Tricyclic heterocycles display diverse sensitivity to the A147T TSPO polymorphism

Article abstract of DOI:10.1016/j.ejmech.2020.112725

The 18 kDa translocator protein (TSPO) is a target for the development of imaging agents to detect neuroinflammation. The clinical utility of second-generation TSPO ligands has been hindered by the presence of a polymorphism, rs6971, which causes a non-co

Full text of DOI:10.1016/j.ejmech.2020.112725

Journal Pre-proof
Tricyclic heterocycles display diverse sensitivity to the A147T TSPO polymorphism
Renee Sokias, Eryn L. Werry, Hei Wun Alison Cheng, James H. Lloyd, Greta Sohler,
Jonathan J. Danon, Andrew P. Montgomery, Jonathan J. Du, Quanqing Gao, David
E. Hibbs, Lars M. Ittner, Tristan A. Reekie, Michael Kassiou
PII:
S0223-5234(20)30697-8
DOI:
https://doi.org/10.1016/j.ejmech.2020.112725
EJMECH 112725
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 29 June 2020
Revised Date: 2 August 2020
Accepted Date: 3 August 2020
Please cite this article as: R. Sokias, E.L. Werry, H.W. Alison Cheng, J.H. Lloyd, G. Sohler, J.J.
Danon, A.P. Montgomery, J.J. Du, Q. Gao, D.E. Hibbs, L.M. Ittner, T.A. Reekie, M. Kassiou, Tricyclic
heterocycles display diverse sensitivity to the A147T TSPO polymorphism, European Journal of
Medicinal Chemistry, https://doi.org/10.1016/j.ejmech.2020.112725.
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O
N
N
N
Heterocyclic
Scaffold
NH
O
A147T TSPO = 159.4 nM
WT TSPO = 76.8 nM
A147T/WT = 2.1
A147T TSPO = 132.0 nM
WT TSPO = 53.0 nM
A147T/WT = 2.5
MeO
N
N
N
N
HN
N
N
A147T TSPO = 199.5 nM
WT TSPO = 41.3 nM
A147T/WT = 4.8
A147T TSPO = 27.3 nM
WT TSPO = 9.3 nM
A147T/WT = 2.9

Tricyclic heterocycles display diverse sensitivity to the A147T TSPO polymorphism
Renee Sokias,#§ Eryn L. Werry,#§ Hei Wun Alison Cheng,◊ James H. Lloyd,# Greta Sohler,# Jonathan J.
Danon,# Andrew P. Montgomery,# Jonathan J. Du,∆ Quanqing Gao,∆ David E. Hibbs,∆ Lars M. Ittner,φ
Tristan A. Reekie# and Michael Kassiou.#*
#School of Chemistry, Faculty of Science and ◊School of Medical Sciences, Faculty of Medicine and
Health, ∆Sydney School of Pharmacy, Faculty of Medicine and Health, The University of Sydney, NSW,
Australia, 2006 φDepartment of Biomedical Sciences, Faculty of Medicine and Health Sciences, 2
Technology Place, Macquarie University, NSW, Australia, 2109
Abstract
The 18 kDa translocator protein (TSPO) is a target for the development of imaging agents to detect
neuroinflammation. The clinical utility of second-generation TSPO ligands has been hindered by the
presence of a polymorphism, rs6971, which causes a non-conservative substitution of alanine for
threonine at amino acid residue 147 (TSPO A147T). Given the complex nature of TSPO binding, and
the lack of non-discriminating high-affinity ligands at both wild type and A147T forms of TSPO, a
series of novel TSPO ligands containing various heterocyclic scaffolds was developed to explore the
pharmacophoric drivers of affinity loss at TSPO A147T. In general,
N-benzyl-N-methyl-substituted
amide ligands showed increased affinity at TSPO A147T, and a pyrazolopyrimidine acetamide
containing this motif displayed low nanomolar binding affinities to both TSPO forms.
Keywords
TSPO, microglia, polymorphism, docking, non-discriminating, structure-activity relationships.
1

Introduction
The 18 kDa translocator protein (TSPO) is a five transmembrane domain protein expressed
ubiquitously throughout the body.1 TSPO expression is predominant in steroid-synthesising tissues
within the periphery.2, 3 Minimal TSPO expression occurs in the non-pathological central nervous
system (CNS),3 but expression is upregulated in activated glia in areas damaged by
neurodegenerative diseases.1, 3 Molecular imaging using TSPO positron emission tomography (PET)
ligands can image areas affected by neuroinflammation in preclinical and clinical studies of
neurodegenerative diseases.4 Furthermore, the upregulation of the TSPO within cancerous cells has
attracted considerable attention as a potential biomarker for neoplastic growth or as a novel protein
target for chemotherapy.5, 6
Development of TSPO ligands has been hindered by a single nucleotide polymorphism (SNP;
rs6971) in the TSPO that results in the replacement of an alanine (Ala; A) with a threonine (Thr; T) at
amino acid residue 147 (A147T).7, 8 Second-generation TSPO PET ligands such as DPA-713, XBD-173
and PBR-28 show three distinct binding patterns to tissue from human donors: high-, mixed-, and
low-affinity binding. The most common TSPO form, Ala/Ala, is associated with a high-affinity binding
phenotype, Ala/Thr leads to mixed-affinity binding, while Thr/Thr displays low-affinity binding.7, 8 The
TSPO protein’s conformational change resulting from this amino acid substitution significantly
influences binding of second-generation TSPO ligands.7-10 The presence of the differing binding
affinities, particularly in patients who are low-affinity binders, complicates the quantitative
assessment of PET data using second-generation ligands. TSPO PET images of low-affinity binders
have a significantly lower signal-to-noise ratio, and the clinical usefulness of this approach is
therefore limited in these patients.11 The first-generation ligand PK-11195 (1) does not show loss of
affinity at A147T TSPO, however its clinical use is complicated by high non-specific binding, high
plasma protein binding and low blood-brain barrier permeability.12 The closely related 4-azaisostere
(2) did not show loss in affinity at A147T TSPO in membranes prepared from human tissue,10 but a
three-fold reduction in PET binding potential in low-affinity binders compared to high-affinity
binders.13 Given this, a TSPO PET ligand that binds with an equally high-affinity to both A147T and
wild type TSPO in vivo is currently elusive.
Discovery of new ligands with improved binding profiles would be facilitated by an
understanding of pharmacophoric features that influence discrimination between wild type and A147T
TSPO. In reported structure-activity relationship (SAR) studies, pendant acetamide moieties were
demonstrated to be key structural requirements for wild type TSPO affinity across various ligand
classes.14 Moreover, the optimum binding affinity to wild type TSPO required two lipophilic
substituents on the terminal acetamide.14, 15 Although aliphatic substituents on the terminal acetamide
are widely reported, recent SAR studies demonstrated that a
enhancing wild type TSPO affinity.15, 16
N-benzyl substituent is crucial for
Additionally, given that numerous ligand classes can bind the TSPO, efforts have also been
directed toward determining whether the heterocyclic scaffold interacts with the binding sites present
on both TSPO isoforms. Scaffolds including carbazoles (3), pyrazolopyrimidines (4),
pyrazolobenzodiazepines (5), and dibenzodiazepines (6) were explored, based on their reported
bioactivity, high affinity, bioavailability, and their synthetic accessibility.17-19
The work herein explores the effect of the heterocyclic scaffold on TSPO binding, while
simultaneously exploring the effect of the acetamide substituents. We report that for ligands which
bind to TSPO, discrimination at A147T TSPO can be reduced by inclusion of an
substitution on the acetamide motif in most cases.
N
-benzyl-N-methyl
2

