Design, synthesis, and biological evaluation of aryl piperazines with potential as antidiabetic agents via the stimulation of glucose uptake and inhibition of NADH:ubiquinone oxidoreductase

Article abstract of DOI:10.1016/j.ejmech.2020.112416

The management of blood glucose levels and the avoidance of diabetic hyperglycemia are common objectives of many therapies in the treatment of diabetes. An aryl piperazine compound 3a (RTC1) has been described as a promoter of glucose uptake, in part through a cellular mechanism that involves inhibition of NADH:ubiquinone oxidoreductase. We report herein the synthesis of 41 derivatives of 3a (RTC1) and a systematic structure-activity-relationship study where a number of compounds were shown to effectively stimulate glucose uptake in vitro and inhibit NADH:ubiquinone oxidoreductase. The hit compound 3a (RTC1) remained the most efficacious with a 2.57 fold increase in glucose uptake compared to vehicle control and micromolar inhibition of NADH:ubiquinone oxidoreductase (IC₅₀ = 27 μM). In vitro DMPK and in vivo PK studies are also described, where results suggest that 3a (RTC1) would not be expected to provoke adverse drug-drug interactions, yet be readily metabolised, avoid rapid excretion, with a short half-life, and have good tissue distribution. The overall results indicate that aryl piperazines, and 3a (RTC1) in particular, have potential as effective agents for the treatment of diabetes.

Full text of DOI:10.1016/j.ejmech.2020.112416

Journal Pre-proof
Design, synthesis, and biological evaluation of aryl piperazines with potential
as antidiabetic agents via the stimulation of glucose uptake and inhibition of
NADH:ubiquinone oxidoreductase
R. Devine, M. Kelada, S. Leonard, D.S.D. Martin, J.M.D. Walsh, C.J. Breen, R.B.
Driver, G.K. Kinsella, J.B.C. Findlay, J.C. Stephens
PII:
S0223-5234(20)30387-1
DOI:
https://doi.org/10.1016/j.ejmech.2020.112416
EJMECH 112416
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 23 August 2019
Revised Date: 29 April 2020
Accepted Date: 29 April 2020
Please cite this article as: R. Devine, M. Kelada, S. Leonard, D.S.D. Martin, J.M.D. Walsh, C.J. Breen,
R.B. Driver, G.K. Kinsella, J.B.C. Findlay, J.C. Stephens, Design, synthesis, and biological evaluation of
aryl piperazines with potential as antidiabetic agents via the stimulation of glucose uptake and inhibition
of NADH:ubiquinone oxidoreductase, European Journal of Medicinal Chemistry (2020), doi: https://
doi.org/10.1016/j.ejmech.2020.112416.
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Title
Design, synthesis, and biological evaluation of aryl piperazines with potential as antidiabetic
agents via the stimulation of glucose uptake and inhibition of NADH:ubiquinone
oxidoreductase.
Authors
R. Devine,a, † M. Kelada,a,† S. Leonard,a,b,† D. S. D. Martin,b J. M. D. Walsh,a C. J. Breen,b,c
R. B. Driver,a G. K. Kinsella,d J. B. C. Findlay,b,e J. C. Stephens.a,c,*
a Department of Chemistry, Maynooth University, Maynooth, Co. Kildare, Ireland
b Department of Biology, Maynooth University, Maynooth, Co. Kildare, Ireland
c Maynooth University Human Health Research Institute, Maynooth University, Maynooth,
Co. Kildare, Ireland
d School of Food Science and Environmental Health, College of Sciences and Health,
Technological University Dublin, Dublin D07 ADY7, Ireland
e School of Biochemistry and Molecular Biology, University of Leeds, Leeds, UK
† Joint first author, alphabetical order
* Corresponding author
Key words
Diabetes, glucose uptake, complex 1, piperazine, pharmacokinetics, structure activity
relationship
Highlights
•
•
•
Forty one aryl piperazines derivatives have been generated and tested in vitro for their
ability to stimulate glucose uptake.
Six of the compounds were reported to effectively stimulate glucose uptake in vitro
and inhibit NADH:ubiquinone oxidoreductase.
The hit compound 3a (RTC1) remained the most efficacious with a 2.57 fold increase
in glucose uptake compared to vehicle control and micromolar inhibition of
NADH:ubiquinone oxidoreductase (IC50 = 27 µM).
•
Preliminary in vitro DMPK and in vivo PK studies suggested that 3a (RTC1) would
not provoke adverse drug-drug interactions, yet be readily metabolised, avoid rapid
excretion, with a short half-life, and have good tissue distribution.
Abstract
The management of blood glucose levels and the avoidance of diabetic hyperglycemia are
common objectives of many therapies in the treatment of diabetes. An aryl piperazine
compound 3a (RTC1) has been described as a promoter of glucose uptake, in part through a
cellular mechanism that involves inhibition of NADH:ubiquinone oxidoreductase. We report
herein the synthesis of 41 derivatives of 3a (RTC1) and a systematic structure-activity-
relationship study where a number of compounds were shown to effectively stimulate
glucose uptake in vitro and inhibit NADH:ubiquinone oxidoreductase. The hit compound 3a
(RTC1) remained the most efficacious with a 2.57 fold increase in glucose uptake compared

to vehicle control and micromolar inhibition of NADH:ubiquinone oxidoreductase (IC50 = 27
µM). In vitro DMPK and in vivo PK studies are also described, where results suggest that 3a
(RTC1) would not be expected to provoke adverse drug-drug interactions, yet be readily
metabolised, avoid rapid excretion, with a short half-life, and have good tissue distribution.
The overall results indicate that aryl piperazines, and 3a (RTC1) in particular, have potential
as effective agents for the treatment of diabetes.
1. Introduction
Type 2 diabetes mellitus (T2DM) has become one of the great modern public health threats in
terms of human, social, and economic costs [1]. T2DM is a chronic condition where blood
glucose levels are raised due to the body’s inability to produce enough of the hormone insulin
or to use insulin effectively. Insulin is an essential hormone produced in the pancreas and
plays a critical role in the transport of glucose from the bloodstream into the body’s cells. A
lack of insulin or a cells insensitivity to insulin leads to elevated blood glucose levels and
hyperglycaemia, which if left unchecked can result in organ damage and the development of
disabling and life-threatening health complications [1,2].
The numbers suffering from diabetes are immense and are increasing at an alarming rate. In
2017, there were ~ 425 million diabetic sufferers over the age of 20, and this number is
conservatively projected to increase to ~629 million in 2045 [1,2]. There are a number of
therapeutic options available, some of which can be administered in the early stages of the
development of T2DM or as a combination therapy with injectables. There are several classes
of hypoglycemic agents found in the clinic, including metformin, sulfonylureas,
thiazolidinediones, DPP-4 inhibitors, SGLT2 inhibitors, GLP-1 receptor agonists and insulin,
but the control of diabetes remains variable and unsatisfactory [2-4]. The therapeutic
regimens, side effects, and possible off-target actions of some of these options are
undesirable and can lead to reduced compliance [5-8]. As a consequence, taking into account
the projected and rapid growth in the number of those suffering with diabetes, there is an
ever-increasing demand for novel anti-diabetic therapies with defined mechanisms of action.
A common objective for many therapies is the management of blood glucose levels and the
avoidance of diabetic hyperglycemia. Different strategies can be employed to achieve this
including the stimulation of glucose uptake from the bloodstream, reducing gluconeogenesis,
and increasing glucose excretion [3,4]. Therapeutics that increase insulin secretion or
increase insulin sensitivity will typically result in an increase in glucose uptake and a
reduction in diabetic hyperglycemia. Diabetic hyperglycemia has also been reported to
aberrantly upregulate complex I activity in the pancreas, which suggest that complex I could
be a promising therapeutic target for diabetes [8,9]. We recently reported the piperazine
containing compounds RTC1 and RTB70, which act through the inhibition of
NADH:ubiquinone oxidoreductase (complex I of the mitochondrial respiratory chain) to
stimulate glucose uptake and restore the glucose handling abilities of diabetic mice [10,11].
The reduction in NADH:ubiquinone oxidoreductase activity provoked by these compounds
leads to a change in cellular ADP:ATP and AMP:ATP ratios resulting in the activation of
AMP-activated protein kinase (AMPK). Once activated, AMPK rapidly restores cellular
energy balance by switching off ATP consuming anabolic pathways and by switching on
ATP generating catabolic pathways involving increased glucose uptake [12]. Further studies
showed that RTC1 augmented the signalling capabilities of insulin in C2C12 cells stimulated
with tumour necrosis factor-alpha (TNF-α), thus preventing TNF-α induced insulin resistance
through action as an insulin sensitizer [13]. As a result, a systematic analysis of these

compounds was conducted to further explore their ability to stimulate glucose uptake and use
as a potential therapeutic strategy for T2DM.
Herein, we report the synthesis and evaluation of derivatives of compound 3a (RTC1). The
study involved the systematic modification of 3a (RTC1) at five sites, A-E (Figure 1), in
order to explore the relationship between chemical structure and biological activity. This
approach allowed us to investigate the importance of the aryl substituent (site A), the nature
of the aryl group (site B), the piperazine (site C), the length and nature of the alkyl chain (site
D), as well as the terminal group (site E) on the ability to stimulate glucose uptake.
Compounds were evaluated for their in vitro ability to stimulate glucose uptake and, in a
number of cases, for the inhibitory activity against NADH:ubiquinone oxidoreductase in
permeabilised mitochondria. A pharmacophore was developed based on the effectiveness of
the compound library at stimulating glucose uptake, with preliminary drug metabolism and
pharmacokinetic (DMPK) studies also conducted with the hit compound 3a (RTC1).
Fig. 1. Chemical structure of 3a (RTC1) and sites A-E for pharmacomodulation.
2. Results and Discussion
2.1 Synthesis of hit compound 3a (RTC1)
A total of 41 derivatives of the hit compound 3a (RTC1) were synthesised as shown in
Scheme 1. The hit compound 3a (RTC1) was generated in a two-step process. The
intermediate aryl piperazine 2a was synthesised in 50% yield from 4-chlorobenzotrifluoride
1a and piperazine by heating to 200 oC under microwave conditions. Subsequent coupling of
2a with the commercially available 4-(2-thienyl)butyric acid generated the desired product,
3a (RTC1), in 86% yield (Scheme 1) [10].

Scheme 1. Synthesis of 3a (RTC1) derivatives. Reagents and conditions: A. (a) HOBt,
TBTU, NEt3, DMF, overnight, rt; (b) Na2CO3, MeCN, N2 atmosphere, 24 h, reflux; (c) EtOH,
formic acid, formaldehyde, 3 h, reflux; (d) from compound 16e, 2-methoxyethanamine,
HOBt, TBTU, NEt3, DMF, 20 h, rt; (e) from compound 3e, PtO2, H2 atmosphere, 16.5 h, rt.
o
B. (f) Piperazine, NMP, 30 mins, 200 C, MW (2a, R = 4-(trifluoromethyl)phenyl, 50%
yield; 2i, R = 2-chloro-4-(trifluoromethyl)phenyl), 63% yield; 2j, R = 2-bromo-4-
(trifluoromethyl)phenyl), 23% yield); (g) (i) bis(2-chloroethyl)amine.HCl, bis(2-
methoxyethyl) ether, 16 h, reflux, (ii) 5% NaOH (aq.), 4 h, rt (2c, R = p-tolyl, 13% yield).
*Other amine/piperazine intermediates were commercially available.
C. (h) HOBt, TBTU, NEt3, DMF, overnight, rt.
D. (i) 4-(2-Thienyl)butyric acid, HOBt, TBTU, NEt3, DMF, overnight, rt.
2.2 Synthesis of derivatives with variations at site A-E.

