Conformationally rigid derivatives of WAY-267,464: Synthesis and pharmacology at the human oxytocin and vasopressin-1a receptors

Article abstract of DOI:10.1016/j.ejmech.2017.10.059

WAY-267,464 (1) and twelve conformationally rigid analogues (3a-f–4a-f) were synthesised, characterised and evaluated in cellular assays with the aim of systematically exploring interactions with the oxytocin receptor (OTR). Each analogue was evaluated in

Full text of DOI:10.1016/j.ejmech.2017.10.059

Accepted Manuscript
Conformationally rigid derivatives of WAY-267,464: Synthesis and pharmacology at
the human oxytocin and Vasopressin-1a receptors
William T. Jorgensen, Damien W. Gulliver, Timothy A. Katte, Eryn L. Werry, Tristan A.
Reekie, Mark Connor, Michael Kassiou
PII:
S0223-5234(17)30861-9
10.1016/j.ejmech.2017.10.059
EJMECH 9851
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 11 July 2017
Revised Date: 18 October 2017
Accepted Date: 20 October 2017
Please cite this article as: W.T. Jorgensen, D.W. Gulliver, T.A. Katte, E.L. Werry, T.A. Reekie, M.
Connor, M. Kassiou, Conformationally rigid derivatives of WAY-267,464: Synthesis and pharmacology
at the human oxytocin and Vasopressin-1a receptors, European Journal of Medicinal Chemistry (2017),
doi: 10.1016/j.ejmech.2017.10.059.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
Investigation of Linker Conformation
O
OH
HN
N
N
N
H
N
N
N
OH
OR
O
WAY-267,464
1
OR
Investigation of H-Bonding
n
Rigidity
Loss of OTR activity
V1aR activity
O

ACCEPTED MANUSCRIPT
Conformationally Rigid Derivatives of WAY-267,464:
Synthesis and Pharmacology at the Human Oxytocin and
Vasopressin-1a Receptors
William T. Jorgensena*, Damien W. Gulliverb*, Timothy A. Kattea, Eryn L. Werryb,
Tristan A. Reekiea, Mark Connorc, Michael Kassioua#.
aSchool of Chemistry, The University of Sydney, Sydney, NSW 2006, Australia; bFaculty
of Health Sciences and School of Medical Sciences (Pharmacology), Bosch Institute, The
University of Sydney, Sydney, NSW 2006, Australia; cThe Australian School of Advanced
Medicine, Macquarie University, NSW 2109, Australia.
*Joint first authors
#corresponding author: michael.kassiou@sydney.edu.au
Keywords: Oxytocin receptor; WAY-267,464; arginine vasopressin 1a receptor;
diazepine.
1

ACCEPTED MANUSCRIPT
Abstract
WAY-267,464 (1) and twelve conformationally rigid analogues (3a-f – 4a-f) were synthesised,
characterised and evaluated in cellular assays with the aim of systematically exploring
interactions with the oxytocin receptor (OTR). Each analogue was evaluated in radioligand
binding displacement assays at both human OTR and arginine vasopressin 1a receptors (V1aR).
Physiological characterisation was determined by whole cell IP1 accumulation assays on stably
transfected human embryonic kidney (HEK) cells. Incorporation of the rigid, optionally
substituted benzene ring abolished OTR activity and diminished V1aR pharmacology when
compared to 1. A general trend was observed in V1aR affinity for the propyl analogues (3d-3f)
which identified the ortho-substituted analogue as the best in series (Ki = 251 nM) followed by a
decrease in affinity through the meta and para- derivatives (3e; Ki = 874 nM and 3f; Ki = 1756
nM respectively). This study confirms the importance of the central pharmacophoric motifs of
WAY-267,464 and illuminates the differences in the binding pocket of the highly conserved OTR
and V1aR.
1. Introduction
Psychological disorders such as depression, anxiety and substance addiction often manifest
concomitantly with severe social dysfunction.[1] This reveals a possible therapeutic paradigm
through the use of pro-social modulators to treat such psychiatric illnesses. Surmounting
evidence over the past two decades has implicated centrally located oxytocin (OT) receptors
and arginine vasopressin (AVP) 1a receptors as viable targets for the improvement of social
cognition.[2],[3] OT facilitates social bonding and pair attachment in humans and animals,[4]
whereas male rats lacking functional V1a receptors (V1aR) display profound anxiety-like
behaviour and a marked reduction in social recognition.[5] It follows that the development of a
centrally penetrating, orally bio-available, selective ligand at the OT receptor (OTR) or V1aR
represents an attractive research goal.
OT and AVP are structurally related cyclic neuropeptides which elicit their modulatory roles via
interaction with four G-protein coupled receptors; the OT, V1a, V1b and V2 receptors.
Unfortunately, the highly conserved sequence homology across each receptor subtype,[6][7]
coupled with the partial co-localisation of the OTR and V1aR within the CNS presents a
significant challenge in the design of selective receptor ligands. Furthermore, appreciable
crosstalk between the endogenous neuropeptides amongst the OT and vasopressin receptor
families, coupled with the poor pharmacokinetic properties inherent to cyclic peptides (short
half-life, poor CNS permeability and unknown metabolomics), limit their use as pharmacological
tools. As such, the identification of selective, centrally penetrating OTR or V1aR ligands is of
2

ACCEPTED MANUSCRIPT
paramount importance towards the development of molecular probes to differentiate the
behavioural roles of each receptor subtype.[8] Our group has been interested in understanding
the intricacies surrounding the functional activity of WAY-267,464 (1), one of the very few non-
peptidic OTR agonists. We have previously published the behavioural profile of WAY-267,464
and found significant pro-social effects (IP, 100 mg/kg)[9] which were later in-part correlated
to centrally located V1aR antagonism rather than its oxytocinergic effects.[10] In 2016, we
reported the pharmacological binding and efficacy profile of 1 along with its first published
structure-activity relationship. WAY-267,464 was shown to be a non-selective OTR
agonist/V1aR antagonist (OTR Ki = 230 nM; V1aR Ki = 27 nM; OTR EC50 = 420 nM; V1aR IC50 = 613
nM).[11]
Whilst the initial disclosure of WAY-267,464 reported the synthesis of multiple analogues,
derivatisation was focused on modifications to the tricyclic western component or the
benzylpiperazine tail moiety. The central benzylurea linkage (Figure 1) was maintained across
the compound library.[12] In 2010, Hubert and colleagues performed a structure activity
relationship analysis of proline 2, a weak OTR partial agonist which shares structural motifs,
including the benzylurea linker, with WAY-267,464. Whilst their work focused on the truncation
of 2 and elaboration through hybridisation with AVPR and OTR ligands, the benzylurea
component was maintained. As such, the majority of our work has looked at identifying the
functional necessity of this central, structurally conserved motif.
Figure 1: The conserved benzylurea moiety across non-peptide OTR agonists (1 & 2) and our efforts to explore its
importance.
3

ACCEPTED MANUSCRIPT
Previous work involved replacing the rigid benzylurea with flexible alkyl tethers of varying
lengths. This modification abolished OTR activity and largely diminished the OTR binding
affinity (Figure 1). Marginal receptor affinity was gained at increasing chain lengths similar in
distance to the lead molecule 1. Whilst deleterious to receptor pharmacology, these
modifications were better tolerated at the V1aR and thus resulted in selective V1aR ligands. To
exemplify the subtleties surrounding the OTR, removal of the hydrogen-bonding capabilities of
the resorcinol functionality through methyl-ether incorporation resulted in a reversal of WAY-
267,464 functional activity (resulting in a weak OTR antagonist; IC50 = 4 µM).[11]
Considering the lack of published structure-activity relationship (SAR) studies surrounding the
WAY-267,464 molecule and the previously observed subtleties surrounding the flexible alkyl
tethered analogues, we now report the synthesis and pharmacological evaluation of 12
conformationally restricted analogues. Several considerations were necessary in the design of
these molecules. Shortened chain lengths limited the number of freely rotatable bonds,
minimising overall molecular flexibility. Phenylpiperazine analogues, 3a-f and 4a-f were
designed to methodically explore optionally restrained analogues of 1 in an attempt to re-
establish the OTR activity which was lost upon the introduction of flexibility. For consistency,
both methyl-ether and phenolic analogues were incorporated to better correlate with the
previous work in a sustained effort towards the identification of a viable OTR pharmacophore
(Figure 2).
Figure 2: Re-introduction of rigidity via incorporation of an optionally substituted benzene ring.
2. Synthetic Chemistry
The synthetic protocols to generate WAY-267,464 on the gram-scale have previously been
described.[11] Furthermore, we have recently reported a large-scale method for the production
of pyrazolo[1,4]diazepine molecules through a novel, Pictet Spengler cyclisation (Scheme
1).[13] Alkoxide mediated nucleophilic aromatic substitution of arylfluoride 6 by 5-
aminopyrazole generated nitrobenzene 7. Palladium catalysed hydrogenation furnished the
4

ACCEPTED MANUSCRIPT
Pictet-Spengler precursor 8 that, upon subjection to formaldehyde and catalytic quantities of
acetic acid, produced the desired diazepine 9.
Scheme 1: Reagents and conditions: (a) KOtBu, THF, 0 °C → RT, 3 h, 77%; (b) Pd/C (10 mol%), H2 (1 atm), EtOAc, RT, 18 h
88%; (c) HCHO, AcOH (cat), MeCN, RT, 18 h, 79%.
The synthesis of the desired analogues begins with the Fischer esterification of the
appropriately substituted bromophenyl alkanoic acids to yield the methyl esters (10 – 15),
followed by a Buchwald-Hartwig cross coupling amination with Boc-piperazine to furnish the
desired phenylpiperazines (16 – 21). Hydroxide mediated hydrolysis in aqueous methanol
allowed access to the alkanoic acid precursors and upon subjection to a PyBOP® mediated
amidation with diazepine 9 generated the amide intermediates (28 – 33). An optimised one-pot
carbamate deprotection-reductive alkylation with either 3,5-dihydroxybenzaldehyde or 3,5-
dimethoxybenzaldehyde produced the desired rigid analogues in sufficient yield for
pharmacological assays (Scheme 2).
5

ACCEPTED MANUSCRIPT
O
HO
a
n
n
Br
Br
O
O
n=1 o; 10
m; 11
n=2 o; 13
m; 14
b
p; 12
p; 15
O
O
c
HO
O
N
N
n
n
O
O
N
N
O
O
n=1 o; 22
m; 23
n=2 o; 25
m; 26
n=1 o; 16
n=2 o; 19
m; 20
m; 17
p; 18
p; 24
p; 27
p; 21
d
HN
O
N
N
O
N
N
n
O
N
n=1 o; 28
m; 29
n=2 o; 31
m; 32
p; 30
p; 33
e
HN
N
N
N
N
n
N
OR
O
RO
R = H, n=1 o; 3a
m; 3b
R = CH3, n=1 o; 4a
4b
m;
p; 3c
n=2 o; 3d
p; 4c
n=2 o; 4d
m; 4e
m;
3e
p; 3f
p; 4f
Scheme 2: (a) H2SO4, MeOH, reflux, 18 h, 90-100%; (b) Boc-piperazine, RuPhos®, Cs2CO3 Pd2(dba)3, PhMe, reflux, 18 h, 55-
i
99%; (c) LiOH (aq), MeOH, reflux, 1 h, 90-97%; (d) PyBOP®, diazepine 9, Pr2NEt, CH2Cl2, rt, 12 h, 67-81%; (e) i. HCl (1M in
dioxane), MeOH, rt, 3 h. ii. 3,5-dihydroxybenzaldehyde or 3,5-dimethoxybenzaldehyde, NMM, MgSO4, NaCNBH3, rt, 18 h,
43-100%.
3. Results & Discussion
The 12 derivatives synthesised were subjected to radioligand displacement assays to determine
selectivity and receptor affinity utilising membranes from HEK 293 cells expressing hOTR and
hV1aR. Comparison of the rigid dimethoxy analogues 4a-f to the lead compound WAY-267,464
and dimethoxy analogue 34 as well as the best in series of the flexible analogues 35,[11] is
summarised in Table 1.
6