Figure 1: Previously reported TSPO ligands 1–2,10 320 and novel TSPO ligands 4–6.
Results and Discussion
Chemistry. Carbazole acetamide ligands 3a–3d have been previously reported20 and were
synthesised according to Scheme 1. Initial
alkylating agent ( -benzyl-2-bromoacetamide or ethyl bromoacetate), in the presence of sodium
hydride afforded intermediates 8 and 9 in excellent yields (81–86%). Subsequent -methylation or
-ethylation of 8 with methyl or ethyl iodide, in the presence of potassium tert-butoxide, furnished
N-alkylation of 9H-carbazole (7) with the appropriate
N
N
N
target ligands 3a and 3b in excellent yields (87–90%). Hydrolysis of methyl ester 9, under basic
conditions, afforded carboxylic acid 10 in good yield (72%). HBTU-mediated coupling of 10 with
diethylamine or dimethylamine furnished ligands 3c and 3d in good yields (76–83%).
N-benzyl-2-
bromoacetamide was synthesised from benzylamine and 2-bromoacetyl bromide (see Experimental
section).
3

Scheme 1. Synthetic route for the carbazole acetamide ligands.a
aReagents and Conditions: a) i) THF, NaH, 0 °C, 30 min; ii)
N-benzyl-2-bromoacetamide or ethyl
bromoacetate, 0 °C–rt, 2 h, 81–86%; b) KOtBu, appropriate iodoalkane, THF, 0 °C–rt, 17 h, 87–90%;
c) LiOH, THF/H2O, ∆, 4 h, 72%; d) HBTU, DIPEA, dimethylamine, DMF, rt, 16 h, 83%.
Pyrazolopyrimidine acetamide ligands 4a–4d were synthesised according to Scheme 2, while
synthesis of ligand 4c was previously reported.21 Commercially available methyl p-anisate (11) was
treated with sodium methoxide, followed by acetonitrile to generate cyanoacetophenone 12.
Deprotonation of 12 to the corresponding enolate, followed by treatment with tert-butyl 2-
bromoacetate, gave compound 13. Condensation of compound 13 with hydrazine monohydrate
afforded the aminopyrazole 14. Further condensation of aminopyrazole 14 with acetylacetone,
followed by basic hydrolysis of the tert-butyl ester furnished the pyrazolopyrimidine core 15. Amide
coupling with the appropriate secondary amine furnished the desired pyrazolopyrimidine acetamide
ligands 4a–4d.
4

Scheme 2. Synthetic route for the pyrazolopyrimidine acetamide ligands.a
aReagents and Conditions: a) MeCN, NaOMe, PhMe, ∆, 18 h, 44%; b) NaOH, tert-butyl 2-
bromoacetate, EtOH, rt, 24 h, 57%; c) NH2NH2.H2O, cat. AcOH, EtOH, ∆, 13 h, 49%; d) i)
Acetylacetone, EtOH, ∆, 5 h; ii) NaOH, EtOH, ∆, 12 h, 92% (over 2 steps); e) PyBOP, DIPEA, NHR1R2,
DMF, 55–86%.
The pyrazolobenzodiazepine acetamide ligands were synthesised from 16,22 according to Scheme 3.
Compound 16 was N-alkylated with the appropriate alkylating agent in the presence of triethylamine
and furnished target pyrazolobenzodiazepine acetamide ligands 5a–5d.
Scheme 3. Synthesis of the pyrazolobenzodiazepine acetamide ligands.a
O
NR1R2
HN
5a R1 = Me, R2 = Bn
a
5b R1 = Et, R2 = Bn
N
N
5c R1 = R2 = Et
N
H
N
5d R1 = R2 = Me
N
N
N
H
16
aReagents and Conditions: a) Et3N, THF, appropriate alkylating agent, rt, 19 h, 76–81%.
The dibenzodiazepine acetamide ligands were synthesised according to Scheme 4. The synthesis of
key dibenzodiazepine intermediate 21 was achieved by first preparing intermediate 19 via
a
palladium-catalysed Buchwald-Hartwig cross-coupling reaction. Subsequent catalytic hydrogenation
with palladium on carbon (Pd/C) afforded intermediate 20. Base-mediated hydrolysis of the methyl
ester to the carboxylic acid followed by amide coupling furnished the desired dibenzodiazepine
intermediate 21. Dibenzodiazepine acetamide ligands 6a–6d were obtained by N-alkylation of 21
with the appropriate alkylating agent in the presence of potassium tert-butoxide.
5

Scheme 4. Synthetic route for the dibenzodiazepine acetamide ligands.a
O
O
O
O
O
O
I
a
b
H
H
N
N
H2N
NO2
NO2
NH2
17
18
19
20
c,d
O
NR1R2
H
6a R1 = Me, R2 = Bn
6b R1 = Et, R2 = Bn
6c R1 = R2 = Et
N
N
e
6d R1 = R2 = Me
HN
HN
21
O
O
aReagents and Conditions: a) Pd(OAc)2, BINAP, Cs2CO3, PhMe, 90 °C, 17 h, 77%; b) Pd/C (10%
w/w), EtOAc, H2 (1 atm), rt, 6 h, 92%; c) LiOH, MeOH/H2O, ∆, 6 h; d) HBTU, DIPEA, DMF, 10 min,
82% (over 2 steps); e) KOtBu, appropriate alkylating agent, THF, 0 °C–rt, 18 h, 83–87%.
Assessment of TPSO binding affinity. The previously reported carbazole acetamide series of
ligands (3a–d) provide an important comparison and are discussed here briefly.20 Ligand 3a,
featuring a methyl and benzyl group at the R1 and R2 positions displayed moderate binding to both
TSPO isoforms (132.0 nM and 53.0 nM for A147T and WT, respectively). Substituting the methyl
group at the R1 position with an ethyl group (3b) decreased binding at both isoforms (459.6 nM and
91.6 nM for A147T and WT, respectively). Ligand 3c, with ethyl substituents at the R1 and R2 position
increased binding at the TSPO wild type (30.1 nM) and showed moderate binding affinity at the TSPO
A147T (176.6 nM). Binding affinity was completely nullified at both TSPO isoforms for ligand 3d,
which features methyl substituents at both R1 and R2 positions. Examining the A147T-to-wild type
ratio shows that the carbazole acetamide series showed discrimination to both TSPO isoforms,
although the degree of discrimination was smallest with the N-benzyl-N-methyl substituent.
The second series of ligands (4a–4d) contained a pyrazolopyrimidine scaffold and examined
the same modifications at the R1 and R2 positions. Ligand 4a, which featured a methyl substituent at
the R1 position and a benzyl group at the R2 position, displayed strong binding at both TSPO isoforms
(27.3 nM and 9.3 nM for A147T and WT, respectively). Substituting the R1 group with an ethyl group
(4b) selectively decreased binding at the A147T TSPO isoform (80.6 nM) without impacting affinity at
wild type TSPO. Ligand 4c, featuring an ethyl substituent at both the R1 and R2, displayed decreased
binding at both isoforms (98.8 nM and 19.5 nM for A147T and WT, respectively), compared to the
benzyl-substituted analogues (4a, 4b). Binding affinity dropped off greatly with incorporation of an
N,N-dimethyl substituent (4d) into the pyrazolopyrimidine scaffold. The pyrazolopyrimidine acetamide
series again showed discrimination between TSPO isoforms.
The third series of ligands (5a–5d) contained a pyrazolobenzodiazepine scaffold and
modifications at the R1 and R2 positions. Ligand 5a, which featured a methyl and benzyl substituent
at the R1 and R2 positions displayed the strongest, or equal strongest, binding affinity at both TSPO
isoforms (199.5 nM and 41.3 nM for A147T and WT, respectively) within the pyrazolobenzodiazepine
6