The first structural modifications made were those concerned with variations at site A, the
substitution pattern of the aryl ring, and were undertaken in order to study the effect and
importance of the CF3 substituent on activity. This involved (i) variation of the para
substituent on the aryl ring (3a-3f); (ii) changing the position of the CF3 substituent (3g), and
(iii) bis substitution of the aryl ring (3h-j) (Table 1). The key intermediate, the aryl
piperazine, was commercially available in some but not all cases. The bis-substituted 1-(2-
chloro-4-(trifluoromethyl)phenyl)piperazine
2i
and
1-(2-bromo-4-
(trifluoromethyl)phenyl)piperazine 2j were both synthesised from the corresponding aryl
chloride and piperazine via an SNAr reaction in NMP under microwave conditions (Scheme
1, (a)). The alkyl substituted 1-(p-tolyl)piperazine 2c could not be generated by this method,
and instead was synthesised from the corresponding aniline and bis(2-chloroethyl)amine via a
substitution/cyclisation sequence (Scheme 1, (b)). The final step in the synthesis of
compounds 3a-j was the coupling reaction between the aryl piperazine and 4-(2-
thienyl)butyric acid, using HOBt and TBTU as coupling reagents (Scheme 1, (c)). This
method gave the desired products in yields ranging from 24 to 86% for the final step.
Compound 3f was generated from the para-nitro substituted 3e via the reduction of the nitro
group using H2 over PtO2 in a 57% yield.
We next explored variations of the aryl ring, site B. To do this, the aryl ring was changed to a
substituted pyridine (5a-c) as well as cyclic and acyclic alkyl groups (5d-e) (Scheme 1, Table
1). In all cases, 5a-e, the corresponding piperazines were commercially available, which
allowed the target compounds to be synthesized in a single coupling reaction with yields
ranging from 68-91%.
Changes at site C allowed us to explore the importance of the piperazine ring. This required
the synthesis of compounds 7a-c, where the piperazine ring was replaced with a piperidine
ring (7a), a hydrazine group (7b), and an NH (7c) (Scheme1, Table 2). The appropriate
starting amine or hydrazine for each target compound were commercially available, with
subsequent coupling using 4-(2-thienyl)butyric acid generating 7a-c in a single step (Scheme
1, 67-90% yield).
Exploration of site D required the synthesis of derivatives with variations in the alkyl chain
linking the piperazine motif and the thiophene heterocycle. This included the generation of
compounds 9a-e, where the changes in the alkyl chain length was investigated through
sequential addition of single methylene units (Scheme 1, Table 3, 44-82% yield). Compounds
9f-g were also synthesised, using the same coupling methodology, where additional
functional groups were added to the chain including a sulfur atom and a carbonyl group
(Scheme 1, Table 3, 63-84% yield). The hit compound 3a (RTC1) contains an amide
carbonyl group adjacent to the piperazine ring. The importance of this amide carbonyl for
biological activity was explored though the synthesis and evaluation of compound 12, which
lacks the amide carbonyl (Scheme 1, Table 3). Compound 12 was synthesised via a three step
process culminating in a SN2 type reaction between 1-(4-(trifluoromethyl)phenyl)piperazine
and the mesylate ester 11, 70% yield. Mesylate ester 11 was generated by the reduction of the
starting carboxylic acid, followed by mesylation of the resulting primary alcohol 10 (see
experimental section).
The importance of a terminal thiophene heterocycle for biological activity, as found in
compound 3a (RTC1), also required investigation. To explore this we removed the thiophene,
replacing it with a methyl group 14a and a pyrazole heterocycle 14b (Scheme 1, Table 4).

Both 14a and 14b were synthesised in a single step from the corresponding carboxylic acid
and 1-(4-(trifluoromethyl)phenyl)piperazine in a 57% and 10% yield respectively.
Finally, we undertook an investigation where variations were made at more than one site.
This allowed us to explore additional chemical space and the effect that multiple structural
variations would have on biological activity. Analogues 16a-f were synthesised with
variations at both sites D and E (Scheme 1, Table 5). Here variations included methyl, furyl,
and ester terminal groups as replacements for the thiophene heterocycle in 3a (RTC1), as
well as variations in chain length. All compounds, except 16g, were synthesised in a single
coupling step (Scheme 1, 31-98% yield). Compound 16g required an additional step where
compound 16e was coupled with 2-methoxyethanamine to give compound 16g (Table 5).
Compound 17 was also generated, where the entire alkyl chain-thiophene ring motif is
removed, using formic acid and formaldehyde (Scheme 1, Table 5, 70% yield). Compound
18, with variations at site C and D, was synthesised in a single step from the commercially
available 4-(4-trifluoromethyl)phenyl)piperidine and 4-oxo-4-(2-thienyl)butanoic acid
(Scheme 1, Table 5, 87% yield). Compounds 19-21 had variations at both B and D sites and
were generated using the same one step process from commercially available starting
(Scheme 1, Table 5, 63-71% yield). Compound 22 was synthesised, with variations at both
the A and C sites, where the CF3 substituent of 3a (RTC1) is removed from the aryl ring and
the piperazine saturated heterocycle is replaced by a piperidine (Scheme 1, Table 5, 98%
yield). Finally, compound 23 was purchased from a commercial supplier, with compound 24
generated by the straight forward esterification of 23.
2.8 In vitro evaluation of glucose uptake by 3a (RTC1) and its derivatives.
With the desired compounds in hand, we next undertook the evaluation of their ability to
stimulate glucose uptake in C2C12 mouse muscle cells using a tritiated version of deoxy-2-
glucose, a derivative of glucose that cannot be metabolized [10, 14]. Results from this assay
are presented as fold change, induced with a 10 μM treatment, compared with the vehicle
control, DMSO (Tables 1-5). The in vitro glucose uptake data revealed that chemical
structure had a significant impact on activity. Full experimental details can be found in the
experimental section and supporting information.
2.8.1 In vitro glucose uptake for site A modifications.
For site A modifications, which entail changes to the substitution of the aryl ring, all results
showed a reduction or loss of glucose uptake activity compared to 3a (RTC1) (Figure 1,
Table 1). Removal of the p-CF3 substituent (3b), or its replacement with the smaller
hydrophobic methyl (3c) substituent, resulted in a complete loss of activity. Replacing the
CF3 substituent with a single fluorine atom (3d), or another electron withdrawing substituent
such as a nitro substituent (3e), resulted in complete loss of activity, as did its replacement
with the electron donating amino substituent (3f). Moving the CF3 substituent from the para
to the ortho position (3g) also resulted in complete loss of activity. The only A type
modifications that resulted in active compounds, albeit with reduced activity, was the
replacement of the p- CF3 substituent with two meta CF3 substituents (3h) or keeping the p-
CF3 substituent and adding an ortho-Cl substituent (3i). From the type A results, it can be
suggested that the presence of a CF3 substituent on the aromatic ring is important for
biological activity, and that a single CF3 substituent at the para position is preferable over
two CF3 substituents. CF3 substituents are well known for the hydrophobic and lipophilic

nature, and can aid transport across cell membranes, as well as contribute to binding in
hydrophobic enzyme pockets [15,16].
2.8.2 In vitro glucose uptake for site B modifications.
For site B modifications changes were made to the aryl ring itself (Figure 1, Table 1). The
employment of a pyridine ring in place of the phenyl ring resulted in compound 5a, which
was inactive in the glucose uptake assay. Compound 5c, containing a 3-chloro-5-
(trifluoromethyl)pyridine in place of the phenyl ring, gave statistically significant activity but
at a low level and considerably lower than 3a (RTC1). Replacing the aromatic ring with an
aliphatic cyclohexyl ring (5d) did generate an active compound but again with activity levels
lower than 3a (RTC1). Replacement of the aryl ring with a simple aliphatic acyclic group
(methyl, 5e) resulted in complete loss of activity. The results for compounds 5a-5e suggest
that the presence of an aryl ring is important for activity and replacement with a pyridine or
aliphatic ring has a detrimental effect on activity levels.
Table 1
In vitro glucose uptake activity of compounds with variations at sites A, 3a-j, and site B, 5a-
e.
Glucose uptake
Compound
R
Active (Y) with
p-value or inactive (N)
Fold change ± SEM
3a
(RTC1)
2.57 ± 0.08
0.96 ± 0.07
1.00 ± 0.42
1.16 ± 0.09
1.06 ± 0.10
1.32 ± 0.12
Y (***)
N
N
N
N
N
3b
3c
3d
3e
3f
0.85 ± 0.02
N
3g

1.42 ± 0.09
1.58 ± 0.15
Y (*)
3h
3i
Y (***)
1.02 ± 0.04
1.00 ± 0.09
1.22 ± 0.10
N
N
N
3j
5a
5b
1.13 ± 0.02
Y (*)
5c
1.29 ± 0.04
0.84 ± 0.03
Y (***)
N
5d
5e
All data are expressed as means ± standard error of the means (SEM) of a number of experiments (n) performed
for that compound. Statistical analysis was performed using one-way analysis of variance (ANOVA) using
GraphPad Prism 5.0 with a threshold of significance defined as a p-value <0.05 (95% confidence in result) or a
one star significance (*), p-value < 0.01 or two stars significance (**), and p-value < 0.0001 or three stars
significance (***). Only compounds which received stars significance in the glucose uptake assay were
considered “active” compounds. Full experimental details can be found in the experimental section and
supporting information.
2.8.3 In vitro glucose uptake for site C modifications.
Three compounds were evaluated with site C modifications (Figure 1, Table 2). Here,
replacement of the piperazine ring with a piperidine ring (7a) resulted in an active compound
with a 2-fold increase in activity compared to the vehicle control. Although a strong result,
the piperidine containing 7a was still less active that 3a (RTC1), which itself generated a 2.5-
fold increase in activity. Surprisingly, replacement of the piperazine ring with a hydrazine
motif (7b) resulted in an active compound, albeit with a considerably lower level of activity
than 3a (RTC1). The third site C compound evaluated was 7c, where the piperazine ring was
replaced by an NH group, creating an amide linkage between the aryl ring and the rest of the
molecule. As one might have expected, this generated a compound that was inactive in the
glucose uptake assay. The set of results generated under site C modifications would suggest
that the presence of a piperazine ring is optimal for activity, but that replacement with a
piperidine ring does not dramatically reduce activity.
Table 2
In vitro glucose uptake activity of compounds 7a-c.

Glucose uptake
Active (Y) with
Compound
Y
p-value or inactive
(N)
Fold change ± SEM
2.14 ± 0.30
Y (***)
7a
1.24 ± 0.05
1.11 ± 0.08
Y (**)
N
7b
7c
All data are expressed as means ± standard error of the means (SEM) of a number of experiments (n) performed
for that compound. Statistical analysis was performed using one-way analysis of variance (ANOVA) using
GraphPad Prism 5.0 with a threshold of significance defined as a p-value <0.05 (95% confidence in result) or a
one star significance (*), p-value < 0.01 or two stars significance (**), and p-value < 0.0001 or three stars
significance (***). Only compounds which received stars significance in the glucose uptake assay were
considered “active” compounds. Full experimental details can be found in the experimental section and
supporting information.
2.8.4 In vitro glucose uptake for site D modifications.
Site D modifications involved the study of the length and nature of the chain connecting the
piperazine ring to the thiophene heterocycle. Here, most of the compounds generated could
effectively stimulate glucose uptake to some degree, although all were less active than 3a
(RTC1) (Figure 1, Table 3). Compounds 9a-9e explored the length of the methylene chain,
with 9a having no methylene chain and 9e having a five carbon chain in place. The results for
9a-9e suggest that the alkyl chain in 3a (RTC1) is important for biological function, as
activity was lost when the chain was completely removed in compound 9a. Activity was also
reduced when the alkyl chain was shortened but not fully removed, as was the case for
compound 9c (not active). Lengthening the alkyl chain (9d and 9e) also resulted in a decrease
in activity, but not a complete loss in activity. One possible explanation is that a certain alkyl
chain length is required for the molecule to bind strongly in an active/binding site. Too short
or too long a chain may result in a poor fit and weak binding. Also, an entropy effect could
also be considered for the longer chain compounds. The longer chain compounds are able to
adopt a larger number of conformations when unbound. As such, upon binding there will be a
larger increase in order, entropy cost, and hence the binding of such molecules may be less
thermodynamically favourable when compare to the shorter chained 3a (RTC1) [17,18].
The importance of the carbonyl group in 3a (RTC1) was explored by generating a derivative
(12) where this functional group was absent. Compound 12 was considerably less active that
3a (RTC1), although activity was not completely eliminated suggesting that the presence of
the carbonyl group was important for activity but not absolutely essential (Table 3).
Compounds 9f and 9g all contained modifications to the methylene chain but kept the
carbonyl group. These included replacing a methylene unit with a sulfur atom (9f) or a
carbonyl group (9g). All were less active than 3a (RTC1), although compound 9f containing
the S atom, showed a 2-fold increase in activity compared to vehicle control. This result
compared favourably with the 2.57-fold increase observed for 3a (RTC1).