ACCEPTED MANUSCRIPT
Table 1: Binding and functional activity of the conformationally restricted dimethoxy derivatives (general structure
4a-f) compared to WAY-267,464 (1), diMeO-WAY (34), and the most potent dimethoxy flexible analogue (35).
O
OR
HN
N
N
N
H
N
N
N
OR
O
H;
CH3;
R =
1 (WAY-267,464)
34
HN
HN
N
O
O
N
N
N
N
N
N
N
N
n
n
O
N
O
O
O
n = 6; 35
4a-f
OTR (nM ± SD)
EC50
V1aR (nM ± SD)
EC50
#
n
Ki
IC50
Ki
IC50
230 ± 31
801 ± 139
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
420 ± 59
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
n/a
27 ± 3
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
613 ± 206
1113 ± 180
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
1
-
-
-
4129 ± 645
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
62 ± 21
34
35*
4a
4b
4c
-
194.7 ± 82.6
6807 ± 642.5
2095 ± 691.9
1910 ± 262.6
1062 ± 143.2
3343 ± 59.30
6250 ± 964.2
6
1
1
1
2
2
2
o
m
p
4d
4e
4f
o
m
p
*The strongest binder of the dimethoxy flexible alkyl analogues at the hV1aR.
Reintroduction of conformity in the central linker of the WAY analogues did not reinstate
functional efficacy or affinity at the OTR. It is likely that the central tolyl linker and/or urea
moiety participate in favourable binding interactions at the OTR active site which are lacking in
both the flexible and conformationally-restricted analogues. The methylation of the phenols did
not generate OTR antagonists, in contrast to the effect seen with methylation of WAY-
267,464.[11]
In regards to V1aR activity, restricting the number of freely rotatable bonds resulted in a
decrease in radioligand displacement. This was observed for both the n = 1 and n = 2 alkyl
ortho/meta/para derivatives (Table 1). In comparison to the dimethoxy flexible analogue (n =
6, 35) which is the strongest V1aR binder of the dimethoxy alkyl analogues (Ki = 195 nM), the
7

ACCEPTED MANUSCRIPT
best dimethyl conformationally-restricted analogue 4d incorporated an ortho-piperazine
substitution pattern (with a binding affinity Ki = 1062 nM). Although these molecules weakly
bind to the V1aR in a homogenised cell membrane assay, they do not have a functional effect on
the Gq pathway of the V1aR. These functional assays are conducted under different experimental
conditions to the binding assays, and so the incongruous behaviour of the molecules between
assays may be explained by a differing effect on receptors in the whole cell versus membrane
environment, or at different temperatures.
It was anticipated that the O-desmethyl analogues would perform better in radioligand binding
assessment considering the increase in binding affinity observed with the phenolic alkyl
derivatives presented previously.[11] It was satisfying to confirm this prediction with a 2-3-fold
increase in affinity at the V1aR compared to the methylated analogues. This further justifies the
requirement of H-bond donation for V1aR affinity. In comparison to the previously synthesised
flexible analogues, the rigid derivatives resulted in a dramatic loss (4-25 fold) in V1aR binding
affinity. This suggests flexibility is an important determinant of V1aR pharmacology (Table 2).
Table 2: Binding and functional activity of the conformationally restricted resorcinolic derivatives (general structure
3a-f) compared to WAY-267,464 (1 and the most potent dihydroxy flexible analogue (36).
OTR (nM ± SEM)
EC50
V1aR (nM ± SEM)
EC50
#
n
-
Ki
IC50
Ki
IC50
230 ± 31
8349 ± 664
>10,000
>10,000
>10,000
>10,000
>10,000
420 ± 59
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
27 ± 3
>10,000
613 ± 206
1
-
-
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
64.1 ± 5.1
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
4667 ± 823
>10,000
>10,000
>10,000
>10,000
>10,000
36*
3a
3b
3c
7
1
1
1
2
2
1938 ± 519.3
642.8 ± 108.3
795.1 ± 228.0
250.7 ± 32.6
874.1 ± 142.4
o
m
p
o
3d
3e
m
8

ACCEPTED MANUSCRIPT
>10,000
>10,000
>10,000
1756 ± 481.9
>10,000
>10,000
3f
2
p
*The strongest binder of the alkyl analogues at the hV1aR.
Interestingly, for the propyl chain analogues, a trend in the substitution pattern could be
observed. The ortho substitution resulted in the highest binding affinity (3d; Ki = 250 nM)
followed by the meta substituted analogue (3e; Ki = 874 nM) and finally the para derivative (3f;
Ki = 1756 nM). This suggests that optimal binding orientation at the V1aR is obtained through a
highly kinked structural conformer which is not replicated at the OTR.
The highest binder of this series of compounds was the O-desmethyl, ortho substituted analogue
3d which had a radioligand binding displacement affinity of 250 nM. Unfortunately, the
functional inhibition of the Gq-pathway of the V1aR by these compounds was not observed in the
current pharmacological assay.
4. Conclusion
The primary focus of this study was to further explore the structural motifs associated with the
unique oxytocinergic and vasopressinergic activity of the non-peptidic WAY-267,464 molecule.
Prior work has shown that replacement of the central tolyl-urea linkage abolished OTR affinity
and was detrimental to V1aR activity. Reducing the number of freely rotatable bonds between
the tricyclic diazepine and the benzylpiperazine resulted in 12 conformationally rigid
analogues. The rigidity of these molecules did not restore OTR binding activity or efficacy which
confirms that the functionality within the WAY-267,464 linker is important for its receptor
pharmacology.
Similarly to the alkyl tethered analogues, removal of the H-bond donating capabilities of the
resorcinol functionality was detrimental to overall receptor pharmacology. Furthermore, a
trend was observed at the V1aR regarding optimal substitution around the central aromatic ring
incorporated to impart rigidity. The 2-piperazinyl substituted (ortho) compound, 3d was the
highest binder of this series. Unfortunately, these rigid analogues failed to produce an improved
pharmacological profile compared to the lead compound in terms of affinity, potency or
selectivity. This work has shown that the central linker components of WAY-267,464 are
imperative for functional activity, efficacy and affinity at the OTR providing further insight into
the OTR pharmacophore. Furthermore, modifications to this central component are better
tolerated at the V1aR and as such, differences in key receptor-ligand interactions between these
highly homologous receptors have been brought to light. Future work will look at the systematic
introduction of the key functional groups that make up the linker moiety of WAY-267,464 to
elaborate on the conclusions presented here.
9

ACCEPTED MANUSCRIPT
5. Experimental Procedures
5.1. General Experimental
Unless otherwise stated, reactions were conducted under positive pressure of a dry nitrogen or
argon atmosphere. Temperatures of 0 °C and –10 °C were obtained by the use of a water/ice
bath or salt/ice bath respectively. Reaction mixture temperatures were reported according to
the oil bath/cooling bath temperature unless otherwise stated. Anhydrous dichloromethane,
triethylamine and diisopropyl ethylamine were obtained by distillation from calcium hydride.
Anhydrous DMF, methanol, THF, and acetonitrile were obtained from a PureSolv MD 7 solvent
purification system (Innovative Technology, Inc.). Unless noted otherwise, commercially
obtained reagents were used as purchased without further purification. Analytical thin-layer
chromatography (TLC) was performed using Merck aluminium backed silica gel 60 F254 (0.2
mm) plates which were visualised with shortwave (254 nm) and/or longwave (365 nm)
ultraviolet (UV) light. Non UV-active products were visualised with potassium permanganate,
vanillin, p-anisaldehyde, ninhydrin or cerium molybdate (“Goofy’s Dip”) stains. Flash
chromatography was performed using Grace Davisil silica gel, pore size 60 Å, 230–400 mesh
particle size. Solvents for flash chromatography were distilled prior to use, or used as purchased
for HPLC grade, with the eluent mixture reported as the volume/volume ratio (v/v).
Melting points were measured with open capillaries using a Stanford Research Systems (SRS)
MPA160 melting point apparatus with a ramp rate of 0.5–2.0 °C/min and are uncorrected.
Infrared absorption spectra were recorded on a Bruker ALPHA FT-IR spectrometer, and the
data are reported as vibrational frequency (cm–1). Nuclear magnetic resonance spectra were
recorded at 298 K unless stated otherwise, using either a Bruker DRX400 (400.1 MHz) or
AVANCE III 500 Ascend (500.1 MHz) spectrometer. The data is reported as the chemical shift (δ
ppm) relative to the solvent residual peak, multiplicity (s = singlet, d = doublet, t = triplet, q =
quartet, m = multiplet, br = broad, dd = doublet of doublets, etc.), coupling constant (J Hz),
relative integral. Low resolution mass spectra (LRMS) were recorded using electrospray
ionisation (ESI) on a Finnigan LCQ ion trap spectrometer. High resolution mass spectra were
run on a Bruker 7T Apex Qe Fourier Transform Ion Cyclotron resonance mass spectrometer
equipped with an Apollo II ESI/APCI/MALDI Dual source by the Mass Spectrometry Facility of
the School of Chemistry at The University of Sydney. Samples run by ESI were directly infused
(150 µL/hr) using a Cole Palmer syringe pump.
Analytical HPLC purity traces were taken on a Waters 2695 Separations module equipped with
Waters 2996 Photodiode Array detector (set at 230, 254 and 271 nm). All samples were eluted
10

ACCEPTED MANUSCRIPT
through a Waters SunFire™ C18 5 µm column (2.1x150 mm) using a flow rate of 0.2 mL/min of
Solvent A: MilliQ water (+0.1% trifluoroacetic acid or 0.1% formic acid) and Solvent B:
acetonitrile (+0.1% trifluoroacetic acid or 0.1% formic acid). This method consisted of gradient
elution (0-100% Solvent A:B over 30 minutes). Data acquisition and processing was performed
with the Waters Empower 2 software. Reported data for all compounds are based on the 254
nm channel.
5.2. Synthetic Procedures
The synthesis of 1-methyl-1,4,5,10-tetrahydrobenzo[b]pyrazolo[3,4-e][1,4]diazepine (9) was
completed as previously reported.[13] All characterisation data matched with those previously
reported.
5.2.1. 1-Methyl-N-(2-nitrophenyl)-1H-pyrazol-5-amine (7)
A magnetically stirred solution of amine 5 (10.0 g, 103 mmol) in THF (250 mL) was treated with
potassium tert-butoxide (23.0 g, 205 mmol) at 0 °C. The resultant suspension was brought to
room temperature and stirred for 1 h before the dropwise addition of o-fluoronitrobenzene (12
mL, 113 mmol). The reaction mixture was stirred for 3 h, quenched with water (300 mL) and
extracted with EtOAc (3 x 150 mL). The combined organic extracts were dried (MgSO4), filtered
and concentrated under reduced pressure. The crude oil was purified by flash column
chromatography (silica, 3:5 v/v EtOAc:hexanes) and concentration of the relevant fractions (Rf
= 0.42 in 1:1 v/v EtOAc:hexanes) afforded the title compound (16.9 g, 77%) as yellow crystals.
1H NMR (300 MHz, Chloroform-d): δ 9.11 (s, 1H, NH), 8.25 (d, J = 8.1 Hz, 1H), 7.56 – 7.54 (m,
1H), 7.52 – 7.38 (m, 1H), 6.96 – 6.84 (m, 1H), 6.78 – 6.74 (m, 1H), 6.20 (d, J = 2.5 Hz, 1H), 3.76 (s,
3H). 13C NMR (75 MHz, Chloroform-d): δ 142.7, 139.0, 136.8, 136.3, 133.4, 126.6, 118.7, 115.8,
101.8, 35.2. IR (diamond cell, neat) νmax: 3297, 3094, 1609, 1572, 1509, 1490, 1340, 1221, 1145,
1075, 1043, 927, 750 cm–1. LRMS (+ESI) m/z: 219 ([M+H]+, 100%). HRMS (+ESI) Found:
(M+Na)+, 241.0697. C10H10N4O2 requires (M+Na)+, 241.0696. MP: 133 – 135 °C.
5.2.2. N1-(1-Methyl-1H-pyrazol-5-yl)benzene-1,2-diamine (8)
A stirred suspension of 1-methyl-N-(2-nitrophenyl)-1H-pyrazol-5-amine (7) (5.50 g, 25.2
mmol) and Pd/C (10% w/w, 630 mg, 0.6 mmol) in MeOH (250 mL) was stirred under an
atmosphere of hydrogen (1 atm) for 18 h after which time the reaction mixture was filtered
through basic alumina and Celite® and residues washed with EtOAc (150 mL). The filtrate was
concentrated under reduced pressure to give the title compound (4.15 g, 88%) as a pink
crystalline solid.
1H NMR (300 MHz, Chloroform-d): δ 7.40 (d, J = 2.0 Hz, 1H), 6.86 (t, J = 7.5 Hz, 1H), 6.75 (d, J =
7.5 Hz, 1H), 6.63 (d, J = 7.8 Hz, 1H), 5.80 (d, J = 2.0 Hz, 1H), 5.33 – 5.28 (m, 1H), 4.00 – 3.36 (m,
3H). 13C NMR (75 MHz, Chloroform-d): δ 142.1, 138.5, 136.6, 132.2, 122.9, 120.3, 117.9, 117.3,
96.7, 34.9. IR (diamond cell, neat) νmax: 3326, 3213, 1554, 1498, 1430, 1384, 1281, 993, 927,
733, 627 cm–1. LRMS (+ESI) m/z: 211 [(M+Na)+, 80%], 189 [(M+H)+, 100%]. HRMS (+ESI)
Found: (M+Na)+, 211.0955. C10H12N4 requires (M+Na)+, 211.0954. MP: 133 – 135 °C.
5.2.3. 1-Methyl-1,4,5,10-tetrahydrobenzo[b]pyrazolo[3,4-e][1,4]diazepine (9)
11