series, although TSPO isoform discrimination was still observed. Incorporation of an
N-benzyl-N-ethyl
R1R2 group into the scaffold (5b) had little impact on affinity compared to 5a, but substituting the
R1R2 group with an
N,N-diethyl group (5c) decreased binding at both TSPO isoforms. Replacement
of the R1R2 group with an N,N-dimethyl group (5d) abolished binding at both isoforms.
The fourth series of ligands (6a–6d) contained a dibenzodiazepinone scaffold and
modifications at the R1 and R2 positions. The best in this series, ligand 6b, featured an ethyl
substituent at the R1 and a benzyl group at the R2 position, displayed moderate binding affinity at
A147T TSPO (99.4 nM) and strong binding to the wild type TSPO (15.8 nM). A comparison between
ligands 6a–6c showed that by substituting the R1 group with a methyl group (6a) or R2 group with
an ethyl group (6c) decreased binding at both TSPO isoforms. This observation differed from the
pyrazolobenzodiazepine series (5), where the N-benzyl-N-methyl substituted acetamide was more
favourable over the N-benzyl-N-ethyl. However, ligand 6a still displayed moderate binding affinities to
both TSPO isoforms (159.4 nM and 76.8 nM for A147T and WT, respectively), and displayed the least
discrimination between the two TSPO forms throughout the dibenzodiazepinone series.
Drawing all this together, two trends regarding the influence of the R1R2 group within each
scaffold series appear. Firstly, with the exception of the pyrazolobenzodiazepine scaffold, decoration
with an
Secondly, incorporation of an
N
-benzyl-N-methyl R1R2 group imparts the least discrimination between TSPO WT and A147T.
N,N-dimethyl group imparts poor binding at both forms of TSPO (3–
6d). The binding pocket that the R1R2 groups access in TSPO is lipophilic23 so it is possible that the
more lipophilic R1R2 groups in the a–c analogues within each scaffold form stronger interactions with
the binding pocket that those of the d analogues. These results are congruent with previous studies
that hypothesize the necessity of a fully occupied lipophilic pocket with a bulky
greater TSPO affinity.24
N-alkyl group for
Drawing trends across scaffolds was more complex. Comparing each R1,R2 substitution across
scaffolds revealed that the pyrazolopyrimidine scaffold afforded the highest affinity at each isoform of
TSPO. However, the complex nature of the heterocycle on binding was revealed by comparing
ligands 3a–c and 5a–c. For
N-benzyl-N-methyl derivatives (3a vs 5a), the pyrazolobenzodiazepine
scaffold was more favourable at the wild type and less favourable at the A147T TSPO isoform,
compared to the carbazole scaffold. In contrast, the pyrazolobenzodiazepine scaffold was more
favourable a both forms of TSPO in
favourable at both TSPO isoforms in
the influence of the heterocycle on affinity is dependent on the identity of the R1 and R2 substituents.
N-benzyl-N-ethyl-substituted derivatives (3b vs 5b), and was less
N,N
-diethyl-substituted derivates (3c vs 5c). This suggests that
7

Table 1. TSPO binding affinities (Ki) using membranes from TSPO wild type and A147T over-
expressing HEK-293T cells. Values represent the mean ± SEM from at least three independent
experiments performed in duplicate.
N
R1 R2
K
i (nM)
A147T:
Wild Type
A147T
Wild Type
O
3a Me Bn 132.0 ± 8.0
3b Et Bn 459.6 ± 83.0
3c Et Et 176.6 ± 41.6
3d Me Me >10 000
53.0 ± 7.0
91.6 ± 1.0
30.1 ± 3.6
>10 000
2.5
5.0
5.9
N/A
NR1R2
N
4a Me Bn 27.3 ± 3.6
4b Et Bn 80.6 ± 4.3
4c Et Et 98.8 ± 2.3
4d Me Me 559.8 ± 59.3
9.3 ± 2.3
2.9
13.0
5.1
6.2 ± 1.0
19.5 ± 3.5
337.9 ± 29.1
1.7
O
5a Me Bn 199.5 ± 10.5
5b Et Bn 226.9 ± 30.7
41.3 ± 8.1
41.3 ± 8.6
4.8
5.5
4.6
N/A
NR1R2
N
5c Et Et 2256.6 ± 335.6 493.0 ± 45.2
5d Me Me >10 000
>10 000
N
N
N
H
O
6a Me Bn 159.4 ± 17.6
6b Et Bn 99.4 ± 9.8
76.8 ± 16.2
15.8 ± 1.3
2.1
6.2
NR1R2
N
6c Et Et 2357.8 ± 412.4 200.5 ± 29.8
6d Me Me >10 000 >10 000
11.8
N/A
NH
O
Docking Studies. Docking studies were used to examine how each heterocyclic scaffold binds to
both WT and A147T TSPO. As the -benzyl-N-methyl series, in general, showed the least
N
discrimination between A147T and wild type TSPO, ligands 3a, 4a, 5a and 6a were chosen for
docking studies. Docking and MM-GBSA ∆G binding scores (obtained by combining molecular
mechanics (MM) terms with a generalised Born and surface area (MM-GBSA) solvent model)25 are
summarised in Table 2.
8