Table 3
In vitro glucose uptake activity of compounds 3a, 9a-g, and 12.
Glucose uptake
Active (Y) with
Compound
Z
Fold change ± SEM
p-value or inactive (N)
0.93 ± 0.07
N
9a
9b
1.33 ± 0.04
1.09 ± 0.08
Y (***)
N
9c
3a
(RTC1)
2.57 ± 0.08
1.78 ± 0.13
Y (***)
Y (***)
9d
9e
1.33 ± 0.05
Y (***)

2.14 ± 0.23
Y (***)
9f
1.19 ± 0.03
1.36 ± 0.09
Y (***)
Y (***)
9g
12
All data are expressed as means ± standard error of the means (SEM) of a number of experiments (n) performed
for that compound. Statistical analysis was performed using one-way analysis of variance (ANOVA) using
GraphPad Prism 5.0 with a threshold of significance defined as a p-value <0.05 (95% confidence in result) or a
one star significance (*), p-value < 0.01 or two stars significance (**), and p-value < 0.0001 or three stars
significance (***). Only compounds which received stars significance in the glucose uptake assay were
considered “active” compounds. Full experimental details can be found in the experimental section and
supporting information.
2.8.5 In vitro glucose uptake for (i) site E modifications and (ii) a combination of both site D
& site E modifications.
Two compounds were evaluated with site E modifications (14a and 14b) (Figure 1, Table 4).
Both compounds were active with statistical significance but were considerably less active
than 3a (RTC1), suggesting that a thiophene ring at site E is not essential for activity but that
its replacement results in reduced activity.
The effect of modifying both sites D & E simultaneously was explored with compounds 16a-
g (Figure 1, Table 4). Here, all compounds except 16d and 16f were active with statistical
significance. However, all compounds were less active that 3a (RTC1), with 16c and 16g
proving most active with a 1.95- and 2.35-fold increase, respectively, compared to the vehicle
control. This compares favourably to the 2.57-fold increase of 3a (RTC1). The results in
Table 4 suggest that structural modification at the D-E sites of the molecule is better tolerated
than those at the A-B sites.
Table 4
In vitro glucose uptake activity of compounds 14a-b and 16a-g.
Glucose uptake
Active (Y) with
Compound
R
Fold change ±
SEM
p-value or inactive (N)
1.28 ± 0.11
Y (**)
14a
1.35 ± 0.11
1.43 ± 0.09
Y (**)
14b
16a
Y (***)

1.38 ± 0.05
1.95 ± 0.09
Y (***)
Y (***)
16b
16c
1.11 ± 0.04
1.38 ± 0.06
N
16d
16e
Y (***)
1.08 ± 0.08
2.35 ± 0.25
N
16f
Y (***)
16g
All data are expressed as means ± standard error of the means (SEM) of a number of experiments (n) performed
for that compound. Statistical analysis was performed using one-way analysis of variance (ANOVA) using
GraphPad Prism 5.0 with a threshold of significance defined as a p-value <0.05 (95% confidence in result) or a
one star significance (*), p-value < 0.01 or two stars significance (**), and p-value < 0.0001 or three stars
significance (***). Only compounds which received stars significance in the glucose uptake assay were
considered “active” compounds. Full experimental details can be found in the experimental section and
supporting information.
2.8.6 In vitro glucose uptake for modifications at a combination of sites.
A further five compounds (18-22) with modifications at more than one site were also
evaluated for their ability to stimulate glucose uptake in vitro (Table 5). As expected, given
the effects of the single modifications, these compounds generated lower levels of glucose
uptake compared to 3a (RTC1). Four of the five compounds gave low, but statistically
significant levels of activity. Compound 22 perhaps generated the most surprising result, as
earlier results for compound 3b, where the CF3 substituent was removed, generated an
inactive compound. However, the activity of compound 22 was quite low with a 1.25 ± 0.12
fold change compared to vehicle control. Compound 3a (RTC1) containing both the CF3 and
a piperazine ring, and compound 7a containing both the CF3 and the piperidine ring,
generated substantially higher levels of activity than compound 22 (2.57 fold change and 2.14
fold change respectively). This would again suggest that the presence of the CF3 is important
for activity.
The starting materials used in the synthesis of 3a (RTC1), namely compounds 23 and 2a,
along with their methylated forms (24 and 17), were also evaluated (Table 5). Both starting
materials showed very low levels of activity, with their methylated forms proving to be
inactive. This set of results would suggest that neither the thiophone alkyl chain (23, 24), nor
the aryl piperazine (2a, 17) alone is sufficient to stimulate glucose uptake, and that a more
complex molecule is required. It also suggests that 3a (RTC1) is not acting as a prodrug, with
cleavage/hydrolysis in vitro generating the starting materials as the active compounds.
Table 5
In vitro glucose uptake activity of compounds 18-22, 23, 2a, 24, and 17.
Compound
Structure
Glucose uptake

Fold change ± SEM Active (Y) with p-
value or inactive (N)
18
19
20
21
22
23
1.31 ± 0.05
1.22 ± 0.06
1.26 ± 0.06
1.31 ± 0.02
1.25 ± 0.12
Y (***)
N
Y (*)
Y (***)
Y (*)
1.16 ± 0.04
1.16 ± 0.06
Y (*)
Y (*)
2a
24
1.09 ± 0.04
0.92 ± 0.07
N
N
17
All data are expressed as means ± standard error of the means (SEM) of a number of experiments (n) performed
for that compound. Statistical analysis was performed using one-way analysis of variance (ANOVA) using
GraphPad Prism 5.0 with a threshold of significance defined as a p-value <0.05 (95% confidence in result) or a
one star significance (*), p-value < 0.01 or two stars significance (**), and p-value < 0.0001 or three stars
significance (***). Only compounds which received stars significance in the glucose uptake assay were
considered “active” compounds. Full experimental details can be found in the experimental section and
supporting information.
2.9 Molecular modelling.
The key features that are responsible for biological function were identified using
pharmacophore model generation (Biovia, Discovery Studio). These features consisted of 4
hydrophobics (covering sites A, B, C and E) and a hydrogen bond acceptor (HBA) feature
covering site D and 4 exclusion volumes defining the shape of the pocket (Figure 2). One

mis-matched feature was permitted for compound alignment to the pharmacophore. For
example, the importance of the carbonyl group in 3a (RTC1) was explored by analogue 12
which was missing this functional group. While compound 12 was considerably less active
that 3a (RTC1), the activity was not completely eliminated suggesting that the presence of
the carbonyl group was important for activity but not absolutely essential. Hence, additional
flexibility was incorporated into the pharmacophore model. A distinction is observed
between the fit to the pharmacophore of a very active compound such as 3a (RTC1) (Figure
2), with a 2.57-fold change in the glucose uptake assay (Table 4), and many inactive
compounds. Hence, the pharmacophore model could be beneficial in future SAR optimisation
of the compounds for the glucose uptake assay.
Fig. 2. Pharmacophore model with four hydrophobic features (in cyan), a hydrogen bond
acceptor feature (in green), and 4 exclusion volumes (in grey) based on the inactive
compounds. Compound 3a (RTC1), with a 2.57 fold change and a p value of < 0.0001 in the
glucose uptake assay, is shown matching the pharmacophore with a fit value of 1.89.
2.10 In vitro evaluation of NADH:ubiquinone oxidoreductase inhibition by 3a (RTC1) and its
derivatives.
Both 3a (RTC1) and 21 (RTB70) have been reported by this group to act through the
inhibition of NADH:ubiquinone oxidoreductase (complex I of the mitochondrial respiratory
chain), stimulating glucose uptake and restoring the glucose handling abilities of diabetic
mice [10]. As such, a number of the most active compounds in this glucose uptake study
were evaluated for their inhibitory activity against NADH:ubiquinone oxidoreductase in
permeabilised mitochondria (Table 6). For comparison, a number of the inactive compounds
were also evaluated in the NADH:ubiquinone oxidoreductase assay (Table 6). All of the
active compounds evaluated were also found to be active in the NADH:ubiquinone
oxidoreductase assay. The compounds that were most active in the glucose uptake assay were
not necessarily the most active in the NADH:ubiquinone oxidoreductase assay (Table 6).
This may be due to the difference between a whole cell assay system and an inhibition assay,
where for the latter the cell membrane and other cellular machinery have been removed.
Furthermore, and to our surprise, all of the inactive compounds evaluated showed some
activity in the NADH:ubiquinone oxidoreductase assay (Table 6). One possible explanation
is that in the whole cell glucose uptake assay, the compounds could not pass the cell
membrane, avoid efflux mechanisms, or survive the cells internal machinery (e.g. hydrolysis,
enzyme catalysed degradation) and hence were inactive [19]. In an inhibition assay, such as

the NADH:ubiquinone oxidoreductase assay, these barriers and challenges are removed, and
this could allow the compounds to access the complex I active site and inhibit its activity. As
such, whole cell assay systems can be useful indicators of in vivo biological potency.
Hollenback et al. have reported a similar trend in their research where they tested substrate
specificity in membrane and whole cell assays [20]. Research by Hernandez et al. also
compared enzyme assays with a whole cell based assay. They concluded that whole cell
based assays have the advantage of only selecting compounds that are able to penetrate cells
and reach the intracellular targets [21].
Table 6
NADH:ubiquinone oxidoreductase assay results for some of the aryl piperazine compounds.
Glucose
Compound
IC50 (μM)
uptake fold
change ± SEM
27
46
15
60
14
6
2.57 ± 0.08
1.58 ± 0.15
3a (RTC1)
3i
7a
2.14 ± 0.30
1.78 ± 0.13
2.14 ± 0.23
1.95 ± 0.09
0.96 ± 0.07
1.06 ± 0.10
0.84 ± 0.03
9d
9f
16c
3b*
3e*
5e*
7c*
17*
133
17
161
92
67
1.11 ± 0.08
0.92 ± 0.07
* Inactive in glucose uptake assay.
2.11 Predicted physiochemical properties.
In general, the compounds are predicted to have low-to-good solubility, good absorption
properties, and high-to-medium predicted blood brain barrier (BBB) penetration (Biovia
Discovery Studio). These ADMET screening results are summarized in Tables 1-7 of the
supplementary information. For example, compound 3a (RTC1) is predicted to have a low
solubility; good absorption and to readily pass the BBB. It is also likely to bind well to
plasma proteins and not to be hepatotoxic.
2.12 Physiochemical properties, in vitro DMPK, and in vivo PK of 3a (RTC1)
A preliminary study was undertaken in order to offer some insight into the physiochemical
properties, in vitro DMPK, and in vivo PK of 3a (RTC1). The results from this preliminary