ACCEPTED MANUSCRIPT
A solution of aniline 99 (1.40 g, 7.84 mmol) and formaldehyde (0.334 mL of a 37% aq. solution,
8.6 mmol) in CH3CN (80 mL) was treated with acetic acid (0.09 mL, 20 mol%) and stirred at
room temperature for 18 h. The resultant solution was concentrated under reduced pressure
and taken up in NaHCO3 (50 mL of a sat. aq. solution) and extracted with EtOAc (3 x 50 mL). The
combined organics were washed with brine (50 mL), dried (MgSO4), filtered and solvent
evaporated under reduced pressure. The crude oil was purified by flash column
chromatography (silica, 1:9 v/v MeOH:CH2Cl2) and concentration of the relevant fractions
(Rf = 0.43 in 1:9 v/v MeOH:CH2Cl2) yielded the title compound (1.24 g, 79%) as an off-white
powder.
1H NMR (300 MHz, DMSO-d6): δ 7.99 (s, 1H), 7.05 (d, J = 7.6 Hz, 1H), 6.97 (s, 1H), 6.81 (d, J = 7.6
Hz, 1H), 6.73 (t, J = 7.6 Hz, 1H), 6.64 (t, J = 7.6 Hz, 1H), 5.36 (s, 1H), 3.86 (s, 2H), 3.67 (s, 3H). 13
C
NMR (75 MHz, DMSO-d6): δ 140.9, 139.5, 135.0, 133.0, 121.7, 120.8, 120.2, 118.9, 101.5, 43.4,
35.0. IR (diamond cell, neat) νmax: 3293, 1560, 1505, 1393, 1318, 761 cm–1. LRMS (+ESI) m/z:
223 [(M+Na)+, 40%], m/z: 201 [(M+H)+, 40%]. HRMS (+ESI) Found: (M+Na)+, 223.0958.
C11H12N4 requires (M+Na)+, 223.0954. MP: 205 – 207 °C.
5.2.4. General procedure for the Fischer esterification of phenyl alkanoic acids[14]:
A stirred solution of the appropriate bromophenyl alkanoic acid (1 eq) in MeOH (25 mL) was
treated with H2SO4 (0.1 eq of a conc. aq. solution) and heated at reflux for 18 h. The resultant
solution was cooled to room temperature and concentrated under reduced pressure. The
resultant oil was diluted with NaHCO3 (50 mL of a sat. aq. solution) and subsequently extracted
with diethyl ether (3 x 25 mL). The combined organics were washed with brine (1 x 100 mL),
dried (MgSO4), filtered and concentrated under reduced pressure to give the desired methyl
ester:
5.2.5. Methyl 2-(2-bromophenyl)acetate (10)
Subjecting 2-bromophenyl acetic acid (1.05 g, 5 mmol) to the above general procedure gave the
title compound (1.06 g, 93%) as a colourless oil.
1H NMR (400 MHz, Chloroform-d): δ 7.58 (d, J = 8.0 Hz, 1H), 7.32 – 7.27 (m, 2H), 7.19 – 7.11 (m,
1H), 3.80 (s, 2H), 3.72 (s, 3H). 13C NMR (101 MHz, Chloroform-d): δ 171.1, 134.3, 133.0, 131.6,
129.0, 127.7, 125.2, 52.3, 41.6. IR (diamond cell, neat) νmax: 1734, 1471, 1435, 1340, 1219, 1158,
1026, 733, 660, 440 cm–1. LRMS (+ESI) m/z: 251/253 [(M+Na)+, 98/100%].
5.2.6. Methyl 2-(3-bromophenyl)acetate (11)
Subjecting 3-bromophenyl acetic acid (1.05 g, 5 mmol) to the above general procedure gave the
title compound (1.11 g, 97%) as a yellow oil.
1H NMR (400 MHz, Chloroform-d): δ 7.44 (t, J = 1.6 Hz, 1H), 7.41 (dt, J = 6.7, 2.1 Hz, 1H), 7.25 –
7.15 (m, 2H), 3.70 (s, 3H), 3.59 (s, 2H). 13C NMR (101 MHz, Chloroform-d): δ 171.4, 136.2, 132.5,
130.4, 130.2, 128.1, 122.7, 52.3, 40.8. IR (diamond cell, neat) νmax: 1734, 1471, 1435, 1340,
1219, 1158, 1026, 733, 660, 440 cm–1. LRMS (+ESI) m/z: 251/253 [(M+Na)+, 94/100%].
5.2.7. Methyl 2-(4-bromophenyl)acetate (12)
Subjecting 4-bromophenyl acetic acid (1.05 g, 5 mmol) to the above general procedure gave the
title compound (1.07 g, 94%) as a colourless oil.
1H NMR (400 MHz, Chloroform-d): δ 7.45 (d, J = 8.4 Hz, 2H), 7.15 (d, J = 8.4 Hz, 2H), 3.69 (s, 3H),
3.58 (s, 2H). 13C NMR (101 MHz, Chloroform-d): δ 171.6, 133.0, 131.8, 131.1, 121.3, 52.3, 40.6.
IR (diamond cell, neat) νmax: 1734, 1471, 1435, 1340, 1219, 1158, 1026, 733, 660, 440 cm–1.
LRMS (+ESI) m/z: 251/253 [(M+Na)+, 95/100%].
12

ACCEPTED MANUSCRIPT
5.2.8. Methyl 3-(2-bromophenyl)propanoate (13)
Subjecting 2-bromophenyl propionic acid (0.560 g, 2.44 mmol) to the above general procedure
gave the title compound (0.583 g, 97%) as a pale yellow oil.
1H NMR (500 MHz, Chloroform-d): δ 7.52 (dd, J = 7.9, 1.1 Hz, 1H), 7.26 – 7.19 (m, 2H), 7.06 (ddd,
J = 8.1, 6.7, 2.4 Hz, 1H), 3.67 (s, 3H), 3.06 (dd, J = 8.3, 7.4 Hz, 2H), 2.65 (dd, J = 8.4, 7.4 Hz, 2H). 13
C
NMR (126 MHz, Chloroform-d): δ 173.2, 139.8, 133.0, 130.5, 128.2, 127.7, 124.4, 51.8, 34.0,
31.5. IR (diamond cell, neat) νmax: 2950, 1734, 1471, 1436, 1364, 1294, 1257, 1195, 1025, 781,
750, 656 cm–1. LRMS (+ESI) m/z: 265/267 [(M+Na)+, 95/100%].
5.2.9. Methyl 3-(3-bromophenyl)propanoate (14)
Subjecting 3-bromophenyl propionic acid (1.15 g, 5 mmol) to the above general procedure gave
the title compound (1.15 g, 95%) as a pale yellow oil.
1H NMR (500 MHz, Chloroform-d): δ 7.34 (d, J = 1.9 Hz, 1H), 7.31 (dt, J = 7.3, 2.0 Hz, 1H), 7.17 –
7.08 (m, 2H), 3.65 (s, 3H), 2.90 (t, J = 7.3 Hz, 2H), 2.60 (dd, J = 8.2, 7.3 Hz, 2H). 13C NMR (126
MHz, Chloroform-d): δ 172.9, 142.9, 131.4, 130.1, 129.4, 127.0, 122.5, 51.7, 35.3, 30.5. IR
(diamond cell, neat) νmax: 2950, 1732, 1596, 1567, 1475, 1436, 1363, 1297, 1196, 1165, 1071,
1028, 809, 780, 731, 687, 665, 428 cm–1. LRMS (+ESI) m/z: 265/267 [(M+Na)+, 95/100%].
5.2.10. Methyl 3-(4-bromophenyl)propanoate (15)
Subjecting 4-bromophenyl propionic acid (1.15 g, 5 mmol) to the above general procedure gave
the title compound (1.10 g, 90%) as a pale yellow oil.
1H NMR (500 MHz, Chloroform-d): δ 7.39 (d, J = 8.4 Hz, 2H), 7.06 (d, J = 8.5 Hz, 2H), 3.65 (s, 3H),
2.89 (t, J = 7.7 Hz, 2H), 2.60 (t, J = 7.7 Hz, 2H). 13C NMR (126 MHz, Chloroform-d): δ 173.0, 139.5,
131.6, 130.1, 120.1, 51.7, 35.4, 30.3. IR (diamond cell, neat) νmax: 2951, 1733, 1488, 1435, 295,
1257, 1195, 1072, 1010, 908, 694, 614 cm–1. LRMS (+ESI) m/z: 265/267 [(M+Na)+, 95/100%].
5.2.11. General procedure for the Buchwald-Hartwig amination of bromophenyl
alkanoates:
A stirred solution of RuPhos® (10 mol%, w/w) and Pd2(dba)3 (5 mol%, w/w) in dry, degassed
PhCH3 (25 mL) was treated with Boc-piperazine (2 eq), Cs2CO3 (1.5 eq), the required
bromophenyl alkanoate (1 eq) and heated at reflux for 18 h. The reaction volume was filtered
through a Celite® pad and the solids retained were washed with EtOAc (3 x 30 mL). The
combined organics were concentrated under reduced pressure and the resultant crude oil was
purified by flash chromatography (silica, Rf = 0.19-0.22 in a 1:5 v/v EtOAc/hexanes solution).
5.2.12. tert-Butyl 4-(2-(2-methoxy-2-oxoethyl)phenyl)piperazine-1-carboxylate (16)
Subjecting methyl 2-(2-bromophenyl)acetate (920 mg, 4 mmol) to the above general procedure
produced the title compound (740 mg, 55%) as a waxy solid (Rf = 0.19 in a 1:5 v/v
EtOAc/hexanes solution).
1H NMR (500 MHz, Chloroform-d): δ 7.29 – 7.24 (m, 2H), 7.14 – 7.10 (m, 2H), 3.71 (s, 2H), 3.69
(s, 3H), 3.53 (br s, 4H), 2.80 (br s, 4H), 1.48 (s, 9H). 13C NMR (126 MHz, Chloroform-d): δ 172.8,
155.0, 151.7, 131.2, 131.1, 128.5, 125.1, 121.4, 79.9, 52.6, 52.0, 44.0 (br), 37.4, 28.6. IR (diamond
cell, neat) νmax: 1735, 1689, 1418, 1365, 1247, 1160, 1001, 913, 864, 767, 728, 647 cm–1. LRMS
(+ESI) m/z: 357 [(M+Na)+, 100%], 691 [(2M+Na)+, 40%].
5.2.13. tert-Butyl 4-(3-(2-methoxy-2-oxoethyl)phenyl)piperazine-1-carboxylate (17)
13