Table 2. Docking and MM-GBSA ∆G binding scores and key interacting residues for ligands docked
into the chosen receptors via extra precision (XP) docking. Residues in blue interact with the ligand
only in the wild type model and residues in green interact with the ligand only in the A147T model.
docking scorea
(kcal/mol)
MMGBSA ∆G Bindb
Ligand
Interacting residuesc
(kcal/mol)
A147T
WT
A147T
WT
3a
4a
-9.602
-5.248
-9.706
-75.43
-67.37
Trp53, Trp95, Phe99, Trp143, Arg24
Arg24, Tyr34, Trp53, Trp95, Phe100,
Trp143
-7.668
-9.479
-85.77
-74.44
-71.98
-92.64
-9.541
5a
Tyr34, Trp53, Trp143, Arg24, Trp95
Ser23, Phe99, Arg24, Trp53, Tyr57, Asn92,
Trp95, Trp143
6a
-12.885
-9.601
-81.22
-77.24
aDocking scores obtained from extra precision Glide docking are an estimate of protein-ligand binding
energies. More negative scores indicate more favourable binding interactions
bMM-GBSA binding energies are approximate free energies of binding. More negative scores indicate
more favourable binding interactions.
cKey interactions residues are taken from the 2D ligand interaction function of Maestro v12.1.
Ligands 3a and 5a had similar docking scores for both WT and A147T TSPO, while a difference of
∼2.5 kcal/mol and ∼3.3 kcal/mol were observed for ligands 4a and 6a respectively. For both 4a and
6a the binding conformation observed was similar in both WT and A147T TSPO. The differences in
the docking scores for both ligands can be attributed to the pyrazolopyrimidine and dibenzodiazepine
scaffolds being slightly distorted in A147T TSPO by Thr147 when compared to the WT. The MM-GBSA
binding scores were similar for each ligand between the WT and A147T TSPO.
Binding poses for each ligand are shown in Figure 2. Each ligand exhibited binding poses where π–π
interactions are observed with Trp53 and Trp95 in both WT and A147T TSPO (5a WT being the only
exception). This network of π–π interactions with the heterocyclic scaffolds appears to be crucial in
orienting the ligands within the predicted binding site. In addition, hydrogen bond interactions of the
acetamide oxygen of each ligand with the indolic NH of Trp143 and Trp153 was also conserved in
both WT and A147T TSPO. The lower Ki observed for the pyrazolopyrimidine scaffold 4a in
comparison to the other heterocyclic scaffolds can be rationalised by the additional π–π interactions
observed in both the WT and A147T TSPO. This interaction is between Phe100 and the
4-methoxyphenyl group at the 2-position of pyrazolopyrimidine scaffold.
9

A147T TSPO
WT TSPO
3a
4a
10

11

5a
6a
Figure 2. 3D and 2D ligand binding depictions of TSPO ligands 3a, 4a, 5a and 6a into the chosen
receptors. Purple lines represent hydrogen bonding, green lines represent π–π stacking and red lines
represent π-cation bonds. The crystal structure of TSPO protein from Bacilus Cereus (PDB ID: 4RYI),
was used as the template for homology modelling.
12

Conclusion
Taken together, these findings highlight the effect the heterocyclic scaffold has on the binding affinity
at both TSPO isoforms. The pyrazolopyrimidine scaffold (4) remains a promising scaffold for TSPO
ligands. Although lipophilic bulk on N,N-disubstituted acetamide heterocyclic scaffolds is essential to
ensure binding to wild type and A147T TSPO, lowered discrimination at A147T TSPO is predominantly
seen with
N-benzyl-N-methyl ligands, compared to N,N-diethyl and N-benzyl-N-ethyl ligands. This
knowledge can be used to inform further development of TSPO ligands with equally high-affinity at
both the TSPO wild type and A147T for use in diagnosis and treatment of neurodegenerative
disorders and several cancer types.
Experimental section
General Experimental
Unless otherwise stated, all solvents and reagents were purchased from commercial sources and used
without further purification. All reagents were weighed out under ambient conditions. All reactions
were performed under nitrogen or argon. Nuclear magnetic resonance spectra were recorded on
1
Bruker Advance DRX 300, 400 or 500 Ascend spectrometers at 300, 400 or 500 MHz H NMR
frequency, 75, 101 or 151 MHz 13C NMR frequency and 282 MHz 19F NMR frequency. The NMR
spectra for several target compounds showed the presence of rotamers as a result of the amide
functionality. For the sake of simplicity, the NMR signals for only one rotamer are reported. Low-
resolution mass spectra were performed on a Finnigan LCQ mass spectrometer. High-resolution mass
spectra were performed on a Bruker 7T Apex Qe Fourier Transform Ion Cyclotron resonance mass
spectrometer equipped with an Apollo II ESI/APCI/MALDI Dual source. Elemental microanalysis was
obtained from the School of Human Sciences at London Metropolitan University, England. High
performance liquid chromatography (HPLC) was performed on the Waters Alliance 2695 apparatus
equipped with Waters 2996 photodiode array detector, set at 254 nm. HPLC data are recorded as
percentage purity and retention time (RT) in minutes. Separation using a SunFireTM C18 column (5
μm, 2.1 x 150 mm) was achieved using water (solvent A) and acetonitrile (solvent B) at a flow rate of
0.2 mL/min. The method consisted of 0% B to 100% B over 30 minutes.
Carbazole Acetamide Ligands
The spectroscopic data for synthesised compounds 8–10 and 3a, 3b and 3d matched previously
reported data.26
The spectroscopic data for synthesised compound 3c matched previously reported data.26
Pyrazolopyrimidine Acetamide Ligands
The spectroscopic data for synthesised compounds 12,13,14 matched previously reported data.27
The spectroscopic data for synthesised compounds 4c matched previously reported data.21
2-(2-(4-methoxyphenyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl)acetic acid (15)
To a solution of 14 (1.52 g, 5.01 mmol, 1.0 equiv.) in ethanol (20 mL) was added acetylacetone
(560 µL, 5.51 mmol, 1.1 equiv.). The reaction mixture was heated to reflux for 5 hours. Sodium
hydroxide (400 mg, 10.02 mmol, 2.0 equiv.) was added, and the reaction mixture heated at reflux for
a further 12 hours. The reaction mixture was cooled to ambient temperature and evaporated to
dryness. The residue was then diluted in dichloromethane/isopropanol (50 mL, 1:1 v/v) and washed
with aqueous hydrochloric acid (1 M, 20 mL). The organic layer was collected, dried over anhydrous
magnesium sulfate and concentrated in vacuo. The desired product 15 was obtained as a colourless
1
solid. Yield 92%. H NMR (300 MHz, CDCl3) δ = 12.37 (br s, 1H), 7.72 (d,
(d,
J = 8.7 Hz, 2H), 7.07
J
= 8.7 Hz, 2H), 6.86 (s, 1H), 3.81 (s, 2H), 3.70 (s, 3H), 2.71 (s, 3H), 2.51 (s, 3H); 13C NMR (75
MHz, CDCl3) δ = 172.67, 159.46, 157.75, 153.15, 147.25, 144.64, 129.08, 125.71, 114.11, 108.56,
13