study are encouraging, however only tentative conclusions are made due to the limitations of
the study. See supporting information for additional experimental detail and supporting data.
Thermodynamic solubility studies revealed the aqueous solubility of 3a (RTC1) to be 0.004
mg/mL (± 0.001 SEM). The low solubility of 3a (RTC1) is believed to be due to the
lipophilic properties of the compound (logP value of 4.79). However, as removal of the CF3
moiety decreased the logP value and consequentially the activity of derivatives, this property
is believed to be central to the activity of 3a (RTC1), as it may facilitate lipid membrane
permeability and promote target engagement. In vitro analysis of P-glycoprotein (P-gp) -
mediated efflux, in hMDR1-MDCK seeded filters, demonstrated 3a (RTC1) to be a highly
permeable compound with a passive permeability (Papp) value of 27.1x10-6 cm/sec (± 1.9 SD),
similar to that of the positive control, propranolol (21.8 x10-6 cm/sec, ± 1.0 SD). Under these
conditions 3a (RTC1) produced an efflux ratio of 0.9, suggesting the compound does not act
as a P-gp substrate. The administration of 3a (RTC1) should therefore not provoke adverse
drug-drug interactions [22] as many observed drug-drug interactions are caused by the
inhibition of both P-gp and the cytochrome P-450 enzymes (CYP450s).
The effect of 3a (RTC1) on recombinant human CYP450 isozymes was also investigated and
demonstrated weak-moderate inhibition of the major human CYP450s and hence a reduced
risk of unwanted drug-drug interactions. CYP1A2 (53.7% inhibition, ± 0.3 SEM), CYP2C9
(74.2% inhibition, ± 3.2 SEM), CYP2C19 (75.5% inhibition, ± 0.5 SEM), CYP2D6 (-2.7%
inhibition, ± 4.9 SEM), CYP3a4 (80.2% inhibition, ± 1.5 SEM), see supporting information.
Hepatocytes and microsomes were then used to gain an understanding of the way in which 3a
(RTC1) would be metabolised. In both species tested, human and mouse, 3a (RTC1) (1 µM)
demonstrated a shorter half-life and faster intrinsic clearance than the control drug,
verapamil, indicating that 3a (RTC1) would be readily metabolised by the liver in vivo (Table
7). In addition to this, 3a (RTC1) demonstrated no adverse effects on primary rat hepatocytes,
nor did it inhibit the hERG channel [10].
Table 7
In vitro assessment of 3a (RTC1) metabolic stability.
Compound Species
Microsome
Cryopreserved Hepatocytes
a
b
a
c
d
T1/2
CLint
T1/2
CLint
EH
(min)
(µL/min/mg)
(min)
(µL/min/106 cells)
Verapamil Human
Mouse
3a (RTC1) Human
Mouse
23
24
12
6
60
57
116
633
288
3
2
3
346
8
0.16
0.29
0.98
0.48
226
125
a
b
c
d
Half-life. Intrinsic clearance (µL/min/mg). Intrinsic clearance (µL/min/106 cells).
Hepatic extraction ratio. n = 2, see supporting information.
Compound 3a (RTC1) exhibited high mouse and human serum protein binding (Table 8),
which would prevent the rapid excretion of the compound. Compound 3a (RTC1) was also
observed to be relatively stable in plasma (80 % stable at 2 h), indicative of a compound that
would demonstrate good in vivo efficacy.
Table 8
Serum protein binding and plasma stability of 3a (RTC1).

Compound
Species
Serum Protein Binding
(% ± SEM)
Plasma Stability T1/2
(min)
808
327
3a (RTC1)
Human
Mouse
99.48 ± 0.04
99.48 ± 0.01
See supporting information.
As with the recent pharmacodynamic study [10], 3a (RTC1) (10 mg/kg) administered
intravenously (IV) or orally to male C57BL/6 mice produced no observable toxic effects.
Following IV administration, 3a (RTC1) had a short half-life of 0.45 h and high plasma
clearance. The compound distributed at high levels into all of the tissues analysed apart from
muscle, following a similar profile to plasma, with moderate to high clearance. Compound 3a
(RTC1) was also rapidly absorbed following oral dosing, with a similar half-life to post IV
administration at 0.76 h. The tissue levels also show a similar distribution profile to the
plasma profile, as was seen post IV dosing. Plasma clearance of 3a (RTC1) was in line with
that of mouse liver blood flow (5400 mL/h/kg), indicating metabolism to be the main route of
clearance (Table 9). While 3a (RTC1) exhibited high mouse and human serum protein
binding, it had moderate bioavailability (21.4%) suggesting the bound portion of 3a (RTC1)
may act as a reservoir or depot from which 3a (RTC1) is slowly released as the unbound free
form.
Table 9
Pharmacokinetic properties of 3a (RTC1) in vivo.
Parameter
Plasma
Orala
Brain
Kidney
Spleen
Liver
IVb Orala IVb
1.1 1.3
Muscle
IVb Orala IVb
Orala
IVb
Orala
1.1
Orala
IVb
T1/2
(h)
Tmax
(h)
Cmax
0.76
0.25
787
520
520
523
0.45 0.46
0.5
0.57
0.76
1
c.n.c
c.n.c
c.n.c
c.n.c
c.n.c
c.n.c
0.52
0.03 0.25 0.03
996 1430 1029
0.25
0.03
552
0.25
1103
1262
1262
1274
0.03 0.25 0.03
587 4875 1833
463 4048 487
463 4048 487
475 4089 531
0.03
165
1637
1224
1224
1275
(ng/mL)
AUClast
(h*ng/mL)
AUCall
(h*ng/mL)
AUCinf
(h*ng/mL)
Cl
240
240
763
763
774
246
246
320
77.3
77.3
107
320
244
253
348
4102
1686
3958
1826
2871
3057
2105
3188
1884
3059
9312
7134
(mL/h/kg)
Vd
(mL/kg)
a Oral and b IV solutions were formulated in 10% DMSO, 10% cremaphor, 80% saline.
c.n.c = could not calculate.
Concentrations of 3a (RTC1) (mean ± SEM) in mouse (C57 Black, ♂) were determined
following PO or IV administration at 10 mg/kg (n= 3). See supporting information.
2.13 Statistical analysis

Data presented herein are expressed as means ± standard error of the means (SEM) of a
number of experiments (n) performed for that compound. Where possible, statistical analysis
was performed using one-way analysis of variance (ANOVA) using GraphPad Prism 5.0 with
a threshold of significance defined as a p-value <0.05 (95% confidence in result) or a one star
significance (*), p-value < 0.01 or two stars significance (**), and p-value < 0.0001 or three
stars significance (***). Only compounds which received stars significance in the glucose
uptake assay were considered “active” compounds.
3. Conclusions
To summarise, the synthesis of 41 derivatives of hit compound 3a (RTC1) and a systematic
structure-activity-relationship study are reported where a number of compounds were shown
to effectively stimulate glucose uptake in vitro and inhibit NADH:ubiquinone
oxidoreductase. The key structural features responsible for biological function were also
identified via the generation of a pharmacophore model. The hit compound 3a (RTC1)
remained the most effective compound of those evaluated, with a 2.57 fold increase in
glucose uptake compared to vehicle control and micromolar inhibition of NADH:ubiquinone
oxidoreductase (IC50 = 27 µM). The presence of a p-CF3 aryl piperazine motif is important
for biological activity, with greater tolerance observed for changes to the alkyl chain and
thiophene heterocycle. Preliminary in vitro DMPK and in vivo PK studies are also described,
where encouraging results suggest that 3a (RTC1) does not act as a P-gp substrate and should
not provoke adverse drug-drug interactions. An initial in vitro metabolic stability study, using
hepatocytes and microsomes, suggested that 3a (RTC1) would be readily metabolised by the
liver. Intravenous (IV) or oral administration of 3a (RTC1) to male C57BL/6 mice produced
no observable toxic effects in our preliminary study. Following IV administration, 3a (RTC1)
had a short half-life of 0.45 h, showed high plasma clearance, and was distributed at high
levels into all of the tissues analysed apart from muscle. While 3a (RTC1) exhibited high
mouse and human serum protein binding, it had moderate bioavailability (21.4 %) suggesting
the bound portion of 3a (RTC1) may act as a reservoir or depot from which 3a (RTC1) is
slowly released as the unbound free form. The overall results suggest that aryl piperazines,
and 3a (RTC1) in particular, have potential as efficacious agents for the treatment of
diabetes.
4. Experimental section
4.1 Chemistry
4.1.1. General information
NMR spectra were recorded using Bruker Ascend 500 spectrometer at 293 K operating at
1
500 MHz for the H nucleus and 126 MHz for the 13C nucleus or a Bruker Advance 300
spectrometer operating at 300 MHz for 1H nucleus and 75 MHz for 13C nucleus. All chemical
shifts were referenced relative to the relevant deuterated solvent residual peaks or TMS.
Chemical shifts were given in ppm downfield from the internal standard and coupling
constants were given in Hz. 13C NMR spectra were recorded with complete proton
decoupling. Microwave reactions were carried out using a CEM Discover Microwave

Synthesizer with a vertically focused floor mounted infrared temperature sensor, external to
the microwave tube. The 10 mL reaction vessels used were supplied from CEM and were
made of borosilicate glass. Melting point analyses were carried out using a Stewart Scientific
SMP11 melting point apparatus and are uncorrected. High-resolution mass spectrometry
(HRMS) was performed in Maynooth University (MU) and the University of Bath (UoB).
HRMS in MU was conducted on an Agilent-LC 1200 Series coupled to a 6210 or 6530
Agilent Time-Of-Flight (TOF) mass spectrometer equipped with an electrospray source in
both positive and negative (ESI+/−) modes. In UoB, HPLC-ESI-TOF analysis was conducted
using an electrospray time-of-flight (MicrOTOF) mass spectrometer (Bruker Daltonik
GmbH, Bremen, Germany), which was coupled to an Agilent HPLC stack (Agilent, Santa
Clara, CA, United States) consisting of Agilent G1312A binary pump with G1329A
autosampler and G1316A column oven. Analyses were performed in ESI positive and
negative mode. Data processing was performed using the Compass Data Analysis software
scripts (Bruker Daltonik GmbH, Bremen, Germany). Infrared spectra were recorded on a
Perkin Elmer Spectrum 100 FT-IR spectrophotometer using a smart endurance single bounce
diamond, attenuated total reflection (ATR) cell or potassium bromide (KBr) disks. Spectra
were recorded in the region of 4000–600 cm−1 and were obtained by the co-addition of 4
scans with a resolution of 4 cm-1. Scintillation counts were obtained using a Wallac
MicroBeta scintillation counter (Perkin Elmer). Reactions were monitored with thin layer
chromatography (TLC) on Merck Silica Gel F254 plates. Developed sheets were visualised
using a portable UVltec CV-006 lamp (λ = 254, 365 nm). Flash column chromatography was
performed using Merck Silica gel 60.
4.1.2 1-(4-(Trifluoromethyl)phenyl)piperazine (2a).
1-Chloro-4-(trifluoromethyl)benzene (1.59 mL, 11.91 mmol) and piperazine (2.00 g, 23.82
mmol) were dissolved in NMP (5 mL) and heated at 200 º C for 30 mins in a microwave
reactor. The reaction mixture was purified using column chromatography (1:9, MeOH:DCM)
to give a white solid, 1.42 g (50%). Rf: 0.2 (9:1, DCM:MeOH); IR (ATR): υ (cm-1) 3257,
1
2836, 1668, 1613, 1323, 1101, 1067; H NMR (500 MHz, CDCl3): δ (ppm) 7.48 (d, J = 8.8
Hz, 2H), 6.92 (d, J = 8.8 Hz, 2H), 3.28 – 3.21 (m, 4H), 3.07 – 3.00 (m, 4H), 2.01 (bs, 1H); 1H
13
NMR data matches literature data [31]; C NMR (126 MHz, CDCl3): δ (ppm) 153.7, 126.4
(q, J = 3.8 Hz), 124.7 (q, J = 270.9 Hz), 120.6 (q, J = 32.6 Hz), 114.6, 49.1, 45.9; 13C NMR
data matches literature data [32]; HRMS calcd for C11H14F3N2 [M + H]+ 231.1104 found
231.1106.
4.1.3 1-(p-Tolyl)piperazine (2c).
p-Toluidine (1.00 g, 9.34 mmol) and bis(2-chloroethyl)amine.HCl (1.66 g, 9.34 mmol) were
dissolved in bis(2-methoxyethyl)ether (20 mL) and heated at reflux for 16 hrs after which the
reaction mixture was allowed to cool to rt. Et2O was added until the precipitation of a brown
solid was complete. The precipitate was collected via vacuum filtration and washed with
Et2O (3 x 20 mL) to give the HCl salt of compound 2c, which was then dissolved in a 5%
aqueous NaOH solution (10 mL) and stirred at rt for 4 hrs. The aqueous layer was extracted
with DCM (10 mL, followed be 3 x 10 mL) and and the combined organic layers were dried
over MgSO4. The solvent was removed under reduced pressure and the residue was purified
using flash chromatography to give a brown solid, 221 mg (13%). Rf: 0.2 (1:9,
1
MeOH:DCM); H NMR (300 MHz, CDCl3): δ (ppm) 7.07 (d, J = 8.4 Hz, 2H), 6.83 (d, J =