ACCEPTED MANUSCRIPT
Subjecting methyl 2-(3-bromophenyl)acetate (920 mg, 4 mmol) to the above general procedure
produced the title compound (910 mg, 68%) as a waxy solid (Rf = 0.21 in a 1:5 v/v
EtOAc/hexanes solution).
1H NMR (500 MHz, Chloroform-d): δ 7.21 (td, J = 8.0, 2.7 Hz, 1H), 6.91 – 6.73 (m, 3H), 3.74 – 3.62
(m, 3H), 3.62 – 3.45 (m, 6H), 3.24 – 3.03 (m, 4H), 1.48 (s, 9H). 13C NMR (126 MHz, Chloroform-
d): δ 172.1, 154.8, 151.6, 135.0, 129.5, 121.2, 117.6, 115.3, 80.0, 52.1, 49.4, 43.5 (br), 41.6, 28.5.
IR (diamond cell, neat) νmax: 1736, 1690, 1601, 1419, 1365, 1239, 1157, 997, 970, 912, 868, 768,
729, 646 cm–1. LRMS (+ESI) m/z: 357 [(M+Na)+, 100%], 691 [(2M+Na)+, 5%].
5.2.14. tert-Butyl 4-(4-(2-methoxy-2-oxoethyl)phenyl)piperazine-1-carboxylate (18)
Subjecting methyl 2-(4-bromophenyl)acetate (920 mg, 4 mmol) to the above general procedure
produced the title compound (897 mg, 67%) as a waxy solid (Rf = 0.22 in a 1:5 v/v
EtOAc/hexanes solution).
1H NMR (500 MHz, Chloroform-d): δ 7.18 (dd, J = 8.5, 2.6 Hz, 2H), 6.88 (dd, J = 8.5, 2.6 Hz, 2H),
3.68 (d, J = 2.4 Hz, 3H), 3.60 – 3.50 (m, 6H), 3.11 (d, J = 5.5 Hz, 4H), 1.48 (s, 9H). 13C NMR (126
MHz, Chloroform-d): δ 172.5, 154.9, 150.4, 130.1, 125.7, 116.8, 80.1, 52.1, 49.5, 44.0 (br), 40.4,
28.5. IR (diamond cell, neat) νmax: 1736, 1690, 1516, 1419, 1365, 1227, 1158, 1000, 915, 810,
768, 574 cm–1. LRMS (+ESI) m/z: 357 [(M+Na)+, 100%].
5.2.15. tert-Butyl 4-(2-(3-methoxy-3-oxopropyl)phenyl)piperazine-1-carboxylate (19)
Subjecting methyl 3-(2-bromophenyl)propanoate (500 mg, 2 mmol) to the above general
procedure produced the title compound (500 mg, 70%) as a yellow oil (Rf = 0.14 in a 1:9 v/v
EtOAc/hexanes solution).
1H NMR (500 MHz, Chloroform-d): δ 7.24 – 7.15 (m, 2H), 7.11 – 7.04 (m, 2H), 3.68 (s, 3H), 3.57
(br s, 4H), 3.08 – 2.97 (m, 2H), 2.83 (br s, 4H), 2.72 – 2.61 (m, 2H), 1.49 (s, 9H).13C NMR (126
MHz, Chloroform-d): δ 173.9, 155.0, 151.5, 136.2, 129.8, 127.5, 124.8, 120.9, 79.9, 52.9, 51.7,
44.9 (broad), 34.9, 28.6, 26.8. IR (diamond cell, neat) νmax: 2975, 2950, 1736, 1691, 1491, 1451,
1418, 1365, 1321, 1246, 1222, 1163, 1120, 1001, 922, 863, 765, 731 cm–1. LRMS (+ESI) m/z:
371 [(M+Na)+, 100%], 719 [(2M+Na)+, 15%].
5.2.16. tert-Butyl 4-(3-(3-methoxy-3-oxopropyl)phenyl)piperazine-1-carboxylate (20)
Subjecting methyl 3-(3-bromophenyl)propanoate (972 mg, 4 mmol) to the above general
procedure produced the title compound (1.01 g, 72%) as a yellow oil (Rf = 0.12 in a 1:9 v/v
EtOAc/hexanes solution).
1H NMR (500 MHz, Chloroform-d): δ 7.18 (dd, J = 9.0, 7.4 Hz, 1H), 6.88 – 6.74 (m, 2H), 6.74 –
6.68 (m, 1H), 3.67 (s, 3H), 3.57 (t, J = 5.2 Hz, 4H), 3.12 (t, J = 5.1 Hz, 4H), 2.91 (t, J = 7.9 Hz, 2H),
2.62 (dd, J = 8.5, 7.2 Hz, 2H), 1.48 (s, 9H). 13C NMR (126 MHz, Chloroform-d): δ 173.4, 154.8,
151.6, 141.7, 129.4, 120.3, 116.8, 114.6, 80.0, 51.7, 49.5, 43.6 (broad), 35.9, 31.4, 28.5. IR
(diamond cell, neat) νmax: 2976, 1735, 1685, 1601, 1478, 1285, 1239, 1163, 1125, 1086, 907,
865, 727, 698, 646 cm–1. LRMS (+ESI) m/z: 371 [(M+Na)+, 100%], 719 [(2M+Na)+, 5%].
5.2.17. tert-Butyl 4-(4-(3-methoxy-3-oxopropyl)phenyl)piperazine-1-carboxylate (21)
Subjecting methyl 3-(4-bromophenyl)propanoate (972 mg, 4 mmol) to the above general
procedure produced the title compound (1.32 g, 95%) as a white solid (Rf = 0.21 in a 1:5 v/v
EtOAc/hexanes solution).
1H NMR (500 MHz, Chloroform-d): δ 7.09 (d, J = 8.6 Hz, 2H), 6.85 (d, J = 8.6 Hz, 2H), 3.65 (s, 3H),
3.56 (t, J = 5.2 Hz, 4H), 3.08 (t, J = 5.2 Hz, 4H), 2.87 (t, J = 7.8 Hz, 2H), 2.58 (dd, J = 8.5, 7.2 Hz, 2H),
1.48 (s, 9H). 13C NMR (126 MHz, Chloroform-d): δ 173.5, 154.8, 149.8, 132.4, 129.1, 116.9, 79.9,
51.6, 49.7, 43.6 (broad), 36.0, 30.2, 28.5. IR (diamond cell, neat) νmax: 2813, 1727, 1677, 1515,
14

ACCEPTED MANUSCRIPT
1454, 1414, 1366, 1333, 1305, 1282, 1191, 1174, 1078, 1044, 999, 981, 931, 913, 846, 827, 692,
542 cm–1. LRMS (+ESI) m/z: 371 [(M+Na)+, 100%]. MP: 88 – 91 °C.
5.2.18. General procedure for the saponification of the methyl esters (16 – 21).
A stirred solution of the appropriate alkanoate (1 eq) in MeOH:H2O (10 mL of a 9:1 v/v solution)
was treated with LiOH (3 eq) and heated at reflux for 1 h. The MeOH was removed under
reduced pressure and the resultant aqueous solution washed with ether (3 x 50 mL). The
aqueous solution was acidified (pH 6) with HCl (4M aq. solution) and extracted with CH2Cl2 (3 x
25 mL), dried (MgSO4), filtered and concentrated under reduced pressure. The resultant solids
were purified by column chromatography (silica, Rf = 0.19-0.22 in a 1:1 v/v EtOAc/hexanes
solution).
5.2.19. 2-(2-(4-(tert-Butoxycarbonyl)piperazin-1-yl)phenyl)acetic acid (22)
Subjecting ester 16 (800 mg, 2.4 mmol) to the above general procedure produced the title
compound (704 mg, 91%) as white crystalline needles (Rf = 0.21 in a 1:1 v/v EtOAc/hexanes
solution).
1H NMR (500 MHz, Chloroform-d): δ 7.45 – 7.07 (m, 4H), 3.75 (s, 2H), 3.63 (br s, 4H), 2.96 (t, J =
5.2 Hz, 4H), 1.49 (s, 9H), OH signal not observed. 13C NMR (126 MHz, Chloroform-d): δ 173.5,
154.7, 149.1, 132.1, 130.0, 129.2, 126.7, 121.4, 80.5, 52.6, 43.5 (broad), 40.4, 28.6. IR (diamond
cell, neat) νmax: 3200, 1733, 1661, 1518, 1438, 1371, 1317, 1255, 1133, 1043, 1002, 910, 804,
761, 651, 604, 529 cm–1. LRMS (+ESI) m/z: 343 [(M+Na)+, 100%]. MP: 141 – 142 °C.
5.2.20. 2-(3-(4-(tert-Butoxycarbonyl)piperazin-1-yl)phenyl)acetic acid (23)
Subjecting ester 17 (800 mg, 2.4 mmol) to the above general procedure produced the title
compound (710 mg, 92%) as a white powder (Rf = 0.19 in a 1:1 v/v EtOAc/hexanes solution).
1H NMR (500 MHz, Chloroform-d): δ 7.23 (dd, J = 15.5, 7.5 Hz, 1H), 6.87 – 6.78 (m, 3H), 3.62 –
3.54 (m, 6H), 3.13 (t, J = 4.7 Hz, 4H), 1.48 (d, J = 2.1 Hz, 9H), OH signal not observed. 13C NMR
(126 MHz, Chloroform-d): δ 176.9, 154.9, 151.6, 134.5, 129.6, 121.4, 117.8, 115.6, 80.0, 49.4,
43.7 (broad), 41.4, 28.6. IR (diamond cell, neat) νmax: 3190, 1728, 1664, 1430, 1367, 1319, 1266,
1155, 1131, 1031, 1004, 935, 834, 770, 752, 678, 600, 535, 459 cm–1. LRMS (+ESI) m/z: 343
[(M+Na)+, 100%]. MP: 136 – 138 °C.
5.2.21. 2-(4-(4-(tert-Butoxycarbonyl)piperazin-1-yl)phenyl)acetic acid (24)
Subjecting ester 18 (800 mg, 2.4 mmol) to the above general procedure produced the title
compound (710 mg, 92%) as yellow cubes (Rf = 0.21 in a 1:1 v/v EtOAc/hexanes solution).
1H NMR (500 MHz, Chloroform-d) δ 7.20 – 7.13 (m, 2H), 6.98 – 6.71 (m, 2H), 3.75 – 3.42 (m,
6H), 3.11 (t, J = 4.6 Hz, 4H), 1.48 (s, 9H). OH signal not observed. 13C NMR (126 MHz,
Chloroform-d): δ 177.4, 154.9, 150.6, 130.3, 125.1, 116.9, 80.1, 49.5, 44.3 (broad), 40.2, 28.6. IR
(diamond cell, neat) νmax: 2977, 1733, 1644, 1433, 1367, 1275, 1241, 1161, 995, 971, 845, 779,
733, 693, 971, 599, 539, 428 cm–1. LRMS (+ESI) m/z: 343 [(M+Na)+, 100%]. MP: 121 – 122 °C.
5.2.22. 3-(2-(4-(tert-Butoxycarbonyl)piperazin-1-yl)phenyl)propanoic acid (25)
Subjecting ester 19 (300 mg, 0.86 mmol) to the above general produced the title compound
(224 mg, 78%) as a white crystalline powder (Rf = 0.31 in a 1:1 v/v EtOAc/hexanes solution).
1H NMR (400 MHz, Methanol-d4): δ 7.23 – 7.19 (m, 1H), 7.19 – 7.15 (m, 1H), 7.12 (dd, J = 8.0, 1.5
Hz, 1H), 7.04 (td, J = 7.3, 1.5 Hz, 1H), 3.56 (t, J = 4.9 Hz, 4H), 3.00 (dd, J = 8.5, 7.1 Hz, 2H), 2.82 (t, J
= 5.0 Hz, 4H), 2.62 (dd, J = 8.5, 7.1 Hz, 2H), 1.48 (s, 9H), OH signal not observed. 13C NMR (101
15

ACCEPTED MANUSCRIPT
MHz, Methanol-d4): δ 173.5, 154.7, 149.1, 132.1, 130.0, 129.2, 126.7, 121.4, 80.5, 52.6, 43.5
(broad), 40.4, 31.6, 28.6. IR (diamond cell, neat) νmax: 3217, 2972, 1729, 1640, 1490, 1364, 1323,
1167, 1148, 1130, 921, 758, 575 cm–1. LRMS (+ESI) m/z: 357 [(M+Na)+, 100%]. MP: 134 – 135
°C.
5.2.23. 3-(3-(4-(tert-Butoxycarbonyl)piperazin-1-yl)phenyl)propanoic acid (26)
Subjecting ester 20 (300 mg, 0.86 mmol) to the above general produced the title compound
(271 mg, 94%) as a red oil (Rf = 0.22 in a 1:1 v/v EtOAc/hexanes solution).
1H NMR (400 MHz, Methanol-d4): δ 7.15 (t, J = 7.9 Hz, 1H), 6.85 (t, J = 2.0 Hz, 1H), 6.80 (ddd, J =
8.2, 2.5, 0.9 Hz, 1H), 6.74 (dt, J = 7.7, 1.1 Hz, 1H), 3.55 (t, J = 5.1 Hz, 4H), 3.09 (t, J = 5.1 Hz, 4H),
2.86 (t, J = 7.7 Hz, 2H), 2.57 (t, J = 7.7 Hz, 2H), 1.48 (s, 9H), OH signal not observed. 13C NMR
(101 MHz, Methanol-d4): δ 176.7, 156.4, 152.9, 143.1, 130.2, 121.5, 118.1, 115.9, 81.3, 50.8, 44.9
(broad), 36.8, 32.3, 28.7. IR (diamond cell, neat) νmax: 2976, 1733, 1691, 1601, 1492, 1423, 1366,
1239, 1160, 997, 957, 836, 774, 734, 696 cm–1. LRMS (+ESI) m/z: 357 [(M+Na)+, 100%].
5.2.24. 3-(4-(4-(tert-Butoxycarbonyl)piperazin-1-yl)phenyl)propanoic acid (27)
Subjecting ester 21 (300 mg, 0.86 mmol) to the above general produced the title compound
(224 mg, 78%) as an off-white powder (Rf = 0.22 in a 1:1 v/v EtOAc/hexanes solution).
1H NMR (400 MHz, Methanol-d4): δ 8.67 (d, J = 8.6 Hz, 2H), 8.46 (d, J = 8.6 Hz, 2H), 5.11 (t, J = 5.1
Hz, 4H), 4.74 – 4.52 (m, 4H), 4.39 (t, J = 7.6 Hz, 2H), 4.10 (t, J = 7.6 Hz, 2H), 1.04 (s, 9H), OH signal
not observed. 13C NMR (101 MHz, Methanol-d4): δ 176.8, 156.4, 151.2, 134.3, 130.0, 118.3, 81.3,
51.1, 45.0 (broad), 36.9, 31.2, 28.7. IR (diamond cell, neat) νmax: 2970, 1692, 1616, 1517, 1478,
1419, 1390, 1364, 1287, 1159, 1116, 1085, 866, 819, 768, 596, 522 cm–1. LRMS (+ESI) m/z: 357
[(M+Na)+, 100%]. MP: 146 – 148 °C.
5.2.25. General procedure for the amidation of diazepine 9.
An ice cold magnetically stirred solution of the required phenylalkanoic acid (1 eq), amine 9 (1
i
eq) and Pr2NEt (2 eq) in CH2Cl2 (5 mL) was treated with PyBOP® (1 eq), allowed to warm to
room temperature and stirring continued for 12 h. The solution was diluted with CH2Cl2 (50
mL) and water (50 mL) and the separated organic phase was subsequently washed with
NaHCO3 (25 mL of a sat. aq. solution) and brine (100 mL) before being dried (MgSO4), filtered,
concentrated and purified via flash column chromatography (silica, Rf = 0.24-0.36 in EtOAc).
5.2.26. tert-Butyl 4-(2-(2-(1-methyl-4,10-dihydrobenzo[b]pyrazolo[3,4-e][1,4]diazepin-
5(1H)-yl)-2-oxoethyl)phenyl)piperazine-1-carboxylate (28)
Subjecting 22 (200 mg, 0.62 mmol) to the above general procedure in CH2Cl2 (5 mL) produced
the title compound (220 mg, 71%) as a white powder (Rf = 0.36 in EtOAc).
1H NMR (500 MHz, Chloroform-d): δ 7.22 – 7.10 (m, 3H), 7.08 (dd, J = 7.9, 1.5 Hz, 1H), 7.03 (dd, J
= 7.7, 1.6 Hz, 1H), 6.98 – 6.82 (m, 4H), 5.90 (br s, 1H), 5.63 (d, J = 14.5 Hz, 1H), 3.80 (d, J = 14.5
Hz, 1H), 3.74 – 3.58 (m, 4H), 3.48 (d, J = 15.5 Hz, 1H), 3.31 (br s, 4H), 2.48 – 2.33 (m, 4H), 1.47 (s,
9H). 13C NMR (126 MHz, Chloroform-d): δ 171.2, 155.0, 151.3, 138.9, 138.7, 136.5, 131.6, 131.0,
130.5, 130.5, 129.0, 127.7, 124.2, 122.4, 120.1, 119.7, 101.8, 80.0, 52.1, 44.0 (broad), 43.5, 37.2,
34.7, 28.6. IR (diamond cell, neat) νmax: 3220, 1633, 1560, 1401, 1249, 1174, 1131, 846, 757,
556, 454 cm–1. LRMS (+ESI) m/z: 525 [(M+Na)+, 100%]. MP: 161 – 163 °C.
5.2.27. tert-Butyl 4-(3-(2-(1-methyl-4,10-dihydrobenzo[b]pyrazolo[3,4-e][1,4]diazepin-
5(1H)-yl)-2-oxoethyl)phenyl)piperazine-1-carboxylate (29)
Subjecting 23 (400 mg, 1.24 mmol) to the above general procedure in CH2Cl2 (10 mL) produced
the title compound (415 mg, 67%) as an off-white solid (Rf = 0.24 in EtOAc).
16

