
B
N
F
F
N
1
1
4 A
7 B
13 A
1
6 B
Scheme 2. Synthesis of quinazolinone derivatives 14 and 17. (i) (Bu
3
Sn) , toluene, 110°C. (ii)
2
PdCl (pph 4-fluorobenzoyl chloride/4-fluorobenzyl bromide, toluene, 110°C. (iii) TFA,
2
3
2
) ,
dichloromethane, rt.. (iv) 2-bromopropane, K
2
CO , 70°C.
3
rd
Table 3. The 3 series of quinazolinone derivatives with IC50 and cLogD7.4 values.
Compds. IC50 (nM)
pIC50 a cLog D7.4b
8.40 ± 0.01 1.82
8.43 ± 0.03 2.76
9.59 ± 0.29 4.80
1
1
1
3
4
7
4.0
3.8
0.3
a
b
, mean values ± SEM; , calculated using ACD Labs
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Ligand conformation analysis
1
We noticed that the methyl attached to the phenyl exhibited double peaks on HNMR spectrum
for all of the quinazolinone derivatives. However, VT (variable-temperature) NMR studies
showed that the double peak merged to one single peak when the temperature was increased to
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
00 °C as demonstrated by compound 4e (Figure S3), suggesting that the quinazolinone
derivatives may have more than one conformation. We also noticed that the methyl hydrogens
did not show a double peak until after the cyclization reaction, as demonstrated by compound 2
showing a single peak while compound 3 had double peaks for the methyl hydrogens. By
examining the 3D structure of the small molecule, we found that the piperidine encountered
steric clashes with the carbonyl in the quinazolinone main core as well as the phenyl group
(
Figure 2A), resulting in the single bond b
was trapped into two conformations during the cyclization step. Moreover, the steric clash
between the piperidine and the phenyl group resulted in the single bond b not being fully
1
not being fully rotatable. Therefore, the piperidine
2
rotatable, thus the methyl attached to the phenyl group may also be trapped into to two opposite
directions. Therefore, four conformers were proposed as shown in Figure 2B. These conformers
are referred to as C1, C2, C3 and C4. To investigate if the proposed conformers are stable ones,
an ab initio calculation was performed. The result showed that all proposed conformers are
1
stable with similar total energies (Table S1). Although the HNMR spectrum showed only two
peaks for the methyl, it is possible that these four conformers may exist at the same time, while
not being visible in the NMR spectrum.
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 2. The conformation study on compound 4e. A: The structure of compound 4e with single
bond b
C4 of 4e calculated by ab initio method; C: The front view of the conformers and the plots of
dihedral angle d (green), d (purple) and d (grey) versus MD simulation time.
1
, b
2
, b
3
and dihedral angle d
1
, d
2
, d indicated; B: Four stable conformers C1, C2, C3 and
3
1
2
3
To further demonstrate how the steric clashes among the piperidine, the carbonyl in the
quinazolinone main core, and the methyl phenyl group constrain the single bond b and b
(Figure 2A), molecular dynamics (MD) simulation was performed. To demonstrate the
rotatability of the single bond b , b and b , the dihedral angles (d , d and d ) over the course of
simulation were plotted and depicted in Figure 2C. For C1 and C3, the dihedral angle d
14
1
2
1
2
3
1
2
3
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o
fluctuates from roughly from -80 to -110 , while for C2 and C4 conformers, the dihedral angle
o
d
1
fluctuates from roughly 80 to 110 . The dihedral angle d
2
also experienced similar fluctuation
in a shallow range. The result indicates that the bond b
steric hindrance. As a reference, the dihedral angle d
what one would expect to see as the single bond b
showed that the bonds b and b are highly constrained due to the steric hindrances and the four
1
and b
2
are highly constrained due to the
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
o
3
fluctuates from -180 to 180 , which is
3
is freely rotatable. The MD study clearly
1
2
conformers are not convertible at room temperature.
Molecular docking study
In an attempt to understand the binding mode of quinazolinone derivatives, molecular docking
studies for compound 4e, 5e, 5g, 5i, and 10b were performed using a homology model of GHS-
36
36
R1a created recently based on the homologous GPCR structures (PDB ID: 4BWB, 3ZEV,
3
6
36
37
38 39
4
BUO, 4BV0, 4GRV, 4EA3 ). To include all the possible conformations of the small
molecules in the docking study, the single bond b and b are treated as freely rotatable bonds. A
1
2
large body of literature has reported that the amine on GHS-R1a ligands is important for binding
affinity as well as functional activity.30–32 An ionic interaction between the positively charged
terminal amine of the agonist and Glu124 on the receptor serves as an anchor point for binding.
The SAR of quinazolinone derivatives clearly demonstrated that the amine on the quinazolinone
derivatives played an important role in binding as evidenced by the conversion of amine to the
amide resulting in a dramatically reduced binding affinity (5j, IC50 > 1µM). The result is
consistent with the previous findings.30–32 Given the importance of the interaction between the
amine and Glu124, the docked poses where the positively charged amine of quinazolinone
derivatives is within 5 Å of the carboxylate of Glu124 were selected.
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9
1
1
1
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1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 3. The complex structures from molecular docking studies using a homology model of
GHS-R1a created based on the homologous GPCR structures (PDB ID: 4BWB,36 3ZEV,36
36
36
37
38
4
BUO, 4BV0, 4GRV, 4EA3 ). A: 4e (purple), 5e (red), 5g (yellow), 5i (green) and 10b
(
cyan) are superimposed and viewed from extracellular side; B: Side view of 4e (purple), 5e
(red), 5g (yellow), 5i (green) and 10b (cyan) in the GHS-R1a; 3D depiction of the binding mode
and surrounding residues for 4e (C), 5g (D), 5i (E), 10b (F). Ligands are shown in purple sticks;
the residues are shown in green sticks except for Glu123 that is shown in yellow. The insert
figures show the closest distances between the methyl carbon/fluorine of quinazolinone
derivatives and phenyl ring in Phe279.
In an attempt to investigate the binding modes of the small molecules with the benzothiazolyl
1
group in the R position (i.e., 4e, 5e, 5g, 5i, 10b), we found that all of these small molecules
displayed similar binding poses, as indicated in Figure 3A,B. Their docking poses are aligned
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well with the amine on the piperidine ring pointing to the carboxylate of Glu124. The small
molecules established strong pi-pi stacking interactions with Tyr106 and Phe279 (Figure
3C,D,E,F). It is worth mentioning that the hydroxyl group of 5i established three H-bonds with
His280, Met213 and Ser217, which may explain its sub-nanomolar binding affinity (Figure 3E).
For compound 10b with fluorophenyl in replacement of methylphenyl group, the fluorine is
pointing at the opposite direction as compared to methyl group in ligand 4e, 5e, 5g and 5i. As
shown in Figure 3C,D,E (insert figures), the methyl groups in all models are pointing away from
Phe279, implying steric hindrances posed by three hydrogens of the methyl group, while fluorine
is involved in the pi-pi stacking interaction with Phe279, as evidenced by a distance of 3.64 Å
(Figure 3F). The enhanced pi-pi stacking interaction with Phe279 may partially contribute to a
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
9
0-fold boost in the binding affinity of compound 10b (0.02 nM) as compared to 5g (1.8 nM). It
has been reported that, in many cases, fluorine substitution resulted in a dramatically increased
binding affinity.40,41 Although the contributions of fluorine substitution to the energetics of
ligand binding are poorly understood, it has been suggested that the boost in binding affinity may
have to do with the changes in desolvation or binding to a “fluorophilic” pocket.42
18F Radiochemistry
Compounds 5b and 5e obtained from the first round of optimization were also identified as lead
candidates as they have low nanomolar binding affinity and moderate cLogD7.4 values. The
radiolabelling through a two-step approach is described in Scheme 3. The bitosylate was first
made from tosylation of ethylene glycol. Nucleophilic substitution of the bi-tosylate with the
[18
[18
F-] anion acquired from the PET cyclotron led to the formation of radioactive compound
F]2-fluoroethyl tosylate. The reaction mixture was diluted with hexane/diethyl ether (3:1) and
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
passed through a Sep-Pak Silica Plus column (Waters) to obtain purified product. The non-decay
1
8
corrected radiochemical yield of [ F]2-fluoroethyl tosylate was around 50%.
OTs
OH
TsO
HO
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
M
eCN
-
1
8
+
F /K222/K
110 C 10 i
m n
o
,
18F
NH
N
N
O
O
N
18F
K CO
2
3
O
T
sO
O
N
R1
R1
M CN
e
o
,
1
40 C 20min
N
-
1
8
uoroe
F]2 fl thyl t yl t
os a e
R1
N
[
R
1
N
18F]5b
[
4
b
F
F
S
S
4e
18F]5
e
[
N
N
1
8
18
Scheme 3. Radio-synthesis of [ F]5b and [ F]5e.