99.22, 55.18, 28.78, 24.19, 16.25; LRMS (-ESI) 310 [(M - H)- 100%]; Found: C, 65.63; H, 5.44; N,
13.46 Calc for C17H17N3O3: C, 65.58; H, 5.50; N, 13.50%.
General Procedure for the Synthesis of 4a–4b and 4d:
A solution of carboxylic acid intermediate 15 (100 mg, 0.321 mmol, 1.0 equiv.), appropriate
secondary amine (1.1 equiv.), triethylamine (50 µL, 0.353 mmol, 1.1 equiv) and PyBOP (180 mg,
0.353 mmol, 1.1 equiv.) in N,N-dimethylformamide (1 mL) was stirred at ambient temperature for 5
hours. The reaction mixture was then diluted with ethyl acetate (10 mL) and saturated aqueous
ammonium chloride (10 mL). The organic layer was collected, washed with water (2 x 10 mL) and
brine (1 x 10 mL). The combined organic extracts were dried over anhydrous magnesium sulfate and
concentrated in vacuo. Unless otherwise stated, purification was achieved by flash column
chromatography, followed by recrystallisation.
N-Benzyl-2-(2-(4-methoxyphenyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl)-N-methylacetamide
(4a)
General procedure was followed using N-benzylmethylamine (45 μL, 0.353 mmol). Purification by
flash chromatography (100% ethyl acetate) and recrystallisation from ethyl acetate/hexane furnished
4b as a yellow crystals. Yield 51%. 1H NMR (500 MHz, CDCl3) δ = 7.80–7.75 (m, 2H), 7.31–7.21
(m, 4H), 7.13–7.11 (m, 1H), 7.00–6.96 (m, 2H), 6.51 (s, 1H), 4.62 (s, 2H), 3.99 (s, 2H), 3.85 (s, 3H),
3.05 (s, 3H), 2.74 (s, 3H), 2.54 (s, 3H); 13C NMR (151 MHz, CDCl3) δ = 171.63, 159.92, 157.70,
155.16, 147.71, 144.94, 137.62, 136.83, 130.05, 128.65, 128.58, 128.15, 127.31, 127.29, 126.35,
126.25, 114.10, 108.20, 100.53, 55.39, 51.39, 35.37, 28.62, 24.73, 17.02; LRMS (+ESI) 437 [(M +
Na)+ 100%], 415 [(M + H)+ 60%]; HPLC τR = 24.61 min (98.2% purity).
N-Benzyl-N-ethyl-2-(2-(4-methoxyphenyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl)acetamide (4b)
General procedure was followed using N-ethylbenzylamine (50 μL, 0.353 mmol). Purification by flash
chromatography (100% ethyl acetate) and recrystallisation from ethyl acetate/ hexane furnished 4b
1
as a yellow solid. Yield 48%. H NMR (500 MHz, CDCl3) δ = 7.72–7.69 (m, 2H), 7.22–7.13 (m,
4H), 7.04–7.02 (m, 1H), 6.93–6.88 (m, 2H), 6.44 (s, 1H), 4.57 (s, 2H), 3.94 (s, 2H), 3.78 (s, 3H),
3.41 (q,
J = 7.1 Hz, 2H), 2.68 (s, 3H), 2.47 (s, 3H), 1.10 (t, J
= 7.1 Hz, 3H); 13C NMR (126 MHz,
CDCl3) δ = 171.23, 159.97, 157.69, 155.23, 147.86, 144.92, 138.25, 137.37, 130.13, 128.60,
128.53, 128.19, 127.21, 126.46, 126.37, 126.27, 114.15, 108.33, 100.86, 55.46, 48.52, 42.17, 28.11,
24.79, 17.07, 13.96; LRMS (+ESI) 451 [(M + Na)+ 100%], 429 [(M + H)+ 60%]; HPLC τR = 25.86
min (97.7% purity).
2-(2-(4-Methoxyphenyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl)-N,N-dimethylacetamide (4d)
General procedure was followed using dimethylamine hydrochloride (58 mg, 0.707 mmol, 2.2 equiv.)
and triethylamine (130 µL, 0.964 mmol, 3.0 equiv.). Purification by flash chromatography
1
(methanol/ethyl acetate 1:9 v/v) furnished 4d as a yellow solid. Yield 80%. H NMR (300 MHz,
CDCl3) δ = 7.75 (d,
J = 8.2 Hz, 2H), 6.98 (d, J = 8.2 Hz, 2H), 6.49 (s, 1H), 3.91 (s, 2H), 3.84 (s,
3H), 3.15 (s, 3H), 2.97 (s, 3H), 2.72 (s, 3H), 2.52 (s, 3H); 13C NMR (75 MHz, CDCl3) δ = 171.17,
159.91, 157.64, 155.17, 147.67, 144.88, 130.03, 126.35, 114.12, 108.32, 100.47, 55.41, 37.78,
36.04, 28.48, 24.76, 17.01; LRMS (+ESI) 361 [(M + Na)+ 100%], 339 [(M + H)+ 50%]; HPLC τR =
19.71 min (96.7% purity).
Pyrazolobenzodiapezine Acetamide Ligands
General Procedure for the Synthesis of 5a–5d:
To a solution of 16 (100 mg, 0.499 mmol, 1.0 equiv.) in tetrahydrofuran (1 mL) was added
triethylamine (140 µL, 0.999 mmol, 2.0 equiv.), followed by the dropwise addition of the appropriate
14