1
8.4 Hz, 2H), 3.14-3.13 (m, 4H), 3.08-3.05 (m, 4H), 2.26 (s, 3H); H NMR data matches
literature data [33]; HRMS calcd for C11H17N2 [M + H]+ 177.1385 found 177.1394.
4.1.4 1-(2-Chloro-4-(trifluoromethyl)phenyl)piperazine (2i).
3-Chloro-4-fluorobenzotrifluoride (100 µL, 0.75 mmol) and piperazine (129 mg, 1.5 mmol)
were dissolved in NMP (3 mL) and heated at 200 º C for 30 mins in a microwave reactor. The
reaction mixture was purified using column chromatography (1:4, MeOH:DCM) to give an
orange oil, 124 mg (63%). Rf: 0.2 (1:9, MeOH:DCM); 1H NMR (500 MHz, CDCl3): δ (ppm)
7.61 (d, J = 1.4 Hz, 1H), 7.47 (dd, J = 8.4, 1.4 Hz, 1H), 7.08 (d, J = 8.4 Hz, 1H), 3.10 (s, 8H),
1
13
2.78 (bs, 1H); H NMR data matches literature data [34]; C NMR (126 MHz, CDCl3): δ
(ppm) 152.5, 128.6, 127.9 (q, J = 3.8 Hz), 125.3 (q, J = 33.3 Hz), 124.7 (q, J = 3.9 Hz), 123.6
(q, J = 272.2 Hz), 120.2, 51.9, 51.4, 50.9, 45.9; HRMS calcd for C11H13ClF3N2 [M + H]+
264.06; found 265.0711.
4.1.5 1-(2-Bromo-4-(trifluoromethyl)phenyl)piperazine (2j).
3-Bromo-4-chlorobenzotrifluoride (200 µL, 1.33 mmol) and piperazine (229 mg, 2.66 mmol)
were dissolved in NMP (2 mL) and heated at 200 º C for 30 mins in a microwave reactor.
Compound 4 was used without further purification. Clear oil, 95 mg (23%, crude yield). Rf:
0.2 (1:9, MeOH:DCM); 1H NMR (300 MHz, CDCl3): δ (ppm) 7.46 (d, J = 7.8 Hz, 1H), 7.25
– 7.20 (m, 2H), 3.10 (m, 8H), 2.95 (bs, 1H); 1H NMR data matches literature data [35].
4.1.6 Method 1: General procedure for amide bond formation.
Carboxylic acid, HOBt, TBTU, anhydrous NEt3 and anhydrous DMF were placed in an oven-
dried Schlenk tube under a N2 atmosphere. The resulting solution was stirred at room
temperature for 15 mins. A second Schlenk tube was prepared containing amine and
anhydrous DMF under a N2 atmosphere. The resulting solution was transferred, via a
cannula, to the first Schlenk tube containing the carboxylic acid. The solution was stirred
under N2 overnight. DMF was removed under reduced pressure and the resulting oil was
acidified (pH = 3) using a 0.1 M aqueous HCl solution. The aqueous mixture was extracted
with DCM (20 mL, followed by 4 x 10 mL). The organic combined layers were washed with
a saturated aqueous solution of Na2CO3 (3 x 20 mL) and brine (3 x 20 mL) and dried over
MgSO4 and the residue was purified using column chromatography.
4.1.7 Method 2: General procedure for amide bond formation.
Carboxylic acid, HOBt, TBTU, NEt3 and DMF were placed in a round-bottom flask and
stirred at room temperature for 15 mins. A second round-bottom flask was prepared
containing amine and DMF. The resulting solution was transferred to the first round-bottom
flask containing the carboxylic acid. The solution was stirred overnight. DMF was removed
under reduced pressure and the residue was purified using column chromatography.

4.1.8 4-(Thiophen-2-yl)-1-(4-(4-(trifluoromethyl)phenyl)piperazin-1-yl)butan-1-one (3a).
Prepared from 1-(4-(trifluoromethyl)phenyl)piperazine 2a (100 mg, 0.43 mmol) and 4-(2-
thienyl)butyric acid (50 µL, 0.34 mmol), using HOBt (58 mg, 0.43 mmol), TBTU (140 mg,
0.43 mmol), NEt3 (100 µL, 0.69 mmol) and DMF (3 mL) and following the general
procedure described in method 2 section 4.1.7 [10]. The reaction mixture was stirred at room
temperature for 21 h. DMF was removed under reduced pressure and the residue was purified
using column chromatography (3:2 EtOAc:n-hexane) to give a white solid, 112 mg (86%).
1
Rf: 0.5 (3:2, EtOAc:n-hexane); IR (ATR): υ (cm-1) 2925, 2853, 1652, 1611, 1328, 1068; H
NMR (500 MHz, CDCl3): δ (ppm) 7.50 (d, J = 8.5 Hz, 2H), 7.13 (d, J = 4.9 Hz, 1H), 6.94 –
6.89 (m, 3H), 6.81 (bs, 1H), 3.77 (m 2H), 3.56 (m 2H), 3.31 – 3.17 (m, 4H), 2.93 (t, J = 7.2
Hz, 2H), 2.40 (t, J = 7.4 Hz, 2H), 2.13 – 2.0 (m, 2H); 13C NMR (126 MHz, CDCl3): δ (ppm)
171.1, 152.9, 144.4, 126.9, 126.6 (q, J = 3.7 Hz), 125.6 (q, J = 271.3 Hz), 124.6, 123.3, 121.2
(q, J = 32.8 Hz), 115.0, 48.3, 48.1, 45.0, 41.1, 32.0, 29.3, 27.0; HRMS calcd for
C19H21F3N2OSNa [M + H]+ 405.1219 found 405.1228. Matches literature data. [10, 35]
4.1.9 1-(4-Phenylpiperazin-1-yl)-4-(thiophen-2-yl)butan-1-one (3b).
Prepared from 1-phenylpiperazine (100 µL, 0.64 mmol) and 4-(2-thienyl)butyric acid (84 µL,
0.58 mmol) using HOBt (86 mg, 0.64 mmol), TBTU (205 mg, 0.64 mmol), NEt3 (129 µL,
0.93 mmol) and DMF (5 mL) and following the general procedure described in method 2
section 4.1.7. The reaction mixture was stirred at room temperature for 16.5 h. DMF was
removed under reduced pressure and the residue was purified using column chromatography
(3:2, EtOAc:n-hexane) to give a dark red oil, 142 mg (78%). Rf: 0.65 (3:2, EtOAc:n-hexane);
1
IR (ATR): υ (cm-1) 2915, 1639, 1597, 1227 (C-N); H NMR (500 MHz, CDCl3): δ (ppm)
7.30 – 7.23 (m, 2H), 7.11 (d, J = 5.1 Hz, 1H), 6.95 – 6.85 (m, 4H), 6.83 – 6.77 (m, 1H), 3.81
– 3.70 (m, 2H), 3.59 – 3.48 (m, 2H), 3.16 – 3.08 (m, 4H), 2.92 (t, J = 7.3 Hz, 2H), 2.43 (t, J =
13
7.3 Hz, 2H), 2.05 (p, J = 7.3 Hz, 2H); C NMR (126 MHz, CDCl3): δ (ppm) 171.0, 151.0,
144.4, 129.2, 126.8, 124.5, 123.2, 120.5, 116.6, 49.7, 49.4, 45.4, 41.5, 32.0, 29.3, 27.0;
HRMS calcd for C18H23N2OS [M + H]+ 315.1526 found 315.1531.
4.1.10 4-(Thiophen-2-yl)-1-(4-(p-tolyl)piperazin-1-yl)butan-1-one (3c).
Prepared from 1-(p-tolyl)piperazine 2c (221 mg, 1.25 mmol) and 4-(2-thienyl)butyric acid
(166 µL, 1.13 mmol) using HOBt (168 mg, 1.25 mmol), TBTU (401 mg, 1.25 mmol),
anhydrous NEt3 (253 µL, 1.82 mmol), and anhydrous DMF (5mL) and following the general
procedure described in method 1 section 4.1.6 The reaction mixture was stirred overnight at
room temperature under a N2 atmosphere. Purified using flash chromatography (1:1
EtOAc:n-hexane) to give an orange oil, 102 mg (24%). Rf: 0.3 (1:1, EtOAc:n-hexane); IR
(neat): υ (cm-1) 2917, 1642, 1439, 1232. 1H NMR (500 MHz, CDCl3): δ (ppm) 7.12 (dd, J =
5.2, 1.2 Hz, 1H), 7.09 (d, 2H, J = 8.6 Hz), 6.92 (dd, J = 5.2, 3.4 Hz, 1H), 6.83 (d, J = 8.6 Hz,
2H), 6.81-6.80 (m, 1H), 3.78-3.76 (m, 2H), 3.56-3.54 (m, 2H), 3.09-3.07 (m, 4H), 2.92 (t, J
= 7.3 Hz, 2H), 2.40 (t, J = 7.3 Hz, 2H), 2.28 (s, 3H), 2.08-2.02 (m, 2H). 13C NMR (126 MHz,
CDCl3): δ (ppm) 171.0, 148.8, 144.4, 130.2, 129.7, 126.8, 124.5, 123.2, 117.0, 50.3, 50.0,
45.5, 41.5, 32.0, 29.3, 27.0, 20.4. HRMS calcd for C19H25N2OS [M + H]+ 329.1682 found
329.1687.

4.1.11 1-(4-(4-Fluorophenyl)piperazin-1-yl)-4-(thiophen-2-yl)butan-1-one (3d).
Prepared from 1-(4-fluorophenyl)piperazine (100 mg, 0.55 mmol) and 4-(2-thienyl)butyric
acid (73 µL, 0.50 mmol) using HOBt (74 mg, 0.55 mmol), TBTU (177 mg, 0.55 mmol),
anhydrous NEt3 (123 µL, 0.80 mmol) and anhydrous DMF (5 mL) and following the general
procedure described in method 1 section 4.1.6. The reaction mixture was stirred at room
temperature for 17 h under a N2 atmosphere. DMF was removed under reduced pressure and
the residue was purified using column chromatography (3:2, EtOAc:n-hexane) to give an off-
white solid , 119 mg (71%). Mp: 84-86 °C; Rf: 0.6 (3:2, EtOAc:n-hexane); IR (KBr): υ (cm-1)
1
2824, 1651, 1511, 1437, 1334, 1203; H NMR (300 MHz, CDCl3): δ (ppm) 7.12 – 7.10 (m,
1H), 7.03 – 6.76 (m, 6H), 3.78 – 3.74 (m, 2H), 3.56 – 3.50 (m, 2H), 3.05 – 3.02 (m, 4H), 2.92
(t, J = 7.2 Hz, 2H), 2.40 (t, J = 7.2 Hz, 2H), 2.09 – 1.99 (m, 2H). 13C NMR (75 MHz,
CDCl3): δ (ppm) 171.0, 157.6 (d, J = 238.3 Hz), 147.6 (d, J = 2.3 Hz), 144.4, 126.8, 124.5,
123.2, 118.5 (d, J = 7.6 Hz), 115.7 (d, J = 22.0 Hz), 50.8, 50.4, 45.5, 41.5, 32.0, 29.3, 27.0;
Anal. calcd for C18H21FN2OS C, 54.74; H, 4.84; N, 6.72; found C, 54.73; H, 4.60; N, 6.58;
HRMS calcd for C18H22FN2OS [M + H]+ 333.1431 found 333.1431.
4.1.12 1-(4-(4-Nitrophenyl)piperazin-1-yl)-4-(thiophen-2-yl)butan-1-one (3e).
Prepared from 1-(4-nitrophenyl)piperazine (150 mg, 0.72 mmol) and 4-(2-thienyl)butyric
acid (95 µL, 0.66 mmol) using HOBt (97 mg, 0.72 mmol), TBTU (232 mg, 0.72 mmol), NEt3
(146 µL, 1.05 mmol), and DMF (6 mL) and following the general procedure described in
method 2 section 4.1.7. The reaction mixture was stirred at room temperature for 22.5 h.
DMF was removed under reduced pressure and the residue was purified using column
chromatography (7:3, EtOAc:DCM) to give an orange solid, 199 mg (84%). Mp: 120-121 °C;
1
Rf: 0.65 (7:3, EtOAc:DCM); IR (ATR): υ (cm-1) 2857, 1648, 1599, 1321, 1163; H NMR
(500 MHz, CDCl3): δ (ppm) 8.14 (dd, J = 9.1, 1.4 Hz, 2H), 7.13 (dd, J = 5.1, 1.1 Hz, 1H),
6.95 – 6.90 (m, 1H), 6.86 – 6.78 (m, 3H), 3.84 – 3.76 (m, 2H), 3.65 – 3.56 (m, 2H), 3.47 –
13
3.39 (m, 4H), 2.93 (t, J = 7.2 Hz, 2H), 2.40 (t, J = 7.2 Hz, 2H), 2.10 – 2.03 (m, 2H); C
NMR (126 MHz, CDCl3) δ (ppm) 171.2, 154.4, 144.3, 139.1, 126.9, 126.0, 124.6, 123.3,
112.9, 47.0, 46.9, 44.7, 40.8, 31.9, 29.2, 26.8; HRMS calcd for C18H21N3O3SNa [M + Na]+
382.1196 found 382.1212.
4.1.13 1-(4-(4-Aminophenyl)piperazin-1-yl)-4-(thiophen-2-yl)butan-1-one (3f).
Prepared from 1-(4-(4-nitrophenyl)piperazin-1-yl)-4-(thiophen-2-yl)butan-1-one 3e (143 mg,
0.39 mmol) dissolved in MeOH (10 mL) in a round bottom flask with PtO2 (20 mg, 0.08
mmol). The resulting mixture was degassed and purged with H2 (x3), stirred vigorously at
room temperature for 16.5 h under an atmosphere of H2 gas and monitored by TLC (7:1,
EtOAc:DCM). The reaction mixture was passed through a bed of Celite and the filtrate was
concentrated under reduced pressure. The residue was purified using column chromatography
(7:1, EtOAc:DCM) to give a brown oil, 75 mg (57%). Rf: 0.4 (7:1, EtOAc:DCM); IR (ATR):
1
υ (cm-1) 3337, 2815, 1638, 1510, 1223; H NMR (500 MHz, CDCl3): δ (ppm) 7.12 (dd, J =
5.1, 1.2 Hz, 1H), 6.92 (dd, J = 5.1, 3.4 Hz, 1H), 6.82 – 6.77 (m, 3H, H2), 6.67 – 6.63 (m,
2H), 3.77 – 3.74 (m, 2H), 3.56 – 3.52 (m, 2H), 2.99 – 2.90 (m, 6H), 2.42 – 2.38 (m, 2H), 2.08

– 2.01 (m, 2H); 13C NMR (126 MHz, CDCl3): δ (ppm) 171.0, 144.5, 144.0, 140.9, 126.8,
124.5, 123.2, 119.3, 116.2, 51.6, 51.2, 45.7, 41.7, 32.1, 29.3, 27.1; HRMS calcd for
C18H24N3OS [M + H]+ 331.1664 found: 331.1669.
4.1.14 4-(Thiophen-2-yl)-1-(4-(2-(trifluoromethyl)phenyl)piperazin-1-yl)butan-1-one (3g).
Prepared from 1-(2-(trifluoromethyl)phenyl)piperazine (83 µL, 0.43 mmol) and 4-(2-
thienyl)butyric acid (57 µL, 0.39 mmol) using HOBt (58 mg, 0.43 mmol), TBTU (139 mg,
0.43 mmol), NEt3 (60 µL, 0.62 mmol) and DMF (4 mL) and following the general procedure
described in method 2 section 4.1.7. The reaction mixture was stirred at room temperature for
22 h. DMF was removed under reduced pressure and the residue was purified using column
chromatography (3:2, EtOAc:n-hexane) to give a colourless oil, 122 mg (82%). Rf: 0.74 (3:2,
1
EtOAc:n-hexane); IR (ATR): υ (cm-1) 2916, 1642, 1312, 1107 cm-1; H NMR (500 MHz,
CDCl3): δ (ppm) 7.64 (d, J = 7.9 Hz, 1H), 7.56 – 7.48 (m, 1H), 7.33 – 7.21 (m, 2H), 7.12 (dd,
J = 5.1, 1.1 Hz, 1H), 6.92 (dd, J = 5.1, 3.4 Hz, 1H), 6.82 (dd, J = 3.3, 0.9 Hz, 1H), 3.76 (bs,
2H), 3.59 – 3.48 (m, 2H), 2.97 – 2.82 (m, 6H), 2.41 (t, J = 7.5 Hz, 2H), 2.06 (p, J = 7.4 Hz,
2H); 13C NMR (126 MHz, CDCl3): δ (ppm) 171.0, 151.7 (d, J = 1.1 Hz), 144.4, 132.8, 127.4
(q, J = 28.7 Hz), 127.2 (q, J = 5.4 Hz), 126.7, 125.3, 124.4, 124.0, 123.9 (q, J = 273.7 Hz),
123.1, 53.7, 53.0, 46.0, 42.0, 32.1, 29.3, 27.0; HRMS calcd for C19H22F3N2OS [M + H]+,
383.1399 found 383.1406.
4.1.15 1-(4-(3,5-bis(Trifluoromethyl)phenyl)piperazin-1-yl)-4-(thiophen-2-yl)butan-1-one
(3h).
Prepared from 1-(3,5-bis(trifluoromethyl)phenyl)piperazine (194 mg, 0.65 mmol) and 4-(2-
thienyl)butyric acid (85 µL, 0.58 mmol) using HOBt (87 mg, 0.65 mmol), TBTU (207 mg,
0.65 mmol), NEt3 (129 µL, 0.92 mmol) and DMF (5 mL) and following the general
procedure described in method 2 section 4.1.7. The reaction mixture was stirred at room
temperature for 14.5 h. DMF was removed under reduced pressure and the residue was
purified using column chromatography (3:2, EtOAc:n-hexane) to give a clear oil, 223 mg
(86%). Mp: 46-49 °C; Rf: 0.78 (3:2, EtOAc:n-hexane); IR (ATR): υ (cm-1) 2835, 1655, 1273,
1120; 1H NMR (500 MHz, CDCl3): δ (ppm) 7.33 (s, 1H), 7.24 (s, 2H), 7.12 (dd, J = 5.0 Hz),
6.96 – 6.89 (m, 1H), 6.81 (s, 1H), 3.86 – 3.76 (m, 2H), 3.65 – 3.55 (m, 2H), 3.34 – 3.21 (m,
13
4H), 2.93 (t, J = 7.2 Hz, 2H), 2.41 (t, J = 7.2 Hz, 2H), 2.11 – 2.02 (m, 2H); C NMR (126
MHz, CDCl3): δ (ppm) 171.1, 151.3, 144.3, 132.5 (q, J = 32.8 Hz), 126.8, 124.6, 123.5 (q, J
= 273.3 Hz), 123.2, 115.1 (m), 112.7 (m), 48.4, 48.3, 44.9, 41.0, 31.9, 29.2, 26.9; Anal. calcd
for C20H20F6N2OS C, 53.33; H, 4.48; N, 6.22; found C, 53.41; H, 4.25; N, 6.27; HRMS calcd
for C20H20F6N2OSNa [M + Na]+ 473.1093 found 473.1107.
4.1.16 1-(4-(2-Chloro-4-(trifluoromethyl)phenyl)piperazin-1-yl)-4-(thiophen-2-yl)butan-1-
one (3i).[35]
Prepared from 1-(2-chloro-4-(trifluoromethyl)phenyl)piperazine 2i (90 mg, 0.34 mmol) and
4-(2-thienyl)butyric acid (46 µL, 0.32 mmol) using HOBt (47 mg, 0.34 mmol), TBTU (111
mg, 0.34 mmol), NEt3 (47 µL, 0.51 mmol) and DMF (3 mL) and following the general
procedure described in method 2 section 4.1.7. The reaction mixture was stirred at room

temperature for 19 h. DMF was removed under reduced pressure and the residue was purified
o
using column chromatography (3:2, EtOAc:Pet. ether (40-60 C)) to give an oil, 111 mg
o
(83%). Mp: 84-86 °C; Rf : 0.7 (3:2, EtOAc:Pet. ether (40-60 C)); IR (ATR): υ (cm-1) 2823,
1624, 1325, 1111, 694; 1H NMR (500 MHz, CDCl3): δ (ppm) 7.65 – 7.62 (m, 1H), 7.48 (dd,
J = 8.4, 1.7 Hz, 1H), 7.13 (dd, J = 5.1, 1.0 Hz, 1H), 7.05 (d, J = 8.4 Hz, 1H), 6.95 – 6.91 (m,
1H), 6.84 – 6.79 (m, 1H), 3.86 – 3.76 (m, 2H), 3.64 – 3.56 (m, 2H), 3.09 – 3.02 (m, 4H), 2.93
(t, J = 7.3 Hz, 2H), 2.42 (t, J = 7.5 Hz, 2H), 2.06 (p, J = 7.4 Hz, 2H); 13C NMR (126 MHz,
CDCl3): δ (ppm) 171.2, 151.6, 144.4, 128.8, 128.0 (q, J = 3.8 Hz), 126.8, 126.0 (q, J = 33.4
Hz), 124.9 (q, J = 3.7 Hz), 124.6, 123.5 (q, J = 272.2 Hz), 123.3, 120.4, 51.2, 50.7, 45.6,
41.6, 32.1, 29.3, 27.0; Anal. calcd for C19H20F3N2OSCl C, 54.74; H, 4.84; N, 6.72; found C,
54.73; H, 4.60; N, 6.58; HRMS calcd for C19H20F3N2OSClNa [M + Na]+ 439.0829 found
439.085.
4.1.17 1-(4-(2-Bromo-4-(trifluoromethyl)phenyl)piperazin-1-yl)-4-(thiophen-2-yl)butan-1-
one (3j).[35]
Prepared from 1-(2-bromo-4-(trifluoromethyl)phenyl)piperazine 2j (95 mg, 0.30 mmol) and
4-(2-thienyl)butyric acid (41 µL, 0.28 mmol) using HOBt (41 mg, 0.30 mmol), TBTU (98
mg, 0.30 mmol), anhydrous NEt3 (62 µL, 0.44 mmol) and anhydrous DMF (2 mL) and
following the general procedure described in method 1 section 4.1.6. The reaction mixture
was stirred at room temperature for 21 h under N2 atmosphere. DMF was removed under
reduced pressure and the residue was purified using column chromatography (1:1,
o
EtOAc:Pet. ether (40-60 C)) to give a clear oil, 60 mg (50%). Rf: 0.8 (1:1 EtOAc:Pet. ether
o
1
(40-60 C)); IR (neat): υ (cm-1) 2914, 1645, 1605, 1432, 1324, 1122, 680; H NMR (500
MHz, CDCl3): δ (ppm) 7.49 (dd, J = 8.3, 0.6 Hz, 1H), 7.28 – 7.24 (m, 1H), 7.22 – 7.20 (m,
1H), 7.14 (dd, J = 5.1, 1.2 Hz, 1H), 6.93 (dd, J = 5.1, 3.4 Hz, 1H), 6.84 – 6.80 (m, 1H), 3.86
– 3.79 (m, 2H), 3.63 – 3.58 (m, 2H), 3.07 – 3.02 (m, 4H), 2.94 (td, J = 7.3, 0.5 Hz, 2H), 2.45
13
– 2.38 (m, 2H), 2.11 – 2.03 (m, 2H). C NMR (126 MHz, CDCl3): δ (ppm) 171.3, 149.2,
144.4, 132.6 (q, J = 1.26 Hz), 131.2, 130.2 (q, J = 32.8 Hz), 126.8, 124.6, 123.7 (q, J = 272.9
Hz), 123.2, 120.8 (q, J = 3.77 Hz), 117.4 (q, J = 3.7 Hz), 51.3, 51.0, 45.6, 41.7, 32.1, 29.3,
27.0; HRMS calcd for C19H20BrF3N2OSK [M + K]+ 499.0063 found 499.0079.
4.1.18 4-(Thiophen-3-yl)-1-(4-(5-(trifluoromethyl)pyridin-2-yl)piperazin-1-yl)butan-1-one
(5a).[35]
Prepared from 1-(5-(trifluoromethyl)pyridin-2-yl)piperazine (99 mg, 0.43 mmol) and 4-(2-
thienyl)butyric acid (50 µL, 0.34 mmol) using HOBt (58 mg, 0.43 mmol), TBTU (140 mg,
0.43 mmol), NEt3 (100 µL, 0.69 mmol) and DMF (3 mL) and following the general
procedure described in method 2 section 4.1.7. The reaction mixture was stirred at room
temperature for 20 h. DMF was removed under reduced pressure and the residue was purified
using column chromatography (3:2 EtOAc:n-hexane) to give a white solid, 119 mg (91%).
Mp: 107-109 °C; Rf: 0.5 (3:2, EtOAc:n-hexane); IR (ATR): υ (cm-1) 2912, 1645, 1609, 1415,
1318, 1106; 1H NMR (500 MHz, CDCl3): δ (ppm) 8.41 (s, 1H), 7.66 (dd, J = 9.0 Hz, J = 2.2
Hz, 1H), 7.14 – 7.10 (m, 1H), 6.94 – 6.91 (m, 1H), 6.82 – 6.80 (m, 1H), 6.64 (d, J = 9.0 Hz,
1H), 3.79 – 3.68 (m, 4H), 3.66 – 3.56 (m, 2H), 3.56 – 3.49 (m, 2H), 2.93 (t, J = 7.4 Hz, 2H),