ACCEPTED MANUSCRIPT
1H NMR (500 MHz, Chloroform-d): 7.23 (td, J = 8.0, 1.6 Hz, 1H), 7.19 (s, 1H), 7.14 (dd, J = 7.9, 1.5
Hz, 1H), 7.05 – 6.95 (m, 2H), 6.82 (dd, J = 8.1, 1.4 Hz, 1H), 6.72 (dd, J = 7.9, 2.3 Hz, 1H), 6.47 (t, J =
2.0 Hz, 1H), 6.30 (d, J = 8.0 Hz, 1H), 5.55 (br s, 1H), 5.60 (d, J = 14.5 Hz, 1H), 3.81 (d, J = 14.5 Hz,
1H), 3.62 (s, 3H), 3.52 (br s, 4H), 3.42 (d, J = 5.5 Hz, 2H), 2.97 (br s, 4H), 1.48 (s, 9H). 13C NMR
(126 MHz, Chloroform-d): δ 170.7, 154.9, 151.2, 139.0, 138.8, 136.6, 135.9, 131.9, 130.8, 129.3,
128.9, 122.5, 121.0, 119.7, 116.9, 115.0, 101.6, 80.1, 49.4, 44.2 (broad), 43.6, 41.8, 34.7, 28.6. IR
(diamond cell, neat) νmax: 3201, 1697, 1627, 1561, 1507, 1408, 1234, 1164, 1122, 1053, 973,
833, 765, 740, 704, 629, 534, 457 cm–1. LRMS (+ESI) m/z: 525 [(M+Na)+, 100%]. MP: 158 – 160
°C.
5.2.28. tert-Butyl 4-(4-(2-(1-methyl-4,10-dihydrobenzo[b]pyrazolo[3,4-e][1,4]diazepin-
5(1H)-yl)-2-oxoethyl)phenyl)piperazine-1-carboxylate (30)
Subjecting 24 (400 mg, 1.24 mmol) to the above general procedure in CH2Cl2 (10 mL) produced
the title compound (520 mg, 81%) as an off-white solid (Rf = 0.22 in EtOAc).
1H NMR (500 MHz, Chloroform-d): 7.28 – 7.24 (m, 1H), 7.17 (s, 1H), 7.14 (dd, J = 7.9, 1.6 Hz, 1H),
7.02 (td, J = 7.5, 1.4 Hz, 1H), 6.92 (dd, J = 8.1, 1.4 Hz, 1H), 6.82 (d, J = 8.7 Hz, 1H), 6.73 (d, J = 8.7
Hz, 2H), 5.78 (br s, 1H), 5.59 (d, J = 14.6 Hz, 1H), 3.82 (d, J = 14.6 Hz, 1H), 3.64 (s, 3H), 3.55 (t, J =
5.2 Hz, 4H), 3.35 (s, 2H), 3.05 (t, J = 5.2 Hz, 4H), 1.47 (s, 9H). 13C NMR (126 MHz, Chloroform-d):
δ 171.0, 154.9, 150.1, 139.1, 138.9, 136.5, 132.0, 130.7, 130.0, 129.3, 126.8, 122.6, 119.9, 116.5,
101.6, 80.1, 49.7, 43.7, 43.6, 40.3, 34.7, 28.6. IR (diamond cell, neat) νmax: 3202, 1639, 1555,
1502, 1390, 1229, 1161, 839, 760, 556 cm–1. LRMS (+ESI) m/z: 525 [(M+Na)+, 100%]. MP: 161 –
163 °C.
5.2.29. tert-Butyl 4-(2-(3-(1-methyl-4,10-dihydrobenzo[b]pyrazolo[3,4-e][1,4]diazepin-
5(1H)-yl)-3-oxopropyl)phenyl)piperazine-1-carboxylate (31)
Subjecting 25 (200 mg, 0.6 mmol) to the above general procedure in CH2Cl2 (5 mL) produced
the title compound (220 mg, 71%) as an off-white solid (Rf = 0.21 in EtOAc).
1H NMR (400 MHz, Chloroform-d) δ 7.23 – 7.16 (m, 2H), 7.11 (ddd, J = 8.0, 7.1, 1.7 Hz, 1H), 7.04
(dd, J = 8.0, 1.6 Hz, 1H), 6.98 (dd, J = 8.2, 1.3 Hz, 1H), 6.97 – 6.84 (m, 4H), 6.32 (br s, 1H), 5.64 (d,
J = 14.5 Hz, 1H), 3.80 (d, J = 14.5 Hz, 1H), 3.68 (s, 3H), 3.40 (t, J = 5.0 Hz, 4H), 2.91 – 2.75 (m, 2H),
2.64 (t, J = 4.8 Hz, 4H), 2.51 – 2.23 (m, 2H), 1.48 (s, 9H). 13C NMR (101 MHz, Chloroform-d): δ
172.1, 155.2, 151.4, 139.0(3), 139.9(9), 136.9, 136.5, 131.9, 130.4, 130.3, 129.1, 127.1, 124.5,
122.5, 120.6, 119.9, 101.7, 79.9, 52.6, 44.3 (broad), 43.2, 34.9, 34.5, 28.6, 27.6. IR (diamond cell,
neat) νmax: 2817, 1635, 1557, 1503, 1392, 1365, 1277, 1246, 1163, 1123, 1087, 1035, 842, 760,
533 cm–1. LRMS (+ESI) m/z: 539 [(M+Na)+, 100%]. MP: 118 – 120 °C.
5.2.30. tert-Butyl 4-(3-(3-(1-methyl-4,10-dihydrobenzo[b]pyrazolo[3,4-e][1,4]diazepin-
5(1H)-yl)-3-oxopropyl)phenyl)piperazine-1-carboxylate (32)
Subjecting 26 (200 mg, 0.6 mmol) to the above general procedure in CH2Cl2 (10 mL) produced
the title compound (241 mg, 78%) as an off-white solid (Rf = 0.24 in EtOAc).
1H NMR (400 MHz, Chloroform-d) δ 7.27 – 7.21 (m, 1H), 7.19 (s, 1H), 7.11 – 7.05 (m, 1H), 7.05 –
7.02 (m, 1H), 7.02 – 6.93 (m, 2H), 6.73 – 6.66 (m, 1H), 6.55 (t, J = 1.9 Hz, 1H), 6.50 (d, J = 7.5 Hz,
1H), 5.83 (br s, 1H), 5.65 (d, J = 14.5 Hz, 1H), 3.81 (d, J = 14.5 Hz, 1H), 3.70 (s, 3H), 3.53 (t, J = 5.2
Hz, 4H), 3.01 (t, J = 5.6 Hz, 4H), 2.80 – 2.63 (m, 2H), 2.41 – 2.22 (m, 2H), 1.48 (s, 9H). 13C NMR
(101 MHz, Chloroform-d): δ 171.8, 154.9, 151.4, 142.5, 139.1, 138.9, 136.5, 132.2, 130.6, 129.2,
129.1, 122.9, 120.4, 120.0, 117.0, 114.3, 101.6, 80.1, 49.5, 43.6 (broad), 43.1, 36.1, 34.8, 32.1,
28.6. IR (diamond cell, neat) νmax: 2817, 1637, 1600, 1557, 1502, 1391, 1365, 1285, 1240, 1161,
1126, 997, 840, 162, 696, 557 cm–1. LRMS (+ESI) m/z: 539 [(M+Na)+, 100%]. MP: 101 – 103 °C.
17