alkylating agent (1.1 equiv.). The reaction mixture was stirred at ambient temperature for 19 hours.
The reaction mixture was evaporated to dryness and diluted with ethyl acetate (5 mL) and water
(2 mL). The organic layer was collected, and the aqueous layer was further extracted with ethyl
acetate (2 x 5 mL). The combined organic extracts were washed with aqueous hydrochloric acid (1 M,
2 mL), followed by water (2 mL). The organic layer was dried over magnesium sulfate and
concentrated in vacuo. Purification by flash chromatography on silica gel column using
methanol/dichloromethane (5:95 v/v) furnished the desired pyrazolobenzodiazepine ligands.
N-Benzyl-N-methyl-2-(1-methyl-4,10-dihydrobenzo[b]pyrazolo[3,4-e][1,4]diazepin-5(1H)-
yl)acetamide (5a)
General procedure was followed using alkylating agent
N-benzyl-2-bromo-N-methylacetamide
(133 mg, 0.549 mmol). The desired product 5a was isolated as a beige powder. Yield 85%. 1H NMR
(300 MHz, CDCl3) δ = 7.28–7.25 (m, 3H), 7.14–6.87 (m, 7H), 5.90 (s, 1H), 4.59 (s, 2H), 4.04 (s,
2H), 3.88 (s, 2H), 3.70 (s, 3H), 2.98 (s, 3H); 13C NMR (75 MHz, CDCl3) δ = 169.81, 140.24,
138.74, 137.36, 136.63, 130.88, 128.69, 128.42, 128.03, 126.41, 125.84, 122.44, 119.26, 101.19,
57.09, 50.75, 47.75, 38.22, 34.36; LRMS (+ESI) 384 ([M + Na]+ 100%); Found: C, 69.83; H, 6.47;
N, 19.36. Calc for C21H23N5O: C, 69.78; H, 6.41; N, 19.38%.
N-Benzyl-N-ethyl-2-(1-methyl-4,10-dihydrobenzo[b]pyrazolo[3,4-e][1,4]diazepin-5(1H)-yl)acetamide
(5b)
General procedure was followed using alkylating agent
N-benzyl-2-bromo-N-ethylacetamide (141 mg,
0.549 mmol). The desired product 5b was isolated as a beige powder. Yield 83%. Rƒ 0.63
1
(methanol/dichloromethane, 1:9 v/v); H NMR (300 MHz, CDCl3) δ = 7.29–7.21 (m, 3H), 7.14–
6.81 (m, 7H), 5.90 (s, 1H), 4.62 (s, 2H), 4.12 (s, 2H), 3.92 (s, 2H), 3.70 (s, 3H), 3.36 (t, J = 7.1 Hz,
2H), 1.07 (q,
J
= 7.1 Hz, 3H); 13C NMR (75 MHz, CDCl3) δ = 170.82, 141.16, 140.22, 138.88,
137.25, 135.96, 128.20, 128.02, 127.14, 126.34, 125.35, 121.47, 119.32, 100.89, 56.02, 50.04,
47.59, 41.49, 34.98, 13.81; LRMS (+ESI) 398 ([M + Na]+ 100%); Found: C, 70.26; H, 6.69; N,
18.71. Calc for C22H25N5O: C, 70.38; H, 6.71; N, 18.65%.
N,N-Diethyl-2-(1-methyl-4,10-dihydrobenzo[b]pyrazolo[3,4-e][1,4]diazepin-5(1H)-yl)acetamide (5c)
General procedure was followed using alkylating agent 2-chloro-N,N-diethylacetamide (75 µL,
1
0.549 mmol). The desired product 5c was isolated as a beige powder. Yield 86%. H NMR (300
MHz, CDCl3) δ = 7.24 (s, 1H), 6.92–6.70 (m, 4H), 6.03 (s, 1H), 4.69 (s, 2H), 3.97 (s, 2H), 3.63 (s,
3H), 3.34 (q,
J = 7.4 Hz, 4H), 1.03 (t, J
= 7.4 Hz, 6H); 13C NMR (75 MHz, CDCl3) δ = 168.90,
142.07, 138.57, 137.66, 131.36, 126.80, 125.09, 122.58, 118.85, 101.17, 56.90, 50.16, 41.20, 40.66,
34.66, 13.61, 12.33; LRMS (+ESI) 336 ([M + Na]+ 60%), 314 ([M + H]+ 100%); Found: C, 65.11;
H, 7.33; N, 22.38. Calc for C17H23N5O: C, 65.15; H, 7.40; N, 22.35%.
N,N-Dimethyl-2-(1-methyl-4,10-dihydrobenzo[b]pyrazolo[3,4-e][1,4]diazepin-5(1H)-yl)acetamide (5d)
General procedure was followed using alkylating agent 2-chloro-N,N-dimethylacetamide (70 mg,
0.549 mmol). The desired product 5d was isolated as a colourless powder. Yield 83%. 1H NMR (300
MHz, CDCl3) δ = 7.26 (s, 1H), 6.91–6.71 (m, 4H), 5.83 (s, 1H), 4.61 (s, 2H), 3.90 (s, 2H), 3.68 (s,
3H), 3.01 (s, 6H); 13C NMR (75 MHz, CDCl3) δ = 169.85, 140.23, 138.43, 137.12, 131.41, 126.57,
125.22, 122.26, 119.30, 101.26, 57.14, 50.39, 36.71, 36.12, 34.59. LRMS (+ESI) 308 ([M + Na]+
50%), 286 ([M + H]+ 100%); Found: C, 63.09; H, 6.68; N, 24.60. Calc for C15H19N5O: C, 63.14; H,
6.71; N, 24.54.
Dibenzodiazepine Acetamide Ligands
Methyl 2-((2-nitrophenyl)amino)benzoate (19)
15

A suspension of 2-iodonitrobenzene (17) (1.00 g, 4.02 mmol, 1.0 equiv.), methyl anthranilate (18)
(520 µL, 4.02 mmol, 1.0 equiv.), BINAP (250 mg, 0.402 mmol, 10 mol%) and caesium carbonate
(2.62 g, 8.03 mmol, 2.0 equiv.) in toluene (20 mL) was degassed with argon sparging for 10
minutes. Palladium acetate (45 mg, 0.201 mmol, 5 mol%) was added under argon. The reaction
mixture was stirred at 80 °C for 18 hours. The reaction mixture was cooled to ambient temperature
and evaporated to dryness. The crude material was diluted with ethyl acetate and filtered through a
pad of Celite®. The pad of Celite® was washed with ethyl acetate and the combined filtrates
concentrated in vacuo. Purification by flash column chromatography on silica gel using ethyl
1
acetate/hexane (1:9 v/v) furnished 19 as an orange powder. Yield 77%. H NMR (400 MHz,
CDCl3) δ = 11.13 (br s, 1H), 8.19 (dd, J = 8.4, 1.6 Hz, 1H), 8.06 (dd, J = 7.9, 1.7 Hz, 1H), 7.67–
7.57 (m, 1H), 7.55–7.49 (m, 1H), 7.46 (ddt, J = 8.6, 7.0, 1.7 Hz, 2H), 7.11–7.01 (m, 1H), 6.96 (ddt,
J = 8.3, 7.0, 1.1 Hz, 1H), 3.99 (s, 3H); 13C NMR (101 MHz, CDCl3) δ = 167.46, 142.30, 139.08,
137.39, 134.65, 133.37, 132.05, 126.64, 121.81, 119.89, 119.03, 118.71, 118.59, 52.30; LRMS
(+ESI) 295 [(M + Na)+ 100%].
Methyl 2-((2-aminophenyl)amino)benzoate (20)
To a mixture of 19 (600 mg, 2.20 mmol, 1.0 equiv.) in ethyl acetate (20 mL) was added palladium on
carbon (60 mg, 10% w/w). The reaction mixture was stirred at ambient temperature for 18 hours
under a hydrogen atmosphere (1 atm). The reaction mixture was filtered through a pad of Celite®
and the pad was washed with ethyl acetate. The combined filtrates were evaporated in vacuo
.
Purification by flash column chromatography on silica gel using ethyl acetate/hexane (1:9 v/v)
1
furnished 20 as a colourless powder. Yield 92%. H NMR (400 MHz, CDCl3) δ = 8.98 (br s, 1H),
7.98 (dd, J = 8.1, 1.7 Hz, 1H), 7.32–7.27 (m, 1H), 7.17–7.09 (m, 2H), 6.85–6.76 (m, 2H), 6.73–6.65
(m, 2H), 3.94 (s, 3H), 3.84 (br s, 2H); 13C NMR (101 MHz, CDCl3) δ = 169.07, 149.54, 143.38,
134.39, 131.41, 127.76, 127.14, 125.93, 118.80, 116.38, 115.92, 113.79, 111.01, 52.30; LRMS
(+ESI) 265 [(M + Na)+ 100%].
5,10-Dihydro-11H-dibenzo[b,e][1,4]diazepin-11-one (21)
A suspension of 20 (400 mg, 1.65 mmol, 1.0 equiv.) and lithium hydroxide (158 mg, 6.60 mmol, 4.0
equiv.) in methanol/water (10 mL, 1:1 v/v) was stirred at reflux for 6 hours. The reaction mixture
was then cooled and the methanol evaporated under reduced pressure. The mixture was diluted with
ethyl acetate (20 mL) and acidified using aqueous hydrochloric acid (1 M, 20 mL). The organic layer
was collected and the aqueous layer further extracted with ethyl acetate (2 x 10 mL). The combined
organic extracts were then dried and evaporated in vacuo to give the desired crude carboxylic acid
intermediate
. The crude material was carried onto the next step without purification.
A solution of the crude carboxylic intermediate, HBTU (922 mg, 2.43 mmol, 1.5 equiv.) and N,N
-
diisopropylethylamine (560 µL, 3.24 mmol, 2.0 equiv.) in N,N-dimethylformamide (5 mL) was stirred
at ambient temperature for 5 minutes. The reaction mixture was then poured into ice-cold water (5
mL) and diluted with ethyl acetate (20 mL). The organic layer was extracted and the aqueous layer
further extracted with ethyl acetate (2 x 10 mL). The combined organic extracts were then washed
with aqueous hydrochloric acid (1 M, 10 mL), an aqueous solution of saturated sodium bicarbonate
(10 mL) and water (10 mL). The organic extract was then dried over magnesium sulfate and
evaporated in vacuo. Purification by flash column chromatography on silica gel using ethyl
acetate/hexane (3:7 v/v) furnished 21 as a yellow powder. Yield 82%. 1H NMR (400 MHz, DMSO)
δ = 9.83 (br s, 1H), 7.84 (br s, 1H), 7.68 (dd, J = 7.9, 1.7 Hz, 1H), 7.34 (ddd, J = 8.1, 7.1, 1.7 Hz,
1H), 7.07–6.82 (m, 6H); 13C NMR (101 MHz, DMSO) δ = 168.38, 150.91, 140.42, 133.66, 132.56,
130.29, 124.95, 123.40, 123.23, 121.73, 121.16, 120.23, 119.52; LRMS (+ESI) 233 [(M + Na)+
100%].
General Procedure for the Synthesis of 6a–6d
16