2.41 (t, J = 7.4, 2H), 2.06 (p, J = 7.4 Hz, 2H); 13C NMR (126 MHz, CDCl3): δ (ppm) 171.3,
160.1, 145.8 (q, J = 4.3 Hz), 144.3, 134.7 (q, J = 3.2 Hz), 126.8, 124.6, 124.5 (q, J = 270.9
Hz), 123.2, 115.9 (q, J = 33.1 Hz), 105.7, 45.0, 44.7, 44.4, 41.0, 32.0, 29.3, 26.9; Anal. calcd
for C18H20F3N3OS C, 56.38; H, 5.26; N, 10.96; found C, 56.64; H,4.95; N, 10.89; HRMS
calcd for C18H21F3N3OS [M + H]+ 384.1352 found 384.1355.
4.1.19 1-(4-(6-Chloro-5-(trifluoromethyl)pyridin-2-yl)piperazin-1-yl)-4-(thiophen-2-
yl)butan-1-one (5b).[35]
Prepared from 1-(6-chloro-5-(trifluoromethyl)pyridin-2-yl)piperazine (218 mg, 0.816 mmol)
and 4-(2-thienyl)butyric acid (100 µL, 0.68 mmol) using HOBt (110 mg, 0.82 mmol), TBTU
(262 mg, 0.82 mmol), anhydrous NEt3 (152 µL, 1.08 mmol) and anhydrous DMF (5 mL) and
following the general procedure described in method 1 section 4.1.6. The reaction mixture
was stirred at room temperature for 20 h under a N2 atmosphere. DMF was removed under
reduced pressure and the residue was purified using column chromatography (3:2,
o
EtOAc:Pet. ether (40-60 C)) to give a beige solid, 192 mg (68%). Mp: 62-64 °C; Rf: 0.48
o
(3:2, EtOAc:Pet. ether (40-60 C)); IR (KBr): υ (cm-1) 2904, 1678, 1641, 1416, 1321, 1120,
698; 1H NMR (500 MHz, CDCl3): δ (ppm) 7.70 (d, J = 8.8 Hz, 1H), 7.13 (dd, J = 5.1, 1.2 Hz,
1H), 6.92 (dd, J = 5.1, 3.4 Hz, 1H), 6.84 – 6.77 (m, 1H), 6.48 (d, J = 8.7 Hz, 1H), 3.79 – 3.66
(m, 4H), 3.64 – 3.45 (m, 4H), 2.93 (t, J = 7.5 Hz, 2H), 2.45 – 2.35 (m, 2H), 2.10 – 2.02 (m,
2H); 13C NMR (126 MHz, CDCl3): δ (ppm) 171.3, 159.0, 147.7, 144.3, 137.8 (q, J = 4.5 Hz),
126.9, 124.6, 123.3, 123.2 (q, J = 271.0 Hz), 113.0 (q, J = 33.6 Hz), 103.3, 44.8, 44.6, 44.2,
40.8, 32.0, 29.2, 26.9; HRMS calcd for C18H20ClF3N3OSCl [M + H]+, 418.0962 found
418.0953.
4.1.20 1-(4-(3-Chloro-5-(trifluoromethyl)pyridin-2-yl)piperazin-1-yl)-4-(thiophen-2-
yl)butan-1-one (5c).[35]
Prepared from 1-(3-chloro-5-(trifluoromethyl)pyridin-2-yl)piperazine (114 mg, 0.43 mol) and
4-(2-thienyl)butyric acid (50 µL, 0.34 mmol) using HOBt (58 mg, 0.43 mmol), TBTU (140
mg, 0.43 mmol), NEt3 (100 µL, 0.69 mmol) and DMF (3 mL) and following the general
procedure described in method 2 section 4.1.7. The reaction mixture was stirred at room
temperature for 19 h. DMF was removed under reduced pressure and the residue was purified
using column chromatography (3:2, EtOAc:n-hexane) to give a solid, 123 mg (87%). Mp: 52-
54 °C; Rf: 0.78 (3:2, EtOAc:n-hexane); IR (ATR): υ (cm-1) 3036, 2852, 1646, 1600, 1438,
1317, 1117, 847; 1H NMR (500 MHz, CDCl3): δ (ppm) 8.40 (s, 1H), 7.79 (s, 1H), 7.14 – 7.10
(m, 1H), 6.95 – 6.89 (m, 1H), 6.83 – 6.78 (m, 1H), 3.81 – 3.74 (m, 2H), 3.60 – 3.53 (m, 2H),
3.51 – 3.44 (m, 4H), 2.93 (t, J = 7.2 Hz, 2H), 2.41 (t, J = 7.4 Hz, 2H), 2.06 (p, J = 7.3 Hz,
2H); 13C NMR (126 MHz, CDCl3): δ (ppm) 171.2, 159.6, 144.4, 143.0 (q, J = 4.2 Hz), 136.1
(q, J = 3.3 Hz), 126.8, 124.5, 123.2 (q, J = 272.3 Hz), 123.2, 121.1, 120.6 (q, J = 33.5 Hz),
48.6, 45.2, 41.3, 32.0, 29.3, 27.0; Anal. calcd for C18H19F3N3OSCl C, 51.73; H, 4.58; N,
10.06; found C, 52.17; H, 4.66; N, 9.89; HRMS calcd for C18H20F3N3OSCl [M + H]+
418.0971 found 418.0963.

4.1.21 1-(4-Cyclohexylpiperazin-1-yl)-4-(thiophen-2-yl)butan-1-one (5d).
Prepared from 1-cyclohexylpiperazine (150 mg, 0.89 mmol) and 4-(2-thienyl)butyric acid
(118 μL, 0.81 mmol) using HOBt (120 mg, 0.89 mmol), TBTU (285 mg, 0.89 mmol), NEt3
(180 μL, 1.29 mmol) and DCM (6 mL) and following the general procedure described in
methoid 2 section 4.1.7. The reaction mixture was stirred at room temperature for 17.5 h.
DCM was removed under reduced pressure and the residue was purified using column
chromatography (9:1, DCM:MeOH) to give a red solid, 213 mg (82%). Mp: 33-35 °C; Rf:
1
0.56 (9:1, DCM:MeOH); IR (ATR): υ (cm-1) 2928, 2851, 1632, 1445; H NMR (500 MHz,
CDCl3): δ (ppm) 7.11 (dd, J = 5.1, 1.0 Hz, 1H), 6.91 (dd, J = 5.1, 3.4 Hz, 1H), 6.79 (dd, J =
3.4, 1.0 Hz, 1H), 3.70 – 3.55 (m, 2H), 3.45 – 3.31 (m, 2H), 2.90 (t, J = 7.3 Hz, 2H), 2.67 –
2.40 (m, 4H), 2.40 – 2.32 (m, 2H), 2.32 – 2.25 (m, 1H), 2.07 – 1.96 (m, 2H), 1.90 – 1.73 (m,
13
4H), 1.69 – 1.56 (m, 1H), 1.31 – 1.14 (m, 4H), 1.14 – 1.04 (m, 1H); C NMR (126 MHz,
CDCl3): δ (ppm) 170.8, 144.5, 126.8, 124.6, 123.1, 63.6, 49.3, 48.7, 45.9, 41.9, 32.0, 29.3,
28.8, 27.1, 26.2, 25.8; HRMS calcd for C18H29N2OS [M + H]+ 321.1995 found 321.2005.
4.1.22 1-(4-Methylpiperazin-1-yl)-4-(thiophen-2-yl)butan-1-one (5e).
Prepared from 1-methylpiperazine (71 µL, 0.65 mmol) and 4-(2-thienyl)butyric acid (85 µL,
0.59 mmol) using HOBt (87 mg, 0.65 mmol), TBTU (207 mg, 0.65 mmol), NEt3 (90 µL, 0.94
mmol), and DCM (4 mL), and following the general procedure described in method 2 section
4.1.7. The reaction mixture was stirred at room temperature for 18 h. DCM was removed
under reduced pressure and the residue was purified using column chromatography (3:2,
EtOAc:n-hexane) to give a yellow oil, 104 mg (70 %). Rf: 0.56 (100% DCM); IR (ATR): υ
(cm-1) 2936, 2792, 1635, 1435, 1289; 1H NMR (500 MHz, CDCl3): δ (ppm) 7.11 (dd, J = 5.1,
1.2 Hz, 1H), 6.91 (dd, J = 5.1, 3.4 Hz, 1H), 6.80 (ddd, J = 3.4, 2.1, 1.2 Hz, 1H), 3.65 – 3.61
(m, 2H), 3.43 – 3.39 (m, 2H), 2.90 (t, J = 7.3 Hz, 2H), 2.38 – 2.33 (m, 6H), 2.29 (s, 3H), 2.02
(p, J = 7.4 Hz, 2H); 13C NMR (126 MHz, CDCl3): δ (ppm) 170.9, 144.5, 126.8, 124.5, 123.1,
55.2, 54.7, 46.0, 45.4, 41.5, 32.0, 29.3, 27.0; HRMS calcd for C13H21N2OS [M + H]+
253.1369 found 253.1367.
4.1.23 4-(Thiophen-2-yl)-1-(4-(4-(trifluoromethyl)phenyl)piperidin-1-yl)butan-1-one
(7a).[35]
Prepared from 4-(4-(trifluoromethyl)phenyl)piperidine.HCl (100 mg, 0.38 mmol) and 4-(2-
thienyl)butyric acid (50 μL, 0.34 mmol) using HOBt (51 mg, 0.38 mmol), TBTU (122 mg,
0.38 mmol), NEt3 (55 µL, 0.54 mmol) and DMF (2 mL) and following the general procedure
described in method 2 section 4.1.7. The reaction mixture was stirred for 22 h at room
temperature. DMF was removed under reduced pressure and the residue was purified using
column chromatography (3:2, EtOAc:n-hexane) to give an off-white solid, 87 mg (67%). Mp:
1
48-49 °C; Rf: 0.7 (3:2, EtOAc:n-hexane); IR (ATR): υ (cm-1) 2850, 1644, 1323, 1112; H
NMR (500 MHz, CDCl3): δ (ppm) 7.56 (d, J = 8.0 Hz, 2H), 7.30 (d, J = 8.0 Hz, 2H), 7.12 (d,
J = 5.1 Hz, 1H), 6.97 – 6.87 (m, 1H), 6.83 – 6.79 (m, 1H), 4.82 (d, J = 13.2 Hz, 1H), 3.92 (d,
J = 13.5 Hz, 1H), 3.10 (t, J = 12.9 Hz, 1H), 2.93 (t, J = 7.3 Hz, 2H), 2.85 – 2.74 (m, 1H), 2.63
(t, J = 12.7 Hz, 1H), 2.41 (t, J = 7.4 Hz, 2H), 2.06 (p, J = 7.4 Hz, 2H), 1.93 – 1.84 (m, 2H),
1.68 – 1.52 (m, 2H); 13C NMR (126 MHz, CDCl3): δ (ppm) 170.9, 149.2, 144.5, 128.9 (q, J =

32.5 Hz), 127.1, 126.8, 125.6 (q, J = 3.8 Hz), 124.5, 124.2 (q, J = 272.3 Hz), 123.1, 46.0,
42.7, 42.2, 33.7, 32.6, 32.2, 29.4, 27.1; HRMS calcd for C20H22F3NOSNa [M + Na]+
404.1266 found 404.1279.
4.1.24 4-(Thiophen-2-yl)-N'-(4-(trifluoromethyl)phenyl)butane hydrazide (7b).
Prepared from (4-(trifluoromethyl)phenyl)hydrazine (195 mg, 1.11 mmol) and 4-(2-
thienyl)butyric acid (149 µL, 1.03 mmol) using HOBt (149 mg, 1.11 mmol), TBTU (356 mg,
1.11 mmol), anhydrous NEt3 (231 µL, 1.66 mmol) and anhydrous DMF (8 mL) and
following the general procedure described in method 1 section 4.1.6. The reaction mixture
was stirred at room temperature for 20 h under a N2 atmosphere. DMF was removed under
reduced pressure and the residue was purified using column chromatography (elution
gradient 1:1 EtOAc:n-hexane to 3:2 EtOAc:n-hexane) to give an off-white solid , 235 mg
(69%). Mp: 110-112 °C; Rf: 0.3 (1:1, EtOAc:n-hexane); IR (KBr): υ (cm-1) 3309, 2963, 1639,
1614, 1333, 1104; 1H NMR (500 MHz, DMSO-d6): δ (pm) 9.82 (d, J = 1.4 Hz, 1H), 8.39 (s,
1H), 7.47 (d, J = 8.6 Hz, 2H), 7.34 (dd, J = 5.1, 1.0 Hz, 1H), 6.97 (dd, J = 5.1, 3.4 Hz, 1H),
6.88 (m, 1H), 6.79 (d, J = 8.5 Hz, 2H), 2.83 (t, J = 7.6 Hz, 2H), 2.26 (t, J = 7.4 Hz, 2H), 1.95
– 1.85 (m, 2H); 13C NMR (126 MHz, DMSO-d6): δ (ppm) 172.2, 153.0, 144.5, 127.5, 126.6
(q, J = 3.8 Hz), 126.1 (q, J = 294.2 Hz), 125.1, 124.1, 118.5 (q, J = 31.9 Hz), 111.8, 32.9,
29.1, 27.7; Anal. calcd for C15H15F3N2OS C, 54.87; H, 4.60; N, 8.53; found C, 54.44; H, 4.39;
N, 8.09; HRMS calcd for C15H16F3N2OS [M + H]+ 330.0975 found 330.096.
4.1.25 4-(Thiophen-2-yl)-N-(4-(trifluoromethyl)phenyl)butanamide (7c)
Prepared from 4-(trifluoromethyl)aniline (69 mg, 0.43 mmol) and 4-(thiophen-2-yl)butanoic
acid (50 μL, 0.34 mmol) using HOBt (58 mg, 0.43 mmol), TBTU (140 mg, 0.43 mmol), NEt3
(100 µL, 0.51 mmol) and DCM (6 mL) and following the general procedure described in
method 2 section 4.1.7. The reaction mixture was stirred at room temperature for 20 h. DCM
was removed under reduced pressure and the residue was purified using column
chromatography (3:2, EtOAc:n-hexane) to give a white solid, 96 mg (90%). Rf: 0.6 (3:2,
1
EtOAc:n-hexane); IR (ATR): υ (cm-1) 3304, 2926, 1670, 1514, 1319, 1125; H NMR (500
MHz, CDCl3): δ (ppm) 7.62 (d, J = 8.6 Hz, 2H), 7.56 (d, J = 8.6 Hz, 2H), 7.29 (bs, 1H), 7.14
(ddd, J = 5.1, 1.2, 0.5 Hz, 1H), 6.93 (dd, J = 5.1, 3.4 Hz, 1H), 6.81 (dd, J = 2.2, 1.1 Hz, 1H),
13
2.94 (t, J = 7.2 Hz, 2H), 2.42 (t, J = 7.4 Hz, 2H), 2.12 (p, J = 7.2 Hz, 2H); C NMR (126
MHz, CDCl3): δ (ppm) 171.0, 143.8, 140.8, 126.9, 126.3 (q, J = 3.8 Hz), 126.1 (q, J = 33.0
Hz), 124.8, 124.1 (q, J = 272.1 Hz), 123.4, 119.3, 36.4, 29.0, 27.0; HRMS calcd for
C15H14NOSNa [M + H]+ 336.1393 found 336.1393.
4.1.26 3-(Thiophen-2-ylthio)propanoic acid (8f).
Thienyl-2-thiol (48 µl, 0.52 mmol), 3-bromopropanoic acid (80 mg, 0.52 mmol) and
NaHCO3 (130 mg, 1.56 mmol) were heated at reflux in EtOH (2 mL). After 5 h the reaction
mixture was allowed to cool to room temperature and the EtOH was removed under reduced
pressure. The residue was dissolved in H2O (2 mL) and washed with Et2O (4 x 10 mL). The
aqueous layer was acidified to pH = 6 with 0.1 M aqueous HCl and extracted with Et2O (4 x
10 mL). The combined organic layers were concentrated under reduced pressure and the

residue was purified using column chromatography (1:9, MeOH:DCM) to give a clear oil, 83
1
mg (85%). Rf: 0.3 (1:9, MeOH:DCM); H NMR (300 MHz, CDCl3): δ (ppm) 10.86 (br s,
1H), 7.37 (dd, J = 5.1, 1.2 Hz, 1H), 7.16 (dd, J = 2.4, 1.2 Hz, 1H), 6.97 – 6.99 (m, 1H), 2.97
1
(t, J = 7.2 Hz, 2H), 2.64 (t, J = 7.2 Hz, 2H); H NMR data matches literature data [31];
HRMS calcd for C7H9O2S2 [M + H]+: 189.0038 found 189.0040.
4.1.27 Thiophen-2-yl (4-(4-(trifluoromethyl)phenyl)piperazin-1-yl) methanone (9a).
Prepared from 1-(4-(trifluoromethyl)phenyl)piperazine 2a (76 mg, 0.33 mmol) and
thiophene-2-carboxylic acid (38 mg, 0.30 mmol) using HOBt (45 mg, 0.33 mol), TBTU (107
mg, 0.33 mmol), NEt3 (75 µL, 0.48 mmol) and DMF (4 mL) and following the general
procedure described in method 2 section 4.1.7. The reaction mixture was stirred for 21 h at
room temperature. DMF was removed under reduced pressure and the residue was purified
o
using column chromatography (1:1, EtOAc:Pet. ether (40-60 C)) to give a solid, 70 mg
(69%). Mp: 126-130 °C; Rf: 0.6 (1:1, EtOAc:Pet.ether (40-60 oC)); IR (ATR): υ (cm-1) 2838,
1606, 1334, 1096; 1H NMR (500 MHz, CDCl3): δ (ppm) 7.55 – 7.45 (m, 3H), 7.35 (d, J = 3.2
Hz, 1H), 7.13 – 7.02 (m, 1H), 6.93 (d, J = 8.6 Hz, 2H), 3.98 – 3.86 (m, 4H), 3.42 – 3.27 (m,
4H); 13C NMR (126 MHz, CDCl3) δ (ppm) 163.7, 152.8, 136.6, 129.1, 129.0, 126.8, 126.5 (q,
J = 3.8 Hz), 124.5 (q, J = 271.3 Hz), 121.3 (q, J = 32.8 Hz), 114.9, 48.2; Anal. calcd for
C16H15F3N2OS C, 56.46; H, 4.44; N, 8.23; found C, 56.72; H, 4.62; N, 8.42; HRMS calcd for
C16H15F3N2OSNa [M + Na]+ 363.0749 found 363.0762.
4.1.28 2-(Thiophen-2-yl)-1-(4-(4-(trifluoromethyl)phenyl)piperazin-1-yl)ethanone (9b).[35]
Prepared from 1-(4-(trifluoromethyl)phenyl)piperazine 2a (100 mg, 0.43 mmol) and 2-
(thiophen-2-yl)acetic acid (48 mg, 0.34 mmol) using HOBt (58 mg, 0.43 mmol), TBTU (138
mg, 0.43 mmol), NEt3 (87 µL, 0.63 mmol), DMF (4 mL) and following the general procedure
described in method 2 section 4.1.7. The reaction mixture was stirred at room temperature for
21 h. Purified using column chromatography (3:2, EtOAc:n-hexane) to give an orange solid,
98 mg (82%). ]. Rf: 0.9 (3:2, EtOAc:n-hexane); IR (ATR): υ (cm-1) 2826, 1614, 1409, 1336,
1229, 1101; 1H NMR (500 MHz, CDCl3): δ (ppm) 7.49 (d, J = 8.7 Hz, 2H), 7.21 (dd, J = 5.1
Hz, J = 1.2 Hz, 1H), 6.97 – 6.88 (m, 4H), 3.96 (s, 2H), 3.84 – 3.78 (m, 2H), 3.72 – 3.64 (m,
2H), 3.29 – 3.23 (m, 2H), 3.21 – 3.12 (m, 2H); 13C NMR (126 MHz, CDCl3): δ (ppm) 168.5,
152.8, 136.2, 127.0, 126.5 (q, J = 3.3 Hz), 126.1, 124.9, 124.6 (q, J = 271.3 Hz), 121.4 (q, J =
32.8 Hz), 115.0, 48.2, 47.9, 45.8, 41.5, 35.2; HRMS calcd for C17H17F3N2O2SNa [M + Na]+
377.0906 found 377.0915.
4.1.29 3-(Thiophen-2-yl)-1-(4-(4-(trifluoromethyl)phenyl)piperazin-1-yl)propan-1-one
(9c).[35]
Prepared from 1-(4-(trifluoromethyl)phenyl)piperazine 2a (81 mg, 0.35 mmol) and 3-
(thiophen-2-yl)propanoic acid (50 mg, 0.32 mmol) using HOBt (47 mg, 0.35 mmol), TBTU
(113 mg, 0.35 mmol), anhydrous NEt3 (71 µL, 0.51 mmol) and anhydrous DMF (5 mL) and
following the general procedure described in method 1 section 4.1.6. The reaction mixture
was stirred at room temperature for 16 h under a N2 atmosphere. DMF was removed under
reduced pressure and the residue was purified using column chromatography (3:2, EtOAc:n-