ACCEPTED MANUSCRIPT
5.2.31. tert-Butyl 4-(4-(3-(1-methyl-4,10-dihydrobenzo[b]pyrazolo[3,4-e][1,4]diazepin-
5(1H)-yl)-3-oxopropyl)phenyl)piperazine-1-carboxylate (33)
Subjecting 27 (200 mg, 0.6 mmol) to the above general procedure in CH2Cl2 (10 mL) produced
the title compound (241 mg, 78%) as an off-white solid (Rf = 0.24 in EtOAc).
1H NMR (400 MHz, Chloroform-d) δ 7.25 – 7.20 (m, 1H), 7.18 (s, 1H), 7.06 – 6.94 (m, 3H), 6.89
(d, J = 8.6 Hz, 2H), 6.74 (d, J = 8.6 Hz, 2H), 6.17 (s, 1H), 5.62 (d, J = 14.6 Hz, 1H), 3.81 (d, J = 14.6
Hz, 1H), 3.69 (s, 3H), 3.57 – 3.47 (m, 4H), 3.02 (t, J = 5.2 Hz, 4H), 2.87 – 2.62 (m, 2H), 2.41 – 2.21
(m, 2H), 1.47 (s, 9H). 13C NMR (101 MHz, Chloroform-d): δ 172.0, 154.9, 149.6, 139.2, 139.1,
136.4, 133.1, 131.9, 130.4, 129.3, 129.2, 122.8, 120.0, 116.8, 101.6, 80.0, 49.8, 43.7 (broad), 43.2,
36.1, 34.8, 30.7, 28.6. IR (diamond cell, neat) νmax: 3200, 1636, 1611, 1558, 1504, 1391, 1365,
1284, 1249, 1230, 1161, 1127, 1000, 839, 762, 557 cm–1. LRMS (+ESI) m/z: 539 [(M+Na)+,
100%]. MP:121 – 123 °C.
5.2.32. General procedure for the one pot carbamate de-protection/reductive amination
of the protected piperazines (28 – 33).
A magnetically stirred solution of the required 1-boc-Piperazine (1 eq) in MeOH (5 mL) was
treated with HCl (1.5 eq of a 4M solution in dioxane) and stirring continued at room
temperature for 3 h. The resultant solution was concentrated under a stream of nitrogen,
followed by reduced pressure. The resultant foams were re-dissolved in MeOH (5 mL) and the
pH adjusted (pH = 6) with N-methylmorpholine (4-8 drops), treated with the appropriate
benzaldehyde (2 eq), MgSO4 (20% w/w) and magnetically stirred at room temperature for 1 h.
The resulting suspension was then treated with NaCNBH3 (1.2 eq) and stirred for a further 18 h.
The solvent was then removed under reduced pressure and the residue dissolved in EtOAc
(20 mL) and NaHCO3 (20 mL of a sat. aq. solution). The separated organic phase was washed
with brine (50 mL) before being dried (MgSO4), filtered and concentrated under reduced
pressure to give off-white solids. The crude solids were purified by flash column
chromatography (silica, EtOAc followed by 5:95 v/v MeOH(sat. with ammonia)/CH2Cl2
solution)(Rf = 0.08-0.23 in EtOAc).
5.2.32.
2-(2-(4-(3,5-Dihydroxybenzyl)piperazin-1-yl)phenyl)-1-(1-methyl-4,10-
dihydrobenzo[b]pyrazolo[3,4-e][1,4]diazepin-5(1H)-yl)ethan-1-one (3a)
Subjecting 28 (100 mg, 0.2 mmol) to the above general procedure using 3,5-
dihydroxybenzaldehyde (55 mg, 0.4 mmol) produced the title compound 3a (64 mg, 61%) as a
white powder (Rf = 0.12 in EtOAc).
1H NMR (500 MHz, MeOD-d4): δ 7.19 (ddd, J = 8.6, 7.2, 1.6 Hz, 1H), 7.16 (s, 1H), 7.13 (ddd, J = 8.6,
7.2, 2.0 Hz, 1H), 7.10 – 7.18 (m, 2H), 6.97 (dd, J = 8.0, 1.2 Hz, 1H), 6.93 (dd, J = 7.6, 1.4 Hz, 1H),
6.90 (dd, J = 8.3, 1.6 Hz, 1H), 6.86 (td, J = 7.4, 1.2 Hz, 1H), 6.31 (d, J = 2.2 Hz, 2H), 6.21 (t, J = 2.2
Hz, 1H), 5.51 (d, J = 14.5 Hz, 1H), 3.78 (d, J = 14.5 Hz, 1H), 3.75 – 3.62 (m, 4H), 3.46 (d, J = 15.6
Hz, 1H), 3.43 – 3.36 (m, 2H), 2.67 – 2.55 (m, 4H), 2.41 (br s, 4H), OH signals and NH signal not
observed. 13C NMR (126 MHz, MeOD-d4): δ 173.7, 159.5, 152.7, 141.6, 140.3, 140.2, 137.3, 132.2,
131.6, 131.0(4), 130.9(5), 130.2, 128.7, 124.8, 123.0, 121.0, 120.9, 109.2, 102.6(5), 102.6(4),
64.0, 54.2, 52.8, 44.8, 37.9, 35.2. IR (diamond cell, neat) νmax: 2920, 1595, 1552, 1494, 1449,
1391, 1292, 1156, 999, 842, 757, 696, 554, 454, 428 cm–1. LRMS (+ESI) m/z: 525 [(M+H)+,
100%], 547 [(M+Na)+, 29%]. HRMS (+ESI) Found: (M+H)+, 525.2609. C30H32N6O3 requires
(M+H)+, 525.2609. MP: 192 – 194 °C. HPLC purity: 99.69%, RT: 14.44 min.
5.2.33.
2-(3-(4-(3,5-Dihydroxybenzyl)piperazin-1-yl)phenyl)-1-(1-methyl-4,10-
dihydrobenzo[b]pyrazolo[3,4-e][1,4]diazepin-5(1H)-yl)ethan-1-one (3b)
Subjecting 29 (100 mg, 0.2 mmol) to the above general procedure using 3,5-
dihydroxybenzaldehyde (55.0 mg, 0.4 mmol) produced the title compound 3b (74 mg, 71%) as
a white powder (Rf = 0.13 in EtOAc).
18

ACCEPTED MANUSCRIPT
1H NMR (500 MHz, DMSO-d6) δ 9.13 (br s, 2H), 8.35 (br s, 1H), 7.34 – 7.24 (m, 2H), 7.21 (dd, J =
7.8, 1.4 Hz, 1H), 7.07 (s, 1H), 7.04 – 6.93 (m, 2H), 6.71 (dd, J = 8.3, 2.4 Hz, 1H), 6.39 (t, J = 2.0 Hz,
1H), 6.34 (d, J = 7.4 Hz, 1H), 6.22 (t, J = 1.7 Hz, 2H), 6.11 (d, J = 2.0 Hz, 1H), 5.40 (d, J = 14.5 Hz,
1H), 3.76 (d, J = 14.5 Hz, 1H), 3.66 (s, 3H), 3.32 (s, 2H), 3.31 – 3.22 (m, 2H), 2.99 (t, J = 5.3 Hz,
4H), 2.46 (br s, 4H). 13C NMR (126 MHz, DMSO-d6): δ 169.4, 158.3, 158.1, 150.8, 139.4, 139.2,
136.1, 135.7, 131.1, 130.3, 128.8, 128.5, 121.2, 119.8, 119.6, 115.7, 113.6, 106.8, 106.7, 100.6,
62.3, 52.6, 48.2, 43.0, 40.0, 35.3. IR (diamond cell, neat) νmax: 3079, 1626, 1598, 1560, 1504,
1447, 1405, 1341, 1294, 1251, 1155, 995, 757, 740, 689, 450 cm–1. LRMS (+ESI) m/z: 525
[(M+H)+, 95%], 547 [(M+Na)+, 100%]. HRMS (+ESI) Found: (M+Na)+, 547.2428. C30H32N6O3
requires (M+Na)+, 547.2428. MP: 192 – 195 °C. HPLC purity: 99.36%, RT: 15.49 min.
5.2.34.
2-(4-(4-(3,5-Dihydroxybenzyl)piperazin-1-yl)phenyl)-1-(1-methyl-4,10-
dihydrobenzo[b]pyrazolo[3,4-e][1,4]diazepin-5(1H)-yl)ethan-1-one (3c)
Subjecting 30 (100 mg, 0.2 mmol) to the above general procedure using 3,5-
dihydroxybenzaldehyde (55.0 mg, 0.4 mmol) produced the title compound 3c (104 mg, quant.)
as a white powder (Rf = 0.08 in EtOAc).
1H NMR (500 MHz, MeOD-d4): δ 7.30 (t, J = 7.8 Hz, 1H), 7.20 (d, J = 7.8 Hz, 1H), 7.18 – 7.14 (m,
2H), 7.03 (t, J = 7.6 Hz, 1H), 6.74 (d, J = 8.2 Hz, 2H), 6.67 (d, J = 8.2 Hz, 2H), 6.34 (d, J = 2.2 Hz,
2H), 6.23 (d, J = 2.2 Hz, 1H), 5.47 (d, J = 14.4 Hz, 1H), 3.82 (d, J = 14.4 Hz, 1H), 3.64 (s, 3H), 3.45
(s, 2H), 3.34 (dd, J = 3.5, 1.8 Hz, 2H), 3.13 (d, J = 5.1 Hz, 4H), 2.63 (t, J = 5.1 Hz, 4H), OH signals
and NH signal not observed. 13C NMR (126 MHz, MeOD-d4): δ 173.4, 159.5, 151.3, 141.5, 140.5,
140.3, 137.3, 132.3, 131.1, 130.5, 130.4, 127.1, 122.8, 121.0, 117.1, 109.1, 102.7, 102.5, 64.0,
54.1, 50.1, 45.0, 41.2, 35.1. IR (diamond cell, neat) νmax: 2922, 2336, 2158, 2016, 1695, 1545,
1499, 1447, 1389, 1345, 1230, 1156, 974, 824, 756, 535, 457 cm–1. LRMS (+ESI) m/z: 525
[(M+H)+, 60%], 547 [(M+Na)+, 100%]. HRMS (+ESI) Found: (M+Na)+, 547.2429. C30H32N6O3
requires (M+Na)+, 547.2428. MP: 182 – 184 °C. HPLC purity: 98.67%, RT: 15.50 min.
5.2.35.
3-(2-(4-(3,5-Dihydroxybenzyl)piperazin-1-yl)phenyl)-1-(1-methyl-4,10-
dihydrobenzo[b]pyrazolo[3,4-e][1,4]diazepin-5(1H)-yl)propan-1-one (3d)
Subjecting 31 (100 mg, 0.19 mmol) to the above general procedure using 3,5-
dihydroxybenzaldehyde (27 mg, 0.2 mmol) produced the title compound 3d (100 mg, 93%) as a
white powder (Rf = 0.09 in EtOAc).
1H NMR (400 MHz, MeOD-d4): δ 7.30 (ddd, J = 8.5, 7.0, 1.6 Hz, 1H), 7.23 (dd, J = 8.2, 1.6 Hz, 1H),
7.17 (s, 1H), 7.16 – 7.12 (m, 1H), 7.10 – 7.04 (m, 2H), 7.02 – 6.89 (m, 1H), 6.92 – 6.83 (m, 2H),
6.39 (d, J = 2.2 Hz, 2H), 6.30 (s, 1H), 5.52 (d, J = 14.5 Hz, 1H), 3.80 (d, J = 14.5 Hz, 1H), 3.74 – 3.70
(m, 5H), 2.85 (t, J = 7.1 Hz, 2H), 2.80 (br s, 8H), 2.47 (t, J = 7.3 Hz, 2H), OH signals and NH signal
not observed. 13C NMR (101 MHz, MeOD-d4): δ 174.4, 159.9, 152.0, 141.4, 140.3, 137.7,
137.2(2), 137.2(1), 132.5, 131.1(2), 131.1(0), 130.3, 128.3, 125.7, 123.1, 121.8, 121.2, 109.6,
103.6, 102.6, 63.3, 54.3, 52.4, 44.5, 35.6, 35.2, 28.2. IR (diamond cell, neat) νmax: 3301, 2931,
1624, 1599, 1559, 1491, 1396, 1351, 1293, 1250, 1224, 1200, 1017, 764, 670, 488 cm–1. LRMS
(+ESI) m/z: 539 [(M+H)+, 100%], 561 [(M+Na)+, 45%]. HRMS (+ESI) Found: (M+Na)+, 561.2587.
C31H34N6O3 requires (M+Na)+, 561.2585. MP: 201 – 203 °C. HPLC purity: 97.24%, RT: 15.70 min.
5.2.36.
3-(3-(4-(3,5-Dihydroxybenzyl)piperazin-1-yl)phenyl)-1-(1-methyl-4,10-
dihydrobenzo[b]pyrazolo[3,4-e][1,4]diazepin-5(1H)-yl)propan-1-one (3e)
Subjecting 32 (100 mg, 0.19 mmol) to the above general procedure using 3,5-
dihydroxybenzaldehyde (27 mg, 0.2 mmol) produced the title compound 3e (58 mg, 54%) as a
white powder (Rf = 0.07 in EtOAc).
1H NMR (400 MHz, MeOD-d4): δ 7.32 – 7.21 (m, 2H), 7.15 (s, 1H), 7.04 – 6.99 (m, 1H), 6.99 – 6.95
(m, 2H), 6.74 (dd, J = 8.1, 2.4 Hz, 1H), 6.51 (t, J = 2.0 Hz, 1H), 6.42 (d, J = 7.4 Hz, 1H), 6.33 (d, J =
19