To a solution of 21 (50 mg, 0.238 mmol, 1.0 equiv.) in tetrahydrofuran (0.2 M) was added potassium
tert-butoxide (40 mg, 0.357 mmol, 1.5 equiv.) at 0 °C and stirred for 30 minutes at ambient
temperature. The reaction mixture was cooled to 0 °C and the appropriate alkylating agent
(1.1 equiv.) was added dropwise. The reaction mixture was warmed to ambient temperature and
stirred for 8 hours. The reaction mixture was quenched with water at 0 °C and then diluted with ethyl
acetate (10 mL). The organic layer was collected and the aqueous layer was further extracted with
ethyl acetate (2 x 5 mL). The combined organic extracts were then washed with brine (2 mL), dried
over magnesium sulfate and concentrated in vacuo. Purification by flash chromatography on silica gel
column using ethyl acetate/hexane (1:1 v/v) furnished the desired dibenzodiazepine ligands.
N-Benzyl-N-methyl-2-(11-oxo-10,11-dihydro-5H-dibenzo[b,e][1,4]diazepin-5-yl)acetamide (6a)
General procedure was followed using alkylating agent,
N-benzyl-2-bromo-N-methylacetamide (63
mg, 0.262 mmol). The desired product 6a was isolated as a colourless powder. Yield 86%. 1H NMR
(400 MHz, CDCl3) δ = 7.91 (dd, J = 7.8, 1.6 Hz, 1H), 7.45–7.25 (m, 7H), 7.12–7.04 (m, 3H) 6.94–
6.92 (m, 1H), 6.82–6.79 (m, 1H), 5.37 (br s, 1H), 4.80 (s, 2H), 4.70 (s, 2H), 3.05 (s, 3H); 13C NMR
(101 MHz, CDCl3) δ = 173.20, 170.37, 150.51, 141.67, 137.01, 134.60, 133.10, 132.55, 128.63,
128.02, 127.35, 126.50, 124.53, 123.37, 122.73, 120.19, 120.05, 118.46, 52.82, 51.44, 34.37; LRMS
(+ESI) 394 [(M + Na)+ 100%]; Found: C, 74.29; H, 5.69; N, 11.26. Calc for C23H21N3O2: C, 74.37;
H, 5.70; N, 11.31.
N-Benzyl-N-ethyl-2-(11-oxo-10,11-dihydro-5H-dibenzo[b,e][1,4]diazepin-5-yl)acetamide (6b)
General procedure was followed using alkylating agent,
N-benzyl-2-bromo-N-ethylacetamide (67 mg,
0.262 mmol). The desired product 6b was isolated as a colourless powder. Yield 92%. 1H NMR (400
MHz, DMSO) δ = 7.88 (dd, J = 9.4, 1.6 Hz, 1H), 7.63–7.60 (m, 1H), 7.42–7.25 (m, 6H), 7.14–6.95
(m, 4H), 6.99–6.65 (m, 1H), 4.73 (s, 2H), 4.61 (s, 2H), 3.36 (q,
J = 7.1 Hz, 2H), 1.17 (t, J = 7.1 Hz,
3H), NH proton not observed
;
13C NMR (101 MHz, DMSO) δ = 168.12, 167.66, 152.34, 144.42,
138.61, 135.07, 132.96, 132.56, 128.85, 127.90, 127.30, 126.02, 124.67, 123.90, 123.76, 121.82,
120.65, 119.25 52.96, 48.15, 41.10, 14.01; LRMS (+ESI) 408 [(M + Na)+ 100%]; Found: C, 74.79;
H, 6.03; N, 10.94. Calc for C24H23N3O2: C, 74.78; H, 6.01; N, 10.90.
N,N-Diethyl-2-(11-oxo-10,11-dihydro-5H-dibenzo[b,e][1,4]diazepin-5-yl)acetamide (6c)
General procedure was followed using alkylating agent, 2-chloro-N,N-diethylacetamide (35 µL, 0.262
mmol). The desired product 6c was isolated as a colourless powder. Yield 83%. 1H NMR (400 MHz,
DMSO) δ = 7.86 (br s, 1H), 7.58 (dd, J = 7.8, 1.6 Hz, 1H), 7.34 (ddd, J = 8.5, 7.2, 1.7 Hz, 1H), 7.18
(dd, J = 7.7, 1.7 Hz, 1H) 7.14–6.92 (m, 5H), 4.58 (s, 2H), 3.38 (t,
J = 7.1 Hz, 4H), 1.20 (q, J = 7.1
Hz, 3H), 1.08 (q,
J
= 7.1 Hz, 3H); 13C NMR (101 MHz, DMSO) δ = 168.01, 167.02, 152.29,
144.36, 135.14, 132.88, 132.51, 125.95, 124.79, 123.86, 123.69, 121.79, 120.58, 119.21 53.16,
41.13, 13.54; LRMS (+ESI) 346 [(M + Na)+ 100%]; Found: C, 70.51; H, 6.49; N, 12.93. Calc for
C19H21N3O2: C, 70.57; H, 6.55; N, 12.99.
N,N-Dimethyl-2-(11-oxo-10,11-dihydro-5H-dibenzo[b,e][1,4]diazepin-5-yl)acetamide (6d)
General procedure was followed using alkylating agent, 2-chloro-N,N-dimethylacetamide (25 µL,
0.262 mmol). The desired product 6d was isolated as a colourless powder. Yield 75%. 1H NMR (400
MHz, DMSO) δ = 7.87 (br s, 1H), 7.52 (dd, J = 7.8, 1.6 Hz, 1H), 7.37–7.31 (m, 1H), 7.19 (d,
J =
7.8 Hz, 1H), 7.18–6.91 (m, 5H), 4.58 (s, 2H), 3.11 (s, 6H); 13C NMR (101 MHz, DMSO) δ =
168.08, 166.98, 151.84, 144.31, 136.01, 132.74, 132.50, 126.19, 124.84, 123.89, 123.79, 122.52,
120.56, 119.22 52.95, 36.69, 36.11; LRMS (+ESI) 318 [(M + Na)+ 100%]; Found: C, 69.18; H,
5.86; N, 14.19. Calc for C17H17N3O2: C, 69.14; H, 5.80; N, 14.23.
17