Next, conditions for radiolabeling of 4b were explored including variables reaction solvent
(DMSO, MeCN), base (KHCO , K CO , Cs CO ), reaction time (15−30 min), reaction
temperature (100−150 °C), and precursor quantity (0.5-3 mg). The best conditions were
3
2
3
2
3
1
8
18
identified. The [ F]fluoroalkylation of amine precursor 4b using intermediate [ F]2-fluoroethyl
18
tosylate yielded the desired ligand [ F]5b with overall decay-corrected yield of 10.3 ± 0.8% and
18
high purity (≥99) (Table 4). Further evidence for successful labeling of 4b to [ F]5b was
provided by a co-injection of a pre-mixed solution of non-radioactive standard 5b and
1
8
radioactive ligand [ F]5b (Figure 4). Conditions for radiolabeling of 5e was also explored;
1
8
radioligand [ F]5e was synthesized with overall decay-corrected yields of 7.0 ± 0.6% and
18
greater than 99% purity. Further evidence for successful labeling of 4e to [ F]5e was also
provided by a co-injection of a pre-mixed solution of the non-radioactive standard 5e and
18
radioactive ligand [ F]5e (Figure 5).
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Table 4. Radiosynthesis Data
1
8
[18F]5e
Radioligand
Radiochemical yield (%) 10.3 ± 0.8 7.0 ± 0.6
Radiochemical purity (%)
≥99 ≥99
[ F]5b
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
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6
0
1
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5
6
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8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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5
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7
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0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Total synthesis time (min) 105 ± 1.7 127 ± 7.1
1000
8
00
00
6
400
200
0
0.4
0.3
0.2
0.1
.0
0
0
1
2
3
4
5
6
7
8
9
10
Time (min)
Figure 4, Stacked HPLC chromatograms (λ = 254 nm) (bottom line) and radio chromatograms
1
8
(
top line) for 5b and [ F]5b
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2
000
500
1
1000
5
00
0
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0
1
.0
.5
.0
0
0
0
1
2
3
4
5
6
7
8
9
10
Time (min)
Figure 5, Stacked HPLC chromatograms (λ = 254 nm) (bottom line) and radio chromatograms
18
(
top line) for 5e and [ F]5e
Conclusion
We have developed three series of fluorine-bearing quinazolinone derivatives with potential for
imaging of GHS-R1a in disease by PET. Their binding affinities with GHS-R1a were evaluated
using a radio ligand competitive binding assay. The SAR investigation resulted in the
identification of two compound (5i, 16) with sub-nanomolar binding affinity and one fluorine-
bearing compound (10b) with picomolar binding affinity (20 pM), representing the highest
binding affinity for GHS-R1a reported to date. A molecular modelling study was carried out to
provide insights into structure-activity relationships with GHS-R1a. Although there are many
1
8
reports in the literature of the nitro group being an efficient leaving group for F nucleophilic
aromatic substitution in the presence of an electron withdrawing group, our attempts to
radiolabel compound 14 and 10b using this strategy were not successful. Two other lead
compounds (5b, IC50 = 20.6 nM; 5e, IC50 = 9.3nM) with low nanomolar binding affinity for
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18
GHS-R1a were successfully F-radiolabeled with high radiochemical purity. Preliminary in vivo
studies are planned with a murine cancer model for both radiolabeled compounds. Further
1
8
development of F-radiolabeled compounds (10b) is currently underway in our laboratories and
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
will be reported in due course.
Experimental Section
All reagents were obtained from Sigma-Aldrich, Fisher Scientific, Ark Pharm Inc or Oakwood
Chemicals. The purity of the compounds was analyzed using analytical UHPLC. These results
are summarized in Table S2 and Figure S4, with all compounds determined to have ≥95% purity.
Acquity UHPLC H-Class system (Waters, Inc.) combined with a Xevo QTof mass spectrometer
was used for analytical UHPLC-MS work. Waters Acquity UHPLC BEH C18 column (2.1 x 50
mm, 1.7 μm) was used. High resolution mass spectra were obtained on a Thermo Scientific DFS
(
Double Focusing Sector) mass spectrometer. NMR data was obtained on the Varian Inova 400
1
25
MHz in methanol-d with the chemical shifts referenced to solvent signals. [ I]-Ghrelin for
4
binding assay was purchased from Perkin Elmer. Cyclotron produced [ F]H
18O was obtained
1
8
2
from the Nordal Cyclotron & PET Radiochemistry Facility at Lawson Health Research Institute,
London, Ontario, Canada. All Sep-Pak cartridges for radiochemistry were purchased from
Waters. Analytical radio-RP-HPLC was performed on a Waters 1525 Binary HPLC pump
containing a Waters 2487 dual λ absorbance detector and gamma detector. AgilentTM RP-C18
column (4.6 x 150 mm, 5 μm) was used for analytical analysis. Agilent RP-C18 column (19 x
1
50 mm, 5 μm) was used for semi-preparative HPLC purification.
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5
6
Synthesis of quinazolinone derivatives
6
-bromo-2-(o-tolyl)-4H-benzo[d][1,3]oxazin-4-one (1)
2
3
The compound was made according to literature procedures with some modifications. Toluoyl
chloride (10.0 g, 64.7 mmol) was added slowly into a solution of 5-bromoanthranilic acid (12.7 g,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
8.8 mmol) and triethylamine (24.6 mL, 176.4 mmol) in dichloromethane (120 mL) at 0 °C. The
reaction mixture was stirred at rt for 16 h. After the solvent was removed under reduced pressure,
water (80ml) and dichloromethane (80ml) were added into the resulting product (yellow oil). The
organic layer was washed with additional 80ml of water, dried over Na
anhydride (50 mL) was added in to the filtrate and the solution was heated at 50 °C for 3 h. The
reaction mixture was cooled to rt, diluted with dichloromethane. Saturated NaHCO was added
slowly. The layer was separated and the organic layer was washed with brine, dried over Na SO
2
SO
4
and filtered. Acetic
3
2
4
and filtered. The filtrate was concentrated under reduced pressure and the crude product was
treated with ethanol and stirred for 10mins. The solid was filtered and washed with additional
ethanol, and then air dried for 3 h to give 13 g of the product in 70% yield. UHPLC-MS (waters)
method: 5-90% acetonitrile (0.1% TFA) in water (0.1% TFA), 3 min run, RT (min) 2.58, calcd
+
m/z 316.0 (MH ), found m/z 316.9.
(R)-tert-butyl
3-((5-bromo-2-(2-methylbenzamido)benzamido)methyl)piperidine-1-
carboxylate (2)
The compound was made according to literature procedure with some modifications.23
Compound 1 (3g, 9.5mmol) was dissolved in toluene (20ml). (R)-1-Boc-3-(aminomethyl)
piperidine (2.4 g, 11.4 mmol) was added and the solution was refluxed at 110 °C for 8h. The
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resulting solution was cooled down to rt. The solvent was removed by around 90% and hexane
(
20ml) was added. The precipitate was filtered out and washed with additional 10ml hexane to
give 4.9g pure product in 97% yield. UHPLC-MS (waters) method: 50-90% acetonitrile (0.1%
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
TFA) in water (0.1% TFA), 3 min run, RT (min) 2.19, calcd m/z 530.2 (MH ), found m/z 530.1.
(
R)-tert-butyl
3-((6-bromo-4-oxo-2-(o-tolyl)quinazolin-3(4H)-yl)methyl)piperidine-1-
carboxylate (3)
Compound 2a (2.50 g, 4.72 mmol) was added to microwave reaction vessel followed by ethylene
glycol (12 mL) and LiOH (0.22mg, 9.17 mmol). The resulting mixture was subjected to
microwave irradiation with stirring for 30 minutes at 150 °C, cooled to rt, and diluted with
dichloromethane (50ml) and water (50ml). The organic layer was washed with brine (50 mL)
twice. The combined organic layer was dried over Mg
pressure. Purification via silica gel column chromatography using a gradient elution from 25 to
0% ethyl acetate in hexanes to yield 1.93g the product in 82% yield. UHPLC-MS (waters)
2
SO and then concentrated under reduced
4
5
method: 50-90% acetonitrile (0.1% TFA) in water (0.1% TFA), 3 min run, RT (min) 1.86, calcd
+
m/z 512.2 (MH ), found m/z 512.1.
(
S)-6-(4-fluorophenoxy)-3-(piperidin-3-ylmethyl)-2-(o-tolyl)quinazolin-4(3H)-one (4a)
23
The compound was made according to literature procedure with some modifications. Under a
nitrogen atmosphere, compound 3a (512 mg, 1.00 mmol), copper(I) iodine (95 mg, 0.05 mmol),
2
,2,6,6-tetramethylheptane-3,5-dione (TMHD) (46 mg, 0.25 mmol), 4-fluorophenol (168mg,
.5mmol) and Cs CO (652 mg, 2.00 mmol) were added to 1-methyl-2-pyrrolidone (2 mL). The
1
2
3
reaction mixture was stirred for 16 h at 120 °C, cooled, and filtered through Celite. The filtrate
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1
1
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1
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2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
was concentrated under reduced pressure and purified via silica gel column chromatography
using a gradient elution from 10 to 20% ethyl acetate in hexanes to give 300mg of the product in
55% yield. The above product (300 mg, 0.55 mmol) was dissolved in dichloromethane (5 mL),
followed by TFA (2 mL). The solution was stirred for 4 h at rt. The solvent was removed and
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
saturated aqueous NaHCO
The combined organic layers were dried with Na
under reduced pressure, and the crude compound was purified by reverse phase chromatography
Isolera One, HS-C18-30g cartridge) with a gradient from 5 to 60 % MeOH in water. It was
3
solution was added. The product was then extracted with EtOAc (2×).
2
SO and filtered, and the solvent was removed
4
(
lyophilized to give desired product as white powder in 85% yield. UHPLC-MS (waters) method:
5-95% acetonitrile (0.1% TFA) in water (0.1% TFA), 3 min run, RT (min) 1.65. HRMS (ES+)
+
1
for C27
H27FN
3
O
2
(MH ), calcd 444.2087, found 444.2094. H NMR (400 MHz, methanol-d ) δ
4
ppm 7.69 (d, J = 8.9 Hz, 1H), 7.60 (d, J = 2.7, 1H), 7.54 (dd, J = 8.9, 2.7 Hz, 1H), 7.46-7.50 (m,
2H), 7.41-7.37 (m, 2H), 7.22 – 7.10 (m, 4H), 4.19 – 3.93 (m, 1H), 3.64 – 3.35 (m, 1H), 2.86-2.83
(
m, 1H), 2.80-2.62 (m, 1H), 2.46-2.37 (m, 1H), 2.23 – 2.07 (m, 4H), 1.80-1.72 (m, 1H), 1.60-
19
1
.27 (m, 3H), 1.06 – 0.90 (m, 1H). F NMR (376 MHz, methanol-d ) δ ppm -118.76 – -118.66
4
(
m)
(
(
S)-6-((5-fluoropyridin-2-yl)oxy)-3-(piperidin-3-ylmethyl)-2-(o-tolyl)quinazolin-4(3H)-one
4b)
The method similar for the preparation of compound 4a was used, except replacing 4-
fluorophenol with 5-fluoro-2-pyridinol. The Boc-protected intermediate was purified via silica
gel column chromatography using a gradient elution from 20 to 30% ethyl acetate in hexane and
gave 44% yield. After Boc deprotection, the crude compound was purified by reverse phase
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chromatography (Isolera One, HS-C18-30g cartridge) with a gradient from 30 to 100 % MeOH
in water. It was lyophilized to give the product in 67% yield. UHPLC-MS (waters) method: 5-95%
+
acetonitrile (0.1% TFA) in water (0.1% TFA), 3 min run, RT (min) 2.01. HRMS (ES ) for
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
1
C
26
H
26FN
4
O
2
(MH ), calcd 445.2040; found 445.2046. H NMR (400 MHz, methanol-d
.04 (d, J = 3.2 Hz, 1H), 7.90 (d, J = 2.7 Hz, 1H), 7.75 – 7.68 (m, 2H), 7.62 (dd, J = 8.9, 2.7 Hz,
1H), 7.53 – 7.46 (m, 2H), 7.44 - 7.37 (m, 2H), 7.15 (dd, J = 8.9, 3.2 Hz, 1H), 4.23 – 3.99 (m, 1H),
4
) δ ppm
8
3
.66 – 3.37 (m, 1H), 2.90 – 2.65 (m, 2H), 2.41 (qd, J = 11.9, 2.8 Hz, 1H), 2.27 – 2.07 (m, 4H),
.85 – 1.72 (m, 1H), 1.64 – 1.24 (m, 3H), 1.10 – 0.90 (m, 1H).
1
(S)-6-((6-fluoropyridin-3-yl)oxy)-3-(piperidin-3-ylmethyl)-2-(o-tolyl)quinazolin-4(3H)-one
(
4c)
The method similar for the preparation of compound 4a was used, except replacing 4-
fluorophenol with 6-fluoropyridin-3-ol. The Boc-protected intermediate was purified via silica
gel column chromatography using elution of 30% ethyl acetate in hexane and gave 44% yield.
After Boc deprotection, the crude compound was purified via aluminum oxide column
chromatography using a gradient elution from 2 to 5% methanol in dichloromethane to give the
product in 95% yield. UHPLC-MS (waters) method: 5-95% acetonitrile (0.1% TFA) in water
+
+
(
0.1% TFA), 3 min run, RT (min) 1.25. HRMS (ESI ) for C26
H26FN
4
O (MH ), calcd 445.2040,
2
1
found 445.2047. H NMR (400 MHz, methanol-d
4
) δ ppm 8.07 (d, J = 2.9 Hz, 1H), 7.79 – 7.72
(
m, 2H), 7.70 (d, J = 2.9 Hz, 1H), 7.63 (dd, J = 8.9, 2.9 Hz, 1H), 7.53 – 7.46 (m, 2H), 7.44 – 7.37
m, 2H), 7.17 (dd, J = 9.2, 2.9 Hz, 1H), 4.22 – 4.00 (m, 1H), 3.65 – 3.38 (m, 1H), 2.86 (d, J =
2.0 Hz, 1H), 2.82 – 2.64 (m, 1H), 2.43 (q, J = 12.0 Hz, 1H), 2.28 – 2.08 (m, 4H), 1.83 – 1.73 (m,
1H), 1.62 – 1.24 (m, 3H), 1.06 – 0.93 (m, 1H).
(
1
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2
2
2
2
2
2
2
2
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3
3
3
3
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3
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4
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6
(
S)-6-((6-((5-fluoropyridin-2-yl)oxy)pyridin-3-yl)oxy)-3-(piperidin-3-ylmethyl)-2-(o-
tolyl)quinazolin-4(3H)-one (4d)
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Compound 4d was synthesized as a side product during the preparation of compound 4c with a
yield of 34%. UHPLC-MS (waters) method: 10-90% acetonitrile (0.1% TFA) in water (0.1%
+
+
TFA), 3 min run, RT (min) 1.48. HRMS (ESI ) for C31
H29FN
5
O (MH ), calcd 538.2254, found
3
1
5
–
38.2255. H NMR (400 MHz, methanol-d
4
) δ ppm 8.10 (s, 1H), 7.99 (d, J = 2.9 Hz, 1H), 7.86
7.79 (m, 1H), 7.72 (d, J = 8.8 Hz, 2H), 7.64 (d, J = 2.9 Hz, 1H), 7.61 (dd, J = 8.8, 2.9 Hz, 1H),
7.52 – 7.45 (m, 2H), 7.44 – 7.36 (m, 2H), 7.20 (d, J = 8.8 Hz, 1H), 7.13 (dd, J = 8.8, 2.9 Hz, 1H),
4
2
.20 – 3.96 (m, 1H), 3.65 – 3.36 (m, 1H), 2.84 (d, J = 11.9 Hz, 1H), 2.81 – 2.62 (m, 1H), 2.47 –
.34 (m, 1H), 2.25 – 2.06 (m, 4H), 1.81 – 1.69 (m, 1H), 1.60 – 1.24 (m, 3H), 1.04 – 0.92 (m, 1H).
(
S)-6-(benzo[d]thiazol-6-yloxy)-3-(piperidin-3-ylmethyl)-2-(o-tolyl)quinazolin-4(3H)-one (4e)
The method similar for the preparation of compound 4a was used, except replacing 4-
fluorophenol with 6-bezothiazolol. The Boc-protected intermediate was purified via silica gel
column chromatography using an elution of 25% ethyl acetate in hexane and gave 14% yield.
After Boc deprotection, the crude compound was purified via reverse phase chromatography
(
Isolera One, HS-C18-30g cartridge) with a gradient from 30 to 100 % MeOH in water. The
product was lyophilized to give white power in 90% yield. UHPLC-MS (waters) method: 5-95%
+
acetonitrile (0.1% TFA) in water (0.1% TFA), 3 min run, RT (min) 1.43. HRMS (ESI ) for
+
1
C
28
H
27
N
4
O
2
S (MH ), calcd 483.1855, found 483.1855. H NMR (400 MHz, methanol-d
4
) δ ppm
9.23 (s, 1H), 8.12 (d, J = 8.9 Hz, 1H), 7.82 (d, J = 2.3 Hz, 1H), 7.75 (dd, J = 8.9, 1.4 Hz, 1H),
7
.71 – 7.68 (m, 1H), 7.67 – 7.61 (m, 1H), 7.54 – 7.47 (m, 2H), 7.46 – 7.39 (m, 2H), 7.35 (dd, J =
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.9, 2.3 Hz, 1H), 4.34 – 4.03 (m, 1H), 3.78 – 3.32 (m, 1H), 3.26 – 3.09 (m, 2H), 2.89 – 2.78 (m,
H), 2.77 – 2.51 (m, 1H), 2.29 – 2.18 (m, 3H), 2.10 – 1.94 (m, 1H), 1.88 – 1.76 (m, 1H), 1.63 –
1
1.31 (m, 2H), 1.23 – 1.05 (m, 1H).
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
S)-6-(4-(4-acetylpiperazin-1-yl)phenoxy)-3-(piperidin-3-ylmethyl)-2-(o-tolyl)quinazolin-
(3H)-one (4f)
4
The method similar for the preparation of compound 4a was used, except replacing 4-
fluorophenol with 1-acetyl-4-(4-hydroxyphenyl)piperazine. The Boc-protected intermediate was
purified via reverse phase chromatography (Isolera One, HS-C18-30g cartridge) with a gradient
from 0 to 100 % MeOH in water and gave 7% yield. After Boc deprotection, the crude
compound was purified via reverse phase chromatography (Isolera One, HS-C18-30g cartridge)
with a gradient from 8 to 80 % MeOH in water. The product was lyophilized to give white power
in 90% yield. UHPLC-MS (waters) method: 5-95% acetonitrile (0.1% TFA) in water (0.1%
+
+
TFA), 3 min run, RT (min) 1.38. HRMS (ESI ) for C33
H38FN
5
O (MH ), calcd 552.2975, found
3
1
5
52.2968. H NMR (400 MHz, methanol-d
4
) δ ppm 7.68 (d, J = 9.3 Hz, 1H), 7.59 – 7.52 (m,
2H), 7.52 – 7.46 (m, 2H), 7.46 – 7.38 (m, 2H), 7.13 – 7.02 (m, 4H), 4.34 – 4.01 (m, 1H), 3.78 –
3
.34 (m, 5H), 3.25 – 3.07 (m, 6H), 2.89 – 2.51 (m, 2H), 2.27 – 2.18 (m, 3H), 2.18 – 2.13 (m, 3H),
.02 (s, 1H), 1.89 – 1.76 (m, 1H), 1.63 – 1.32 (m, 2H), 1.23 – 1.06 (m, 1H).
2
(S)-3-((1-(2-fluoroethyl)piperidin-3-yl)methyl)-6-(4-fluorophenoxy)-2-(o-tolyl)quinazolin-
(3H)-one (5a)
4
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Compound 4a (160 mg, 0.36 mmol), 1-fluoro-2-idoethane (313 mg, 1.80 mmol), and K
2
CO
3
(
200 mg, 1.45 mmol) were combined in acetonitrile (10ml) and heated to 70 °C for 8 h. The solid
was filtered off, and the filtrate was concentrated under reduced pressure. Saturated aqueous
NaHCO solution was added, and the product was extracted with EtOAc (2×). The combined
organic layers were dried with Na SO and filtered. The solvent was removed under reduced
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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9
0
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0
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7
8
9
0
3
2
4
pressure, and the crude product was purified via silica gel column chromatography (2% MeOH
in dichloromethane) to give 80mg of the product in 45% yield. UHPLC-MS (waters) method:
0-60% acetonitrile (0.1% TFA) in water (0.1% TFA), 3 min run, RT (min) 1.93. HRMS (ES+)
2
+
1
for C29
H
30
F
2
N
3
O
2
(MH ), calcd 490.2306, found 490.2308. H NMR (400 MHz, methanol-d ) δ
4
ppm 7.70 (d, J = 8.9 Hz, 1H), 7.62 (d, J = 2.9 Hz, 1H), 7.56 (dd, J = 8.9, 2.9 Hz, 1H), 7.51 –
7
4
.46 (m, 2H), 7.42 – 7.37 (m, 2H), 7.23 – 7.11 (m, 4H), 4.56 – 4.46 (m, 1H), 4.44 – 4.35 (m, 1H),
.14 – 4.04 (m, 1H), 3.57 – 3.43 (m, 1H), 2.84 – 2.51 (m, 4H), 2.23 (d, J = 2.5 Hz, 3H), 2.00 –
1.84 (m, 2H), 1.82 – 1.22 (m, 4H), 0.93 – 0.76 (m, 1H).
(
S)-3-((1-(2-fluoroethyl)piperidin-3-yl)methyl)-6-((5-fluoropyridin-2-yl)oxy)-2-(o-
tolyl)quinazolin-4(3H)-one (5b)
The method similar for the preparation of compound 5a was used, except replacing compound
4
a with 4b. The compound was purified by reverse phase chromatography (Isolera One, HS-
C18-30g cartridge) with a gradient from 30 to 100 % MeOH in water. The product was
lyophilized to give white power in 72% yield. UHPLC-MS (waters) method: 5-95% acetonitrile
+
(
(
0.1% TFA) in water (0.1% TFA), 3 min run, RT (min) 1.46. HRMS (ESI ) for C28
H
29
F
2
N
4
O
2
+
1
MH ), calcd 491.2259, found 491.2260. H NMR (400 MHz, methanol-d ) δ ppm 7.99 (d, J =
4
3.2 Hz, 1H), 7.86 (d, J = 2.7 Hz, 1H), 7.71 – 7.63 (m, 2H), 7.57 (dd, J = 8.8, 2.7 Hz, 1H), 7.50 –
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.42 (m, 2H), 7.39 – 7.33 (m, 2H), 7.10 (dd, J = 9.0, 3.2 Hz, 1H), 4.48 (dd, J = 11.5, 5.3 Hz, 1H),
.36 (dd, J = 11.5, 5.3 Hz, 1H), 4.14 – 4.01 (m, 1H), 3.58 – 3.41 (m, 1H), 2.75 (t, J = 11.0 Hz,
1H), 2.71 – 2.45 (m, 3H), 2.20 (d, J = 2.1 Hz, 3H), 1.90 (t, J = 11.0 Hz, 2H), 1.80 – 1.21 (m, 4H),
1
1
1
1
1
1
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1
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2
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4
4
4
4
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5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
19
0
2
.92 – 0.71 (m, 1H). F NMR (376 MHz, methanol-d
4
) δ ppm -134.69 – -135.69 (m, 1F), -
17.01 – -218.50 (m, 1F)
(
S)-3-((1-(2-fluoroethyl)piperidin-3-yl)methyl)-6-((6-fluoropyridin-3-yl)oxy)-2-(o-
tolyl)quinazolin-4(3H)-one (5c)
The method similar for the preparation of compound 5a was used, except replacing compound
4
a with 4c. The crude compound was purified by reverse phase chromatography (Isolera One,
HS-C18-30g cartridge) with a gradient from 5 to 100 % MeOH in water. The product was
lyophilized to give white power in 45% yield. UHPLC-MS (waters) method: 5-95% acetonitrile
+
(
0.1% TFA) in water (0.1% TFA), 3 min run, RT (min) 1.39. HRMS (ESI ) for C28
H
29
F
2
N
4
O
2
+
1
(MH ), calcd 491.2259, found 491.2259. H NMR (400 MHz, methanol-d
4
) δ ppm 8.07 (d, J =
3
7
4
.0 Hz, 1H), 7.78 – 7.72 (m, 2H), 7.69 (d, J = 2.9 Hz, 1H), 7.62 (dd, J = 8.9, 2.9 Hz, 1H), 7.52 –
.46 (m, 2H), 7.44 – 7.37 (m, 2H), 7.16 (dd, J = 8.8, 3.0 Hz, 1H), 4.56 – 4.46 (m, 1H), 4.46 –
.33 (m, 1H), 4.11 (dt, J = 13.9, 7.0 Hz, 1H), 3.64 – 3.43 (m, 1H), 2.86 – 2.53 (m, 4H), 2.23 (d, J
=
2.9 Hz, 3H), 2.01 – 1.84 (m, 2H), 1.85 – 1.23 (m, 4H), 0.93 – 0.75 (m, 1H). 19F NMR (376
MHz, methanol-d
4
) δ ppm -73.85 (s, 1F), -217.04 – -218.61 (m, 1F)
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