ACCEPTED MANUSCRIPT
2.2 Hz, 2H), 6.21 (t, J = 2.2 Hz, 1H), 5.50 (d, J = 14.5 Hz, 1H), 3.79 (d, J = 14.5 Hz, 1H), 3.70 (s, 3H),
3.44 (s, 2H), 3.12 – 2.99 (m, 4H), 2.68 (t, J = 7.1 Hz, 2H), 2.61 (t, J = 5.0 Hz, 4H), 2.47 – 2.18 (m,
2H), 2 OH signals and 1 NH signal not observed. 13C NMR (101 MHz, MeOD-d4): δ 174.1, 159.6,
152.7, 142.9, 141.5, 140.6, 140.3, 137.2, 132.7, 131.1, 130.4, 130.0, 123.3, 121.3, 121.0, 117.4,
115.4, 109.1, 102.7, 102.3, 64.0, 54.1, 50.1, 44.5, 37.1, 35.2, 32.9. IR (diamond cell, neat) νmax
:
2820, 1599, 1559, 1501, 1446, 1396, 1342, 1296, 1248, 1151, 1021, 996, 833, 763, 671, 628,
452 cm–1. LRMS (+ESI) m/z: 539 [(M+H)+, 100%], 561 [(M+Na)+, 28%]. HRMS (+ESI) Found:
(M+H)+, 539.2767. C31H34N6O3 requires (M+H)+, 539.2765. MP: 214 – 216 °C. HPLC purity:
97.48%, RT: 16.02 min.
5.2.37.
3-(4-(4-(3,5-Dihydroxybenzyl)piperazin-1-yl)phenyl)-1-(1-methyl-4,10-
dihydrobenzo[b]pyrazolo[3,4-e][1,4]diazepin-5(1H)-yl)propan-1-one (3f)
Subjecting 33 (100 mg, 0.19 mmol) to the above general procedure using 3,5-
dihydroxybenzaldehyde (27 mg, 0.2 mmol) produced the title compound 3f (92 mg, 85%) as a
white powder (Rf = 0.12 in EtOAc).
1H NMR (400 MHz, MeOD-d4): δ 7.34 – 7.22 (m, 2H), 7.15 (s, 1H), 7.02 – 6.96 (m, 2H), 6.81 (d, J =
8.7 Hz, 2H), 6.78 – 6.70 (m, 2H), 6.31 (d, J = 2.2 Hz, 2H), 6.19 (t, J = 2.2 Hz, 1H), 5.50 (d, J = 14.5
Hz, 1H), 3.79 (d, J = 14.5 Hz, 1H), 3.70 (s, 3H), 3.40 (s, 2H), 3.09 (t, J = 5.0 Hz, 4H), 2.66 (t, J = 5.0
Hz, 2H), 2.59 (t, J = 5.0 Hz, 4H), 2.47 – 2.18 (m, 2H). OH signals and NH signal not observed. 13
C
NMR (101 MHz, MeOD-d4): δ 174.2, 159.5, 151.0, 141.5, 140.5(2), 140.5(0), 137.3, 133.7, 132.7,
131.1, 130.4, 129.8, 123.2, 121.2, 117.5, 109.0, 102.6, 102.4, 64.1, 54.1, 50.5, 44.5, 37.0, 35.2,
31.6. IR (diamond cell, neat) νmax: 2920, 1598, 1558, 1502, 1447, 1395, 1349, 1295, 1248, 1230,
1149, 998, 829, 761, 743, 706 cm–1. LRMS (+ESI) m/z: 539 [(M+H)+, 33%], 561 [(M+Na)+,
100%],. HRMS (+ESI) Found: (M+H)+, 539.2767. C31H34N6O3 requires (M+H)+, 539.2765. MP:
191 – 193 °C. HPLC purity: 99.32%, RT: 15.80 min.
5.2.38.
2-(2-(4-(3,5-Dimethoxybenzyl)piperazin-1-yl)phenyl)-1-(1-methyl-4,10-
dihydrobenzo[b]pyrazolo[3,4-e][1,4]diazepin-5(1H)-yl)ethan-1-one (4a)
Subjecting 28 (100 mg, 0.2 mmol) to the above general procedure using 3,5-
dimethoxybenzaldehyde (47.0 mg, 0.4 mmol) produced the title compound 4a (64.0 mg, 43%)
as a white powder (Rf = 0.21 in EtOAc).
1H NMR (500 MHz, Chloroform-d): δ 7.15 (d, J = 6.4 Hz, 1H), 7.13 – 7.04 (m, 3H), 7.00 – 6.95 (m,
1H), 6.92 – 6.87 (m, 2H), 6.85 – 6.80 (m, 2H), 6.52 (d, J = 2.3 Hz, 2H), 6.38 (t, J = 2.3 Hz, 1H), 6.15
(br s, 1H), 5.61 (d, J = 14.6 Hz, 1H), 3.87 – 3.75 (m, 7H), 3.73 – 3.60 (m, 2H), 3.59 (s, 3H), 3.52 –
3.43 (m, 2H), 2.67 – 2.58 (m, 4H), 2.52 – 2.22 (m, 4H). 13C NMR (126 MHz, Chloroform-d): δ
171.4, 160.9, 151.2, 139.1, 138.7, 136.4, 135.3, 131.5, 130.5, 130.3, 130.1, 128.9, 127.5, 123.6,
122.2, 119.7, 107.2, 106.9, 101.6, 99.3, 66.9, 55.5, 53.3, 51.8, 43.5, 36.9, 34.8. IR (diamond cell,
neat) νmax: 2810, 1640, 1595, 1556, 1502, 1451, 1391, 1292, 1202, 1151, 1063, 1011, 912, 833,
728, 454 cm–1. LRMS (+ESI) m/z: 553 [(M+H)+, 100%], 575 [(M+Na)+, 20%],. HRMS (+ESI)
Found: (M+Na)+, 575.2743. C32H36N6O3 requires (M+Na)+, 575.2741. MP: 98 – 100 °C. HPLC
purity: 98.81%, RT: 17.44 min.
5.2.39.
2-(3-(4-(3,5-Dimethoxybenzyl)piperazin-1-yl)phenyl)-1-(1-methyl-4,10-
dihydrobenzo[b]pyrazolo[3,4-e][1,4]diazepin-5(1H)-yl)ethan-1-one (4b)
Subjecting 29 (100 mg, 0.2 mmol) to the above general procedure using 3,5-
dimethoxybenzaldehyde (47.0 mg, 0.4 mmol) produced the title compound 4b (81.0 mg, 73%)
as a white powder (Rf = 0.20 in EtOAc).
1H NMR (500 MHz, Chloroform-d): δ 7.19 (td, J = 8.1, 1.4 Hz, 1H), 7.16 (s, 1H), 7.12 (dd, J = 7.8,
1.5 Hz, 1H), 7.03 – 6.92 (m, 2H). 6.84 (dd, J = 8.1, 1.4 Hz, 1H), 6.69 (dd, J = 8.2, 2.5 Hz, 1H), 6.53
20

ACCEPTED MANUSCRIPT
(d, J = 2.3 Hz, 2H), 6.42 (t, J = 2.0 Hz, 1H), 6.37 (t, J = 2.3 Hz, 1H), 6.28 (d, J = 7.4 Hz, 1H), 5.98 (br
s, 1H), 5.58 (d, J = 14.5 Hz, 1H), 3.79 (br s, 7H), 3.58 (s, 3H), 3.49 – 3.46 (m, 2H), 3.42 (s, 2H),
3.11 – 2.98 (m, 4H), 2.54 (t, J = 5.0 Hz, 4H). 13C NMR (126 MHz, Chloroform-d): δ 170.8, 160.8,
151.3, 140.7, 139.1, 138.9, 136.4, 135.7, 131.7, 130.6, 129.2, 128.7, 122.2, 120.2, 119.8, 116.2,
114.4, 107.0, 101.5, 99.2, 63.1, 55.4, 53.2, 49.0, 43.6, 41.8, 34.7. IR (diamond cell, neat) νmax
:
2811, 1632, 1596, 1558, 1502, 1446, 1389, 1316, 1246, 1202, 1150, 1061, 944, 834, 763, 694,
441 cm–1. LRMS (+ESI) m/z: 553 [(M+H)+, 100%], 575 [(M+Na)+, 40%]. HRMS (+ESI) Found:
(M+Na)+, 575.2740. C32H36N6O3 requires (M+Na)+, 575.2741. MP: 101 – 103 °C. HPLC purity:
99.49%, RT: 17.50 min.
5.2.40.
2-(4-(4-(3,5-Dimethoxybenzyl)piperazin-1-yl)phenyl)-1-(1-methyl-4,10-
dihydrobenzo[b]pyrazolo[3,4-e][1,4]diazepin-5(1H)-yl)ethan-1-one (4c)
Subjecting 30 (100 mg, 0.2 mmol) to the above general procedure using 3,5-
dimethoxybenzaldehyde (47.0 mg, 0.4 mmol) produced the title compound 4c (71.0 mg, 64%)
as a white powder (Rf = 0.21 in EtOAc).
1H NMR (500 MHz, Chloroform-d): δ 7.21 (td, J = 7.7, 1.6 Hz, 1H), 7.16 (s, 1H), 7.11 (dd, J = 7.8,
1.6 Hz, 1H), 6.98 (td, J = 7.5, 1.3 Hz, 1H), 6.91 (dd, J = 8.1, 1.3 Hz, 1H), 6.77 (d, J = 8.3 Hz, 2H),
6.68 (d, J = 8.3 Hz, 2H), 6.52 (d, J = 2.4 Hz, 2H), 6.36 (t, J = 2.4 Hz, 1H), 6.30 (br s, 1H), 5.58 (d, J =
14.5 Hz, 1H), 3.81 (d, J = 14.5 Hz, 1H), 3.78 (s, 6H), 3.57 (s, 3H), 3.48 (s, 2H), 3.34 (s, 2H), 3.10 (t,
J = 5.0 Hz, 4H), 2.56 (t, J = 5.0 Hz, 4H). 13C NMR (126 MHz, Chloroform-d): δ 171.1, 160.8, 150.1,
140.6, 139.2, 139.1, 136.4, 131.8, 130.5, 129.7, 129.2, 125.9, 122.2, 119.9, 115.8, 107.0, 101.5,
99.1, 63.1, 55.4, 53.1, 49.3, 43.6, 40.3, 34.7. IR (diamond cell, neat) νmax: 2810, 1595, 1556, 1502,
1429, 1386, 1225, 1202, 1150, 1056, 993, 918, 833, 729 cm–1. LRMS (+ESI) m/z: 553 [(M+H)+,
100%], 575 [(M+Na)+, 80%],. HRMS (+ESI) Found: (M+Na)+, 575.2740. C32H36N6O3 requires
(M+Na)+, 575.2741. MP: 102 – 104 °C. HPLC purity: 95.41%, RT: 17.55 min.
5.2.41.
3-(2-(4-(3,5-Dimethoxybenzyl)piperazin-1-yl)phenyl)-1-(1-methyl-4,10-
dihydrobenzo[b]pyrazolo[3,4-e][1,4]diazepin-5(1H)-yl)propan-1-one (4d)
Subjecting 31 (100 mg, 0.19 mmol) to the above general procedure using 3,5-
dimethoxybenzaldehyde (32 mg, 0.2 mmol) produced the title compound 4d (103 mg, 93%) as
a white powder (Rf = 0.19 in EtOAc).
1H NMR (400 MHz, MeOD-d4): δ 7.27 – 7.16 (m, 2H), 7.12 (s, 1H), 7.08 (ddd, J = 8.5, 6.6, 2.1 Hz,
1H), 7.04 – 6.96 (m, 2H), 6.92 (ddd, J = 7.9, 6.6, 1.9 Hz, 1H), 6.86 – 6.76 (m, 2H), 6.53 (d, J = 2.3
Hz, 2H), 6.39 (t, J = 2.3 Hz, 1H), 5.48 (d, J = 14.4 Hz, 1H), 3.76 (s, 6H), 3.73 – 3.68 (m, 1H), 3.66 (s,
3H), 3.45 (d, J = 2.8 Hz, 2H), 2.92 – 2.60 (m, 6H), 2.56 – 2.29 (m, 6H), 1 NH signal not observed.
13C NMR (101 MHz, MeOD-d4): δ 174.3, 162.3, 152.6, 141.4, 140.6, 140.3, 137.6, 137.2, 132.5,
131.1, 131.0, 130.2, 128.2, 125.2, 123.0, 121.7, 121.1, 108.6, 102.6, 100.3, 64.2, 55.7, 54.7, 53.4,
44.4, 35.6, 35.2, 28.4. IR (diamond cell, neat) νmax: 2934, 1634, 1594, 1557, 1503, 1448, 1429,
1392, 1347, 1316, 1293, 1248, 1221, 1202, 1149, 1061, 1010, 992, 933, 832, 759, 745, 693, 487
cm–1. LRMS (+ESI) m/z: 567 [(M+H)+, 100%], 589 [(M+Na)+, 10%]. HRMS (+ESI) Found: (M+H)+,
567.3078. C33H38N6O3 requires (M+H)+, 567.3078. MP: 104 – 106 °C. HPLC purity: 99.61%, RT:
17.76 min.
5.2.42.
3-(3-(4-(3,5-Dimethoxybenzyl)piperazin-1-yl)phenyl)-1-(1-methyl-4,10-
dihydrobenzo[b]pyrazolo[3,4-e][1,4]diazepin-5(1H)-yl)propan-1-one (4e)
Subjecting 32 (100 mg, 0.19 mmol) to the above general procedure using 3,5-
dimethoxybenzaldehyde (32 mg, 0.2 mmol) produced the title compound 4e (88 mg, 78%) as a
white powder (Rf = 0.20 in EtOAc).
1H NMR (400 MHz, MeOD-d4): δ 7.31 – 7.21 (m, 2H), 7.13 (s, 1H), 7.03 – 6.97 (m, 1H), 6.97 – 6.93
(m, 2H), 6.75 – 6.68 (m, 1H), 6.58 – 6.51 (m, 2H), 6.50 (t, J = 2.1 Hz, 1H), 6.40 (d, J = 2.1 Hz, 2H),
21

ACCEPTED MANUSCRIPT
5.49 (d, J = 14.5 Hz, 1H), 3.78 – 3.70 (m, 7H), 3.69 (s, 3H), 3.50 (s, 2H), 3.11 – 2.98 (m, 4H), 2.68
(ddt, J = 16.4, 14.5, 7.3 Hz, 2H), 2.59 (t, J = 5.0 Hz, 4H), 2.45 – 2.16 (m, 2H), NH signal not
observed. 13C NMR (101 MHz, MeOD-d4): δ 174.1, 162.3, 152.6, 142.9, 141.4, 140.6, 140.5, 137.2,
132.7, 131.1, 130.4, 130.0, 123.2, 121.2, 121.0, 117.3, 115.4, 108.5, 102.3, 100.3, 64.0, 55.7, 54.1,
50.1, 44.5, 37.1, 35.2, 32.9. IR (diamond cell, neat) νmax: 2935, 2813, 1635, 1595, 1557, 1501,
1445, 1429, 1394, 1348, 1315, 1293, 1245, 1148, 1057, 993, 831, 761, 694, 664 cm–1. LRMS
(+ESI) m/z: 567 [(M+H)+, 100%], 589 [(M+Na)+, 12%]. HRMS (+ESI) Found: (M+H)+, 567.3078.
C33H38N6O3 requires (M+H)+, 567.3078. MP: 100 – 102 °C. HPLC purity: 96.14%, RT: 18.00 min.
5.2.43.
3-(4-(4-(3,5-Dimethoxybenzyl)piperazin-1-yl)phenyl)-1-(1-methyl-4,10-
dihydrobenzo[b]pyrazolo[3,4-e][1,4]diazepin-5(1H)-yl)propan-1-one (4f)
Subjecting 33 (100 mg, 0.19 mmol) to the above general procedure using 3,5-
dimethoxybenzaldehyde (32 mg, 0.2 mmol) produced the title compound 4f (106 mg, 94%) as a
white powder (Rf = 0.21 in EtOAc).
1H NMR (400 MHz, MeOD-d4): δ 7.35 – 7.20 (m, 2H), 7.14 (s, 1H), 7.00 – 6.95 (m, 2H), 6.80 (d, J =
8.7 Hz, 2H), 6.78 – 6.65 (m, 2H), 6.53 (d, J = 2.3 Hz, 2H), 6.39 (t, J = 2.3 Hz, 1H), 5.49 (d, J = 14.5
Hz, 1H), 3.76 (s, 7H), 3.70 (s, 3H), 3.50 (s, 2H), 3.09 (t, J = 5.1 Hz, 4H), 2.65 (td, J = 7.4, 5.0 Hz,
2H), 2.60 (t, J = 5.0 Hz, 4H), 2.44 – 2.15 (m, 2H), NH signal not observed. 13C NMR (101 MHz,
MeOD-d4): δ 174.2, 162.3, 150.9, 141.5, 140.5, 137.3, 133.7, 132.7, 131.1, 130.4, 130.0, 129.9,
123.2, 121.2, 117.5, 108.5, 102.4, 100.3, 64.1, 55.7, 54.1, 50.4, 44.5, 37.0, 35.2, 31.6. IR (diamond
cell, neat) νmax: 2933, 2814, 1635, 1307, 1557, 1513, 1502, 1453, 1430, 1393, 1350, 1316, 1295,
1241, 1226, 1202, 1149, 1056, 993, 919, 830, 760, 744, 695, 538 cm–1. LRMS (+ESI) m/z: 567
[(M+H)+, 40%], 589 [(M+Na)+, 100%],. HRMS (+ESI) Found: (M+Na)+, 589.2898. C33H38N6O3
requires (M+Na)+, 589.2898. MP: 111 – 113 °C. HPLC purity: 95.56%, RT: 17.82 min.
5.3. Pharmacological Evaluation
5.3.1. Generation of OT and V1a receptor-expressing human embryonic kidney (HEK)
cells.
The Flp-In™ system (Life Technologies, Carlsbad, CA, USA) was used to establish cell lines
expressing the human OT and V1aR. pOG44 plasmids (Life Technologies) and pcDNA5/FRT
plasmids containing human OT or V1aR cDNA were synthesised by GenScript and were
propagated in endonuclease and recombinase-deficient E. coli (BIO-85027, Bioline, London,
UK). Competent E. coli were transformed with plasmid DNA (50 ng) by heat shock at 42°C.
Cultures were then incubated for 16 h at 37°C on Luria-bertani (LB) agar plates (Life
Technologies) containing 100 μg/mL ampicillin (Sigma-Aldrich, St. Louis, MO, USA). Ampicillin-
resistant colonies were isolated and inoculated into 5 mL of LB broth containing 50 μg/mL
ampicillin. This culture was incubated for 16 h at 37°C, and pcDNA5/FRT and pOG44 DNA
purified using the Plasmid Midiprep System (Promega, Madison, WI, USA) according to
manufacturer’s protocols. Plasmid integrity was verified by performing a restriction enzyme
digest using the restriction endonuclease FspI (New England Biolabs, Ipswich, MA, USA)
(Appendix 1). Flp-In™ T-REx™ HEK293 cells used for transfection were maintained in
Dulbecco’s Modified Eagle Medium (DMEM) (Invitrogen, Carlsbad, CA, USA) supplemented with
10% heat-inactivated foetal bovine serum (FBS) (Invitrogen), blasticidin (15 μg/mL; Sigma-
Aldrich), penicillin-streptomycin (100U; Sigma-Aldrich) and zeocin™ selection reagent (100
μg/mL; Life Technologies). Cells were co-transfected with pcDNA5 plasmid containing human
OT or V1aR cDNA sequences, alongside pOG44 plasmid containing Flp-recombinase cDNA.
Transfections were undertaken using the non-liposomal transfection reagent FugeneHD®
(Promega) according to manufacturer’s protocols. Selection of receptor-positive clones was
achieved by treatment with the antibiotic hygromycin (80 μg/mL; Invitrogen). Surface receptor
expression was verified using immunocytochemistry according to the method of Werry et
22

ACCEPTED MANUSCRIPT
al.[15] Stably transfected cell lines were subsequently maintained in 10% DMEMcontaining
hygromycin (80 μg/mL), penicillin/streptomycin (100U) and blasticidin (15 μg/mL). To induce
receptor expression, cells were incubated with tetracycline (2 μg/mL) for 48 h prior to
membrane preparation and functional assays. This concentration of tetracycline was selected
based on previously demonstrated efficacy in inducing receptor expression.[16]
5.3.2. Membrane Preparation.
OTR and V1aR-expressing HEK293 cells were detached from culture dishes using PBS with 5 mM
EDTA and centrifuged at 1200 g for 5 min. The supernatant was removed and cells resuspended
in homogenization buffer (50 mM HEPES, 5 mM EDTA, 5 mM MgCl2, pH 7.4) prior to
homogenization using an Ultra-Turrax homogeniser (IKA, Wilmington, NC, USA). Resulting
homogenates were centrifuged twice at 48000 g, 4 °C for 30 min and membrane pellets
resuspended in 50 mM Tris-HCl, 5 mM MgCl2, pH 7.4. Final protein concentration was calculated
using the BCA protein-assay method (Bio-Rad, Hercules, CA, USA) according to manufacturer’s
protocols. Membranes were stored at –80 °C.
5.3.3. Competition Radioligand Binding.
Binding affinity of WAY-267,464 and derivatives was indexed by competitive displacement of
[3H]-oxytocin or [3H]-vasopressin at Kd concentrations. Membranes (50 μg/well) from OT or V1a
receptor-expressing HEK293 cells were incubated in a final volume of 200 μL containing [3H]-
oxytocin (10 nM) or [3H]-vasopressin (2 nM) alongside competing compounds (0.1 nM - 100
μM) in reaction buffer (50 mM Tris-HCl, 5 mM MgCl2, pH 7.4). Reactions were incubated for 90
min at 4°C to reach equilibrium, and terminated by rapid filtration over glass fibre filters
(Whatman GF/A 1.6 μM), and washing with ice-cold reaction buffer. Radioactivity was detected
after soaking filters in Microscint 0 using a Microbeta2 2450 microplate-reader (Perkin Elmer).
Nonspecific binding was determined in the presence of 1 μM cold oxytocin or vasopressin
(Sigma-Aldrich), respectively.
5.3.4. HTRF-IP1 Accumulation Assays.
OTR or V1aR-expressing-HEK293 cells were seeded onto clear, poly-L-lysine (100 μg/mL)-
coated 384 well plates at a density of 8.75 x 103 cells per well. Levels of receptor activation
induced by compounds were assessed at concentrations ranging from 1 nM - 100 μM using the
HTRF-IP-One kit (CisBio International, Bagnols-sur-Cze, France), according to the
manufacturer’s protocol. For agonist assessment, cells were incubated with compounds for 1 h
prior to the addition of Ab-Cryptate and IP1-d2. The ligand concentration that induced a 50%
maximal response (EC50) was used to evaluate functional effects across compounds. For
antagonist assessment, cells were pre-incubated with test compounds (1 nM - 100 μM)
solubilized in stimulation buffer for 30 min prior to the addition of an EC70 concentration of
vasopressin (25 nM) mixed with compounds or DMSO control (0.1%). Cells were then incubated
for a further 1 h, and Ab-Cryptate and IP1-d2 added. The ligand concentration that inhibited
50% of vasopressin-induced response (IC50) was used to evaluate antagonistic functional
effects across compounds.
Acknowledgements
Work performed at The University of Sydney and presented herein was supported in part by the
National Health and Medical Research Council.
6. References
23

ACCEPTED MANUSCRIPT
[1] H. Hecht, D.v. Zerssen, C. Krieg, J. Pössl, H.-U. Wittchen, Anxiety and depression: Comorbidity,
psychopathology, and social functioning, Compr. Psychiatry, 30 (1989) 420-433.
[2] E.R. de Kloet, M. Joels, F. Holsboer, Stress and the brain: from adaptation to disease, Nat. Rev.
Neurosci., 6 (2005) 463-475.
[3] C.S. Carter, A.J. Grippo, H. Pournajafi-Nazarloo, M.G. Ruscio, S.W. Porges, Oxytocin,
vasopressin and sociality, Prog. Brain. Res., 170 (2008) 331-336.
[4] A. Theodoridou, A.C. Rowe, I.S. Penton-Voak, P.J. Rogers, Oxytocin and social perception:
oxytocin increases perceived facial trustworthiness and attractiveness, Horm. Behav., 56
(2009) 128-132.
[5] I.F. Bielsky, S.-B. Hu, K.L. Szegda, H. Westphal, L.J. Young, Profound Impairment in Social
Recognition and Reduction in Anxiety-Like Behavior in Vasopressin V1a Receptor Knockout
Mice, Neuropsychopharmacology, 29 (2003) 483-493.
[6] B. Chini, M. Manning, Agonist selectivity in the oxytocin/vasopressin receptor family: new
insights and challenges, Biochem. Soc. Trans., 35 (2007) 737-741.
[7] G. Gimpl, F. Fahrenholz, The oxytocin receptor system: structure, function, and regulation,
Physiol. Rev., 81 (2001) 629-683.
[8] M. Manning, S. Stoev, B. Chini, T. Durroux, B. Mouillac, G. Guillon, D.N. Inga, L. Rainer, Peptide
and non-peptide agonists and antagonists for the vasopressin and oxytocin V1a, V1b, V2 and
OT receptors: research tools and potential therapeutic agents, in: Prog. Brain Res., Elsevier,
2008, pp. 473-512.
[9] C. Hicks, W. Jorgensen, C. Brown, J. Fardell, J. Koehbach, C.W. Gruber, M. Kassiou, G.E. Hunt,
I.S. McGregor, The Nonpeptide Oxytocin Receptor Agonist WAY 267,464: Receptor-Binding
Profile, Prosocial Effects and Distribution of c-Fos Expression in Adolescent Rats, J.
Neuroendocrinol., 24 (2012) 1012-1029.
[10] C. Hicks, L. Ramos, T.A. Reekie, R. Narlawar, M. Kassiou, I.S. McGregor, WAY 267,464, a non-
peptide oxytocin receptor agonist, impairs social recognition memory in rats through a
vasopressin 1A receptor antagonist action, Psychopharmacology (Berl.), 232 (2015) 2659-
2667.
[11] W.T. Jorgensen, D.W. Gulliver, E.L. Werry, T. Reekie, M. Connor, M. Kassiou, Flexible
analogues of WAY-267,464: Synthesis and pharmacology at the human oxytocin and
vasopressin 1a receptors, Eur. J. Med. Chem., 108 (2016) 730-740.
[12] P. Hudson, G. Pitt, R. Batt, M. Roe, Piperazines as Oxytocin Agonists, in: PCT (Ed.) Patent
fetcher, Ferring B.V, International Application, 2005.
[13] T.A. Katte, T.A. Reekie, W.T. Jorgensen, M. Kassiou, The Formation of Seven-Membered
Heterocycles under Mild Pictet-Spengler Conditions: A Route to Pyrazolo[3,4]benzodiazepines,
J. Org. Chem., 81 (2016) 4883-4889.
[14] E. Fischer, A. Speier, Darstellung der Ester, Ber. Dtsch. Chem. Ges., 28 (1895) 3252-3258.
[15] E.L. Werry, G.J. Liu, M.R. Bennett, Glutamate-stimulated ATP release from spinal cord
astrocytes is potentiated by substance P, Journal of Neurochemistry, 99 (2006) 924-936.
[16] A. Knapman, M. Santiago, Y.P. Du, P.R. Bennallack, M.J. Christie, M. Connor, A Continuous,
Fluorescence-based Assay of µ-Opioid Receptor Activation in AtT-20 Cells, Journal of
Biomolecular Screening, 18 (2013) 269-276.
24

ACCEPTED MANUSCRIPT
Highlights:
•
•
•
•
Synthesis of 12 analogues of WAY-267,464
Investigated the ideal linker conformation for OTR and V1aR pharmacophore
Modifications abolished OTR pharmacology and reduced V1aR affinity in comparison to lead
All analogues are investigated in competitive binding assays and functionally evaluated.