Biological Evaluation
Radioligand binding. Binding affinities (Ki) to wild type and A147T TSPO were measured as per our
published protocol.20 Briefly, membranes were prepared from HEK293T cells stably transfected with
wild type and A147T TSPO by homogenisation, using an Ultra-Turrax hand-held homogeniser. These
cells have previously been validated as an in vitro model of low- and high- affinity TSPO binders.20
Wild type (20 µg/well) and A147T (5 µg/well) TSPO membranes were diluted in 50 mM Tris HCl (pH
7.4), and were incubated at 4 ºC for 90 min with ~ Kd concentration of [3H] PK 11195 (10 nM; Perkin
Elmer) and test compounds (0.3 nM–10 µM). A high concentration (1 µM) of unlabelled PK 11195 was
used to measure non-specific binding, which was less than 10% of total binding. Filtration through a
96-well glass-fibre filter plate (Millipore) was used to terminate reactions. Plates were then washed 8
times with ice-cold 50 mM Tris HCl. Radioactivity was read in a Microbeta2 2450 Microplate Counter
(Perkin Elmer) after addition of Microscint 0. Data were analysed using Graphpad Prism 6.0
(GraphPad), applying a four-parameter non-linear regression curve fit to calculate Ki values. Data are
expressed as mean ± SEM from at least three independent experiments.
Target protein preparation. The WT and A147T TSPO homology models generated in our previous
study,20 were prepared using preparation and refinement protocols, directed by the Protein
Preparation Wizard28 embedded in Maestro v12.1 (Schrödinger, LLC, New York, USA). This process
includes assigning bond orders, adding hydrogen atoms, and creating zero order bonds to metals and
disulphide bonds. The hydrogen bond network within the protein was also optimised with all het
groups within the receptor grid bounding box previously removed and the protein structure minimised
to a root mean square deviation (RMSD) of 0.3 Å using the OPLS3 force field.29, 30
Ligand preparation. All ligands were prepared using the LigPrep v4.9 module to generate possible
stereoisomers of the ligands as well as generating all potential ionisation states at pH 7±2. Confgen31
v4.7 was also used to generate up to 64 different conformations for each ligand.
33
Receptor grid generation. The Receptor Grid Generation tool in Glide v8.432,
as used to
characterise the binding site for the docking studies. Binding sites were defined by a 20 Å3 bounding
box centred at the ligand within the active site of each homology model. A Coulomb-van der Waals
scaling factor of 1.0 for receptor van der Waals radii was applied to protein atoms with a partial
charge of less than 0.25 e and a similar factor of 0.8 was applied to ligand atoms with a partial
charge cutoff of less than 0.15 e. Rotations of hydroxyl and thiol groups were not allowed.
Docking studies. The ligands were docked into the receptor grids with Glide v8.4.32, 33 All docking
studies were carried out using the Extra Precision (XP)34 scoring function to refine binding energy
estimates. All ligands were docked with flexible states to allow sampling of the effect of nitrogen
inversion, changing ring conformations and non-planar amide functional groups were penalised.
Prime MM-GBSA calculations,35 which combines molecular mechanics (MM) terms, and a generalised
Born and surface area (GBSA) solvent model,25 were utilised to calculate the free energy of binding
for the ligands. The output poses from Glide XP docking were used as the basis for these calculations.
The calculations were performed using the variable-dielectric generalised Born (VSGB)36 solvation
model and OPLS3 force field.30 The docked ligand and protein residues within 10 Å of the ligand were
allowed to be flexible, with all other atoms remaining rigid.
Author Contributions:
18

R.S. and E.L.W. are co-first-authors§, contributing equally to this work, carrying out synthesis and
affinity measurements, respectively. H.W.A.C assisted with the pharmacological screening while
J.H.L., J.J.D., G.S., T.A.R. and M.K. assisted with the design and synthesis of ligands. The transfected
HEK293 cells were provided by L.M.I. A.P.M., J.J.D., Q.G. and D.E.H. provided the TSPO docking
studies for ligands 3a, 4a, 5a and 6a. All authors contributed to the writing and proof-reading of the
manuscript.
Corresponding Author
* M.K. Telephone: +61 2 9351 2745. Fax: +61 2 9351 3329. Email: michael.kassiou@sydney.edu.au
Funding Sources
The work was supported by the National Health and Medical Research Council of Australia
(APP1132524, 1136241 and 1154692).
ACKNOWLEDGEMENT
The authors wish to also thank Dr Sheng Hua and Dr Donna Lai for assistance with the Bosch
Molecular Biology Facility.
ABBREVIATIONS USED
AcOH, acetic acid, MeCN, acetonitrile; PyBOP, benzotriazol-1-yl-oxytripyrrolidinophosphonium
hexafluorophosphate; BINAP, 2,2′-bis(diphenylphosphino)-1,1′-binaphthalene; Cs2CO3, caesium
carbonate; d, doublet; DIPEA, diisopropylethylamine; DMF, dimethylformamide; EtOH, ethanol;
EtOAc, ethyl acetate; h, hours; HEK, human embryonic kidney; HPLC, high-performance liquid
chromatoragraphy; H2, hydrogen gas;
MeOH, methanol; µM, micromolar; nm, nanomolar; HBTU, N,N,N′,N′-tetramethyl-
K
i, inhibitor constant; kDa, kilodalton; LiOH, lithium hydroxide;
-(1H-benzotriazol-
O
1-yl)uronium hexafluorophosphate; Pd(OAc)2, palladium acetate; Pd/C, palladium on carbon; PET,
positron emission tomography; KOtBu, potassium tert-butoxide; rt, room temperature; s, singlet;
NaOH, sodium hydroxide; NaH, sodium hydride; NaOMe, sodium methoxide; SD, standard deviation;
SAR, structure-activity relationship; Pd(PPh3)4, tetrakis(triphenylphosphine)palladium(0); t, triplet;
THF, tetrahydrofuran; PhMe, toluene; TSPO, translocator protein; Et3N, triethylamine.
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Highlights:
•
•
•
•
Optimal binding to both TSPO isoforms is dependent on both the heterocyclic scaffold and
substituents on the acetamide motif.
The nature of the pyrazolopyrimidine scaffold was found to be a promising scaffold for TSPO
ligands.
Lowered discrimination at A147T TSPO is predominantly seen with
ligands.
N-benzyl-N-methyl
Docking studies were used to examine how each heterocyclic scaffold binds to both WT and
A147T TSPO.

Declaration of interests
☒ 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.
☐The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests: