Exploiting the Biginelli reaction: Nitrogen-rich pyrimidine-based tercyclic α-helix mimetics

Article abstract of DOI:10.1016/j.tet.2015.12.044

Several rationally designed pyrimidine-based scaffolds intended to mimic the spatial projection of the i, i+3, and i+7 residues of an α-helix and also possess improved aqueous solubility were prepared. A Biginelli-oxidation process was used to form the central pyrimidine ring of these scaffolds, which was subsequently manipulated to form pyrimidine-based tercyclic α-helix mimetics. A pyrimidine-based scaffold designed to mimic the α-helical BH3 domain of the pro-apoptotic Bak protein was also prepared as a putative inhibitor of the Bcl-x_L/Bak protein-protein interaction. The pyrimidine-based tercyclic α-helix mimetics, and the putative Bcl-x_L inhibitor, were assessed using a luminescence competition assay, however, none displayed inhibitory activity against Bcl-x_L or Mcl-1.

Full text of DOI:10.1016/j.tet.2015.12.044

Accepted Manuscript
Exploiting the Biginelli reaction: nitrogen-rich pyrimidine-based tercyclic alpha-helix
mimetics
Zelong Lim, Peter J. Duggan, Soo San Wan, Guillaume Lessene, Adam G. Meyer,
Kellie L. Tuck
PII:
S0040-4020(15)30292-1
10.1016/j.tet.2015.12.044
TET 27370
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 2 October 2015
Revised Date: 9 December 2015
Accepted Date: 21 December 2015
Please cite this article as: Lim Z, Duggan PJ, San Wan S, Lessene G, Meyer AG, Tuck KL, Exploiting
the Biginelli reaction: nitrogen-rich pyrimidine-based tercyclic alpha-helix mimetics, Tetrahedron (2016),
doi: 10.1016/j.tet.2015.12.044.
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ACCEPTED MANUSCRIPT
X
N
N
O
N
N
X = N
X = CH
O
+
Y
H N
NH2
2
N
N
N
O
N
N
O
O
O
R
O
R
H
CO Et
2
X
N
O
NH
N
N
NH
X = N
X = CH

ACCEPTED MANUSCRIPT
Exploiting the Biginelli reaction: nitrogen-rich pyrimidine-based
tercyclic alpha-helix mimetics
a,b
b,c
d,e
d,e,f
b
Zelong Lim, Peter J. Duggan, Soo San Wan, Guillaume Lessene, Adam G. Meyer* ,
a
Kellie L. Tuck*
a
School of Chemistry, Monash University, Clayton, Victoria 3800, Australia.
CSIRO Manufacturing, Bag 10, Clayton South, Victoria 3169, Australia.
School of Chemical and Physical Sciences, Flinders University, South Australia 5042, Australia.
ACRF Chemical Biology Division, The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade,
b
c
d
Parkville Victoria 3052, Australia.
e
Department of Medical Biology, The University of Melbourne, Parkville Victoria 3010, Australia.
Department of Pharmacology and Therapeutics, The University of Melbourne, Parkville Victoria 3010,
f
Australia.
Corresponding authors. Tel.: +61 3 99054510; fax: +61 3 99054597; e-mail address: Kellie.Tuck@monash.edu
(
K. L. Tuck); Tel.: +61 3 95452476; fax: +61 3 95452376 e-mail address: Adam.Meyer@csiro.au (A. G. Meyer)
Abstract
Several rationally designed pyrimidine-based scaffolds intended to mimic the spatial
projection of the i, i + 3, and i + 7 residues of an α-helix and possess improved aqueous
solubility were prepared. A Biginelli-oxidation process was used to form the central
pyrimidine ring of these scaffolds, which was subsequently manipulated to form pyrimidine-
based tercyclic α-helix mimetics. A pyrimidine-based scaffold designed to mimic the α-
helical BH3 domain of the pro-apoptotic Bak protein was also prepared as a putative inhibitor
of the Bcl-x /Bak protein-protein interaction. The pyrimidine-based tercyclic α-helix
L
mimetics, and the putative Bcl-x inhibitor, were assessed using a luminescence competition
L
assay, however, none displayed inhibitory activity against Bcl-x or Mcl-1.
L
Keywords: Biginelli reaction; pyrimidine; alpha-helix mimetics; Bcl-x inhibitor
L
1
. Introduction
Protein-protein interactions (PPIs) play an essential role in a wide spectrum of biological
1
, 2
processes.
These PPIs have been recognised over the past decade to be potential
therapeutic targets for a variety of disease states, and consequently the development of
chemical probes to investigate these interactions has been examined by a number of research
groups. A recent review by Arkin describes the progress that has been made with small
3
molecule therapeutics to inhibit PPIs. In the Protein Data Bank (PDB), 62% of PPIs have an
4
, 5
α-helix at the interface, and often the key interactions involve the i, i + 3 (or i + 4) and i + 7
6
7-16
amino acid side chains found on a same ‘face’ of an α-helix. We and others
have been
interested in developing non-peptidic small molecules that are type III α-helix mimetics, i.e.,
molecules that mimic the spatial projection of key residues on an α-helix, but without the
5
, 17-20
peptide backbone. A number of recent reviews have summarised outcomes in this area.
Hamilton and co-workers pioneered work in this area with their terphenyl scaffold mimetics
7
-
1
0
(
e.g., 1 and 2, Fig. 1), with the concept extended to more aqueous soluble trispyridylamide
1
4
scaffold mimetics (e.g., 3 and 4, Fig. 1). The compounds 1-4 were shown to disrupt the Bcl-
x /Bak interaction, an important PPI involved in the apoptosis of cancer cells, with low
L
2
1
micromolar to nanomolar activity. A potent small molecule inhibitor of Bcl-xL (and Bcl-2)
is Navitoclax (ABT-263),
2
2, 23
which has been evaluated in Phase II clinical trials for the
treatment of lung cancer, where it exhibited a clinical benefit with future trials to investigate
2
4, 25
26
combination therapies.
ABT-199, a successor to ABT-263, is also a Bcl-2 inhibitor, and
2
7
is currently in Phase III clinical trials for the treatment of chronic lymphocytic leukaemia.
1

ACCEPTED MANUSCRIPT
a) CO H
b)
NH2
2
O
R1
O
N
N
H
O
O
H
R1 1 (R1 = naphthyl), K = 114 nM
R2 3 (R1 = R2 = phenyl), K = 1.6
M
i
i
2
(R1 = phenyl), K = 1.82
M
N
N
4 (R1 = R2 = i-Pr), Ki = 2.3
M
i
O
O
N
O
CO H
2
O
9, 10
14
Fig. 1. α-Helix mimetics reported by Hamilton et al.: a) terphenyl scaffold;
Inhibitory values are for the disruption of the Bcl-x /Bak interaction.
b) trispyridylamide scaffold.
L
In an effort to substantially improve the aqueous solubility of terphenyl-based mimetics,
Rebek and co-workers explored the use of a pyridazine as the central ring of the scaffold; two
2
8
mimetics relevant to this work are the oxazole-pyridazine-piperazine 5 (Fig. 2a) and
2
9
oxazole-pyridazine-oxazole 6 (Fig. 2b). It was shown that whilst these compounds did
*
disrupt the Bcl-x /Bak interaction , their affinity for Bcl-x was less than that of the terphenyl
L
L
analogue, compound 1. The design and synthesis of 2,5-terpyrimidinylenes as α-helix
mimetics has been reported by McLaughlin and co-workers (e.g., compound 7, Fig. 2c),
3
0, 31
although their ability to disrupt PPIs was not described.
28, 29
Fig. 2. a) and b) Relevant pyridazine-based α-helix mimetics reported by Rebek and co-workers;
terpyrimidinylene mimetic.
c) a 2,5-
3
0, 31
With the aim of improving the aqueous solubility and Bcl-x inhibitory activity of previously
L
published α-helix mimetics, we chose to use pyrimidine as the central ring of new tercyclic
scaffolds, so as to afford nitrogen-rich mimetics able to project residues in a similar manner
to the i, i + 3 and i + 7 positions of an α-helix. The pyrimidine ring system is commonly
found in nature (e.g., adenine), and can be readily accessed from dihydropyrimidinones,
3
2
which in turn can be synthesised in a one-pot procedure using the Biginelli reaction. The
Biginelli reaction is an acid-catalysed, three-component reaction between urea, a β-ketoester,
and an aldehyde (typically an aromatic aldehyde) to give a 3,4-dihydropyrimidin-2-(1H)-one
8
, which can then be readily converted to the corresponding pyrimidine 9. We envisaged that
pyrimidine 9, by virtue of it containing two chemical handles, would serve as a key
intermediate that could be subsequently manipulated to enable the rapid synthesis of
pyrimidine-based tercyclic α-helix mimetics 10 (Scheme 1).
*
Determined with an in vitro fluorescence polarization assay at a concentration of 10 µM.
2

ACCEPTED MANUSCRIPT
Scheme 1. Synthesis of pyrimidine 9 using a Biginelli-oxidation process, and the subsequent formation of
pyrimidine-based tercyclic α-helix mimetics 10.
Guided by an in silico screening process, we report herein the synthesis of several novel
nitrogen-rich tercyclic α-helix mimetics that feature a pyrimidine as the central ring.
Polyalanine mimetics were initially investigated to provide uniformity across the scaffolds
and to abridge in silico calculation complexity. Compounds 11, 12, 13 and 14 (Fig. 3a) were
deemed as being suitable synthesis candidates, and the putative Bcl-x inhibitor 15 (Fig. 3b)
L
was also designed and prepared, and all were evaluated as inhibitors of the Bcl-x /Bak PPI.
L
Fig. 3. Novel pyrimidine-based tercyclic mimetics described in this paper: a) polyalanine α-helix mimetics; b)
putative Bcl-x inhibitor.
L
2
. Results and discussion
2
.1 In silico studies
In silico modelling of a range of nitrogen-rich, pyrimidine-based tercyclic scaffolds as
polyalanine α-helix mimetics was performed by using molecular mechanics conformational
6
searches followed by ab initio calculations, in a similar manner to that previously reported.
For each low energy ab initio conformer, the distances between the carbon atoms of each side
chain methyl group were measured to determine if it was a suitable α-helix mimetic. This is
illustrated for compound 12, where the distances between the carbon atoms of each side chain
methyl group were measured to determine if they matched those between the key i, i + 3 and i
+
7 side chains of a polyalanine α-helix (Fig. 4a). Finally, a root-mean-square deviation
(RMSD) was then calculated for the low energy conformer of the compound overlayed with a
polyalanine α-helix (Fig. 4c). Based on these in silico screening studies it was concluded that
†
compounds 11, 12, 13 and 14 were worthy of synthesis and further investigation.
†
The ab initio optimised low energy conformers of compounds 11 and 13, and the low energy conformers of
compounds 11 and 13 overlayed with a polyalanine α-helix are included in the Supplementary Data.
3

ACCEPTED MANUSCRIPT
Fig. 4. a) An ab initio optimised low energy conformer of compound 12; b) polyalanine α-helix with i, i + 3 and
i + 7 R-groups highlighted in green; c) low energy conformer of compound 12 (grey) overlayed with a
polyalanine α-helix (RMSD: 0.90).
2
.2 Synthesis
The most synthetically accessible pyrimidine precursor proved to be the 4-methyl substituted
pyrimidinone 19, derived from the Biginelli reaction between urea (16), ethyl acetoacetate
(
17) and acetaldehyde (18). Often metals or solid supported catalysts are required to achieve
33-36
acceptable yields when aliphatic aldehydes are used in Biginelli reactions;
however, in
this case compound 19 was formed using classical conditions in a moderate yield of 51%
(
Scheme 1). Although a pyrimidine core substituted with a hydrogen atom at the 4-position
3
7-39
would have been preferable, in our hands and others,
the use of formaldehyde or alternate
formaldehyde sources resulted in poor yields of the subsequent Biginelli product. Synthesis
of the key pyrimidine triflate 21 (which contains two chemical handles that can be
manipulated to allow the incorporation of various nitrogen-based heterocycles) was
accomplished by initial oxidation of the pyrimidinone 19 to give the pyrimidinol 20, followed
by reaction with triflic anhydride (Scheme 2).
O
16
O
OH
OTf
H
2
N
NH
2
a
b
c
HN
O
NH
O
N
O
N
N
O
N
O
O
+
O
O
H
O
O
17
18
19
20
21
Scheme 2. Reagents and conditions: a) EtOH, cat. HCl(aq), rt, 15 h, 51%; b) HNO (70% w/w), rt, 1 h, 72%; c)
3
Tf O, py, DCM, rt, 16 h, 78%.
2
With key functionalised pyrimidine 21 in hand, a number of biaryl intermediates were
synthesised via microwave-assisted Suzuki cross-coupling reactions. Thus, pyrimidine triflate
2
1 was cross-coupled with o-tolylboronic acid, using Pd(PPh ) as the catalyst, to afford the
3 4
‡
phenyl-pyrimidine biaryl 22 in good yield. Likewise, reaction of triflate 21 with (4-
methylpyridin-3-yl)boronic acid gave biaryl product 23. Synthesis of phenyl- and pyridine-
§
‡
Conventional conditions (reflux, 40 h) gave the product in a 56% yield, whereas microwave irradiation (130
°
C, 25 min) resulted in an 84% yield of the desired product.
§
2
3 was unfortunately contaminated with triphenylphosphine oxide that could not be removed by column
chromatography or trituration.
4

ACCEPTED MANUSCRIPT
pyrimidine-oxadiazoles 11 and 12 was then accomplished by heating the biaryl esters,
compounds 22 and 23 respectively, in methanolic hydrazine monohydrate followed by
treatment of the acylhydrazides 24 and 25 with triethylorthoacetate (Scheme 3). The
analytical and spectroscopic data for 11 and 12 were in full accord with the assigned
structures, with the structure of phenyl-pyrimidine-oxadiazole 11 unambiguously confirmed
by single crystal X-ray crystallographic analysis.
Scheme 3. Reagents and conditions: a) Pd(PPh ) , Na CO , 3:1 dioxane/H O, µW, 130 °C, 25 min, 22 84%, 23
3
4
2
3
2
4
3%; b) 1:1 EtOH/NH NH .H O, reflux, 36 h, 24 35%, 25 88%; c) CH (OC H ) , reflux, 6 h, 11 49%, 12 52%.
2 2 2 3 2 5 3
For the synthesis of the 2-methylpiperazine analogues 13 and 14, esters 22 and 23 were
saponified to give phenyl-pyrimidine carboxylic acid 26 and pyridine-pyrimidine carboxylic
acid 27, respectively. Reaction of carboxylic acid 26 with (±)-tert-butyl 2-methylpiperazine-
1
-carboxylate in the presence of amide coupling reagents, followed by Boc group
deprotection, gave the desired phenyl-pyrimidine-piperazine compound 13 in good yield.
When subjected to the same amide coupling reaction, carboxylic acid 27 failed to react fully
with (±)-tert-butyl 2-methylpiperazine-1-carboxylate (despite the use of excess amounts of
coupling reagents and/or increased reaction times). Furthermore, Boc-protected pyridine-
pyrimidine-piperazine 29 could not be separated from (±)-tert-butyl 2-methylpiperazine-1-
carboxylate, and as such the crude reaction mixture was directly treated with TFA. Pyridine-
pyrimidine-piperazine 14 was obtained in only a 7% yield (over the final two steps) following
purification by reverse phase HPLC (Scheme 4).
Scheme 4. Reagents and conditions: a) 2:1 EtOH/2 M NaOH, reflux, 3 h, 26 quant., 27 87%; b) (±)-tert-butyl 2-
methylpiperazine-1-carboxylate, HOBt, EDC, DIPEA, DCM, rt, 18 h, 28 65%; c) TFA, DCM, 13 quant., 14 7%
(
two steps).
We then turned to the preparation of a putative Bcl-x inhibitor via mimicry of its Bak
L
protein partner. It was envisaged that such an inhibitor would bind to the Bcl-x protein, thus
L
4
0
allowing Bak to dimerise and initiate an apoptotic cascade. It has been documented that the
critical binding residues on Bak are Val74, Leu78, Ile81 and Ile85; all located on one face of
41
an α-helix. By installing key binding amino acid residues from the Bak protein onto one of
our pyrimidine-based tercyclic scaffolds, we hoped to form a Bak protein mimetic that would
5

ACCEPTED MANUSCRIPT
closely match the spatial orientation of key amino acid residues, and possess affinity for Bcl-
xL. Due to ease of synthesis, functionalisation of the phenyl-pyrimidine-piperazine scaffold
was selected. Following an in silico study, the corresponding leucine-isoleucine-leucine
analogue (compound 15), was chosen as a candidate for synthesis and investigation as a
putative Bcl-x inhibitor (Fig. 5).
L
Fig. 5. a) Structure of the putative Bcl-xL inhibitor 15; b) An ab initio low energy conformer of compound 15
(
grey) overlayed with the α-helical BH3 domain of Bak bound to Bcl-xL (PDB: 1BXL); only the key
contributing side chains of Val74, Leu78, Ile81 and Ile85 are shown.
Synthesis of putative Bcl-x inhibitor 15 is outlined in Scheme 5. Doubly functionalised
L
pyrimidine 34 was synthesised in four steps from commercially available 3-methylpentan-1-
4
2
ol (30). Oxidation of 30, followed by its participation in a Biginelli reaction, gave the
pyrimidinone 32, which was itself oxidised by ceric ammonium nitrate (CAN) to give the
**
pyrimidinol 33. Treatment of pyrimidinol 33 with triflic anhydride gave the corresponding
triflate 34 in good yield. Formation of the boronic ester coupling partner 38 was
accomplished in three synthetic steps from 2-bromobenzaldehyde (35). The first step, a
4
3,44
Wittig reaction using isopropyltriphenylphosphonium iodide, gave the olefin 36,
which
was hydrogenated to the corresponding isobutyl derivative 37, which was finally subjected to
Miyaura boronation conditions to afford the required pinacol borane 38. A Suzuki cross-
coupling reaction between triflate 34 and boronic ester 38 gave, following ester hydrolysis,
carboxylic acid 39, which was subjected to amide coupling with the protected piperazine
4
5
††
4
0, to afford putative Bcl-x inhibitor 15 after Boc group deprotection.
L
*
*
Attempts to oxidise the pyrimidinone 32 with nitric acid were unsuccessful; increasing reaction time and
temperature did not result in formation of the pyrimidinol 33.
†
†
Attempts to form a pyridyl-pyrimidine ester analogue of compound 39 from a cross-coupling reaction
between 4-isobutyl-3-pyridylboronic acid pinacol ester and triflate 34 were unsuccessful.
6

ACCEPTED MANUSCRIPT
Scheme 5. Reagents and conditions: a) PCC, DCM, rt, 1 h, 94%; b) urea (16), ethyl acetoacetate (17), cat.
HCl(aq), EtOH, 72 h, 56%; c) CAN, 3:2 acetone/H O, 24 h, 47%; d) Tf O, pyridine, DCM, 90%; e)
2
2
(
CH ) CHP(C H ) I, n-BuLi, THF, 2 h, quant.; f) PtO , EtOH, 1 atm. H , 3 h, 87%; g) bis(pinacolato)diboron,
3 2 6 5 3 2 2
Pd(dppf)Cl , DCM, KOAc, dioxane, 85 °C, 18 h, 65%; h) Na CO , Pd(PPh ) , 3:1 dioxane/H O, µW, 130 °C,
2
2
3
3 4
2
4
5
3
5 min, 71%; i) 2:1 EtOH/2 M NaOH, 3 h, quant.; j) (S)-tert-butyl 2-isobutylpiperazine-1-carboxylate (40),
EDCI, HOBt, DIPEA, DCM, 14 h, 51%; k) TFA, DCM, 4 h, 91%.
2
.3 Biological testing
The Bcl-x inhibitory activities of pyrimidine-based tercyclic α-helix mimetics 11−14, and
L
putative Bcl-x inhibitor 15, were investigated using a competition bead-based luminescence
L
®
assay (AlphaScreen ). This technology is different from that used by Hamilton and co-
9
, 10, 14
24, 25
workers,
discovery of selective and potent inhibitors of Bcl-xL.
previously been tested using a Bak BH3 domain peptide giving an IC value of 0.122 µM,
or Rebek and co-workers,
but it is well established in the literature for the
4
6, 47
The validity of the assay has
5
0
which is in agreement with the previously reported K value (0.120 µM) obtained from a
d
48
saturation titration experiment. In this assay the inhibitory effect of the compounds was
tested against Bcl-x and Mcl-1, as well as in a counter screen assay in which no protein was
L
bound to the beads. This additional control allows the assessment of non-selective
4
9, 50
background binding to the bead or compounds interfering with the assay technology.
Disappointingly, all compounds tested showed no inhibitory activity against Bcl-x or Mcl-1
L
at the highest concentration of 300 µM.
3
. Conclusion
In summary, several nitrogen-rich, pyrimidine-based scaffolds intended to possess improved
aqueous solubility were designed, and when evaluated by an in silico process were found to
mimic the spatial projection of the i, i+3, and i+7 residues of an α-helix. The central
pyrimidine ring of these scaffolds was synthesised by means of a Biginelli-oxidation process
to form key functionalised pyrimidine 21, which was subsequently manipulated to form
phenyl- and pyridine-pyrimidine-oxadiazoles 11 and 12, and phenyl- and pyridine-
pyrimidine-piperazines 13 and 14. Functionalisation of the phenyl-pyrimidine-piperazine
scaffold with residues designed to mimic the amino acid sequence leucine-isoleucine-leucine
7

ACCEPTED MANUSCRIPT
gave the α-helical Bak BH3 domain mimetic 15, a putative inhibitor designed to disrupt the
Bcl-x /Bak PPI. The pyrimidine-based tercyclic α-helix mimetics 11-14, and the putative
L
inhibitor 15, were assessed as Bcl-x and Mcl-1 inhibitors using a luminescence competition
L
assay, however, none displayed inhibitory activity at the highest concentration tested. Future
work will concentrate on refining this class of pyrimidine-based tercyclic α-helix mimetics, in
an effort to obtain Bcl-x inhibitory activity.
L
4
. Experimental
4
.1 General Experimental. Starting materials and reagents, with the exception of boronic
acids and esters, were purchased from Sigma-Aldrich, Merck or Acros Organics and were
used as received from the manufacturer. Boronic acids and esters were purchased from Boron
Molecular and were used as received from the manufacturer. (S)-tert-Butyl 2-
isobutylpiperazine-1-carboxylate was synthesised according to reference (45). Solvents were
dried, when necessary. Silica gel used for chromatography was 40-63 µm purchased from
Merck or Grace Davison. Analytical thin layer chromatography (TLC) was performed on
Merck Silica Gel 60 F254 aluminium sheets coated with silica gel containing UV254
fluorescent indicator and visualised using UV light and/or dipped in vanillin, ninhydrin or
potassium permanganate dip. All microwave irradiation reactions were carried out on a
Biotage-EXP Microwave synthesis system, operating at a frequency of 2450 MHz with
continuous irradiation power from 0 to 300 W. All reactions were carried out in 5 or 20 mL
oven-dried Biotage microwave vials sealed with an aluminium/Teflon crimp top, which can
be exposed to a maximum of 250 °C and 20 bar internal pressure. The reaction temperature
was measured by an IR sensor on the outer surface of the process vial. NMR spectra were
1
recorded using Bruker Avance DPX-400 spectrometers (400 MHz for H NMR and 100 MHz
1
3
ǂ
for C NMR spectra), referenced to residual solvent signal. Denotes signals only observed
in 2D NMR. . Electrospray ionisation mass spectrometry (ESI) was performed on a Waters
Q-TOF II or Shimadzu LCMS-2010EV mass spectrometer. Electron ionisation mass
spectrometry (EI) was performed on a ThermoQuest MAT95XP or ThermoQuest MAT95XL.
Atmospheric-pressure chemical-ionisation mass spectrometry (APCI) was performed on a
Thermo Scientific Q-Exactive FTMS. LCMS was performed using a Phenomenex Mercury
MS Aqua C 3µm column with internal diameter of 2.0 mm and length of 20 mm. High
1
8
resolution mass spectra (HRMS) were recorded on a Waters Q-TOF II, ThermoQuest
MAT95XP, ThermoQuest MAT95XL or Thermo Scientific Q-Exactive FTMS mass
spectrometer. Infrared spectra were recorded on an Agilent Cary 630 FTIR either neat or in
−
1
solution (CHCl ).Infrared band frequencies are reported in wavenumbers (cm ) and
3
intensities reported qualitatively as strong (s), medium (m), weak (w) and/or broad (b).
Melting points were measured on a Stuart Scientific SMP3 melting point apparatus. X-ray
crystallography was performed on a Nonius Kappa CCD. Elemental microanalyses were
performed by the Campbell Microanalytical Laboratory, University of Otago, Dunedin, New
Zealand.
4
.2 Computational chemistry
Conformational searches and ab initio optimizations were performed as previously
6
described.
4
4
.3 Synthesis
.3.1 Ethyl 4,6-dimethyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (19).
To a solution of urea (16) (1.20 g, 20.0 mmol) in ethanol (30 mL) that had been stirred at
6
0 °C for 10 min was added ethyl acetoacetate (17) (2.60 g, 20.0 mmol). The reaction
8

ACCEPTED MANUSCRIPT
mixture was cooled in ice and acetaldehyde (18) (1.12 mL, 20.0 mmol) and 4 drops of conc.
HCl(aq) were added. After stirring for 7 h the reaction mixture was allowed to warm to room
temperature and stirred for an additional 8 h. After removal of the volatiles in vacuo the
residue was washed with cold H O (3 x 15 mL) affording the product as a white solid (2.02 g,
2
1
5
1%). m.p.: 192.6–192.8 °C. H NMR (CDCl ) δ: 8.12 (br s, 1 H), 5.80 (br s, 1 H), 4.41 (m, 1
3
1
3
H), 4.17 (m, 2 H), 2.27 (s, 3 H), 1.27 (m, 6 H). C NMR (CDCl ) δ: 166.0, 154.5, 146.4,
3
+
+
1
02.8, 60.1, 47.7, 23.7, 18.7, 14.5. LRMS (ESI ): m/z 617.3 [3M+Na] (33%), 419.2
+
+
+
[
2M+Na] (100%), 221.1 [M+Na] (22%). HRMS (ESI ): m/z calculated for C H N O Na
9 14 2 3
+
[
M+Na] : 221.0902, found: 221.0893. IR (neat): ν 3235m, 3107m, 2977m, 1699s, 1654s,
max
−
1
1
227s, 1099s, 1080s cm .
4
.3.2 Ethyl 2-hydroxy-4,6-dimethylpyrimidine-5-carboxylate (20).
To a stirred solution of HNO (70% w/w, 20 mL) at 0 °C was added ethyl 4,6-dimethyl-2-
3
oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (19) (4.86 g, 24.5 mmol) over 25 min. The
reaction mixture was then allowed to warm to room temperature and stirred for an additional
3
0 min. Crushed ice was then added to the reaction mixture and it was neutralised to pH 7
with solid K CO . The mixture was extracted with DCM (3 x 20 mL) and the organic layers
2
3
dried over MgSO . Removal of the solvent in vacuo afforded the product as a yellow solid
4
1
(
3.48 g, 72%). R = 0.24 (5% methanol in DCM). m.p.: 147.6 °C (decomp.). H NMR (CDCl )
f
3
13
δ: 13.62 (br s, 1 H), 4.33 (q, J = 7.2 Hz, 2 H), 2.53 (s, 6 H), 1.35 (t, J = 7.2 Hz, 3 H). C
‡
‡
+
·+
NMR (CDCl ) δ: 165.4, 158.2, 111.4, 61.6, 14.3. LRMS (EI ): m/z 196.1 [M] (42%),
3
·
+
+
+
1
68.1 [M−C H ] (18%), 151.1 [M−C H O·] (100%). HRMS (EI ): m/z calculated for
2 4 2 5
·
+
C H N O [M] : 196.0842, found: 196.0842. Microanalysis: calculated for C H N O : C,
9
12
2
3
9
12
2
3
5
–
7
5.09%; H, 6.16%; N, 14.28% Found: C, 54.89%; H, 6.10%; N, 14.43%. IR (neat): νmax 3400
2500bs, 1655s, 1607s, 1560s, 1413s, 1368s, 1277s, 1127m, 1105m, 1049s, 1008s, 858s,
−
1
96s, 671s cm .
4
.3.3 Ethyl 4,6-dimethyl-2-(((trifluoromethyl)sulfonyl)oxy)pyrimidine-5-carboxylate
(21).
A solution of ethyl 2-hydroxy-4,6-dimethylpyrimidine-5-carboxylate (20) (244 mg, 1.25
mmol) in dry DCM (15 mL) was cooled to −5 °C before dry pyridine (0.20 mL, 2.50 mmol)
was added and the mixture stirred for 5 min. To the reaction mixture was added
trifluoromethanesulfonic anhydride (0.32 mL, 1.88 mmol) dropwise, before the mixture was
allowed to warm to room temperature and stirred for 16 h. The mixture was then washed with
1
M HCl(aq) (3 x 15 mL), the organic phase dried over MgSO and the volatiles removed in
4
vacuo. The residue was then purified via silica gel column chromatography (10% ethyl
acetate in hexane) to afford the desired product as a yellow oil (322 mg, 78%). R = 0.34 (10%
f
1
ethyl acetate in hexane). H NMR (CDCl ) δ: 4.46 (q, J = 7.2 Hz, 2 H), 2.59 (s, 3 H), 1.42 (t,
J = 7.2 Hz, 3 H). C NMR (CDCl ) δ: 170.0, 166.0, 157.1, 126.6, 118.5, (q, J = 318.6 Hz),
2.6, 23.0, 14.2. F NMR (CDCl ) δ: −73.5. LRMS (EI ): m/z 328.0 [M] (49%), 313.0
M−CH ·] (35%), 300.0 [M−C H ] (28%), 283.0 [M−C H O·] (100%). HRMS (EI ): m/z
3
1
3
3
C-F
·+
1
9
+
6
[
3
+
·+
+
+
3 2 4 2 5
32
·+
calculated for C H N O F S [M] : 328.0335, found: 328.0331.
1
0
11
2
5 3
4
.3.4 Ethyl 4,6-dimethyl-2-(o-tolyl)pyrimidine-5-carboxylate (22).
Method 1:
A mixture of ethyl 4,6-dimethyl-2-(((trifluoromethyl)sulfonyl)oxy)pyrimidine-5-carboxylate
(
21) (0.20 g, 0.61 mmol), Na CO (0.13 g, 12.2 mmol), o-tolylboronic acid (0.10 g, 0.73
2 3
‡
‡
13
The pyrimidine methyl signals and the quaternary carbons at C4 and C6 were not observed in the JMOD
C
NMR spectrum, nor in the 2D HSQC or HMBC NMR experiments. Various temperatures and solvent
conditions were also trialled. These signals were, however, observed for the subsequent compound (21).
9

ACCEPTED MANUSCRIPT
mmol) and tetrakis(triphenylphosphine) palladium (35 mg, 30 µmol) in toluene/H O (5:1, 15
2
mL) was heated at reflux for 40 h. The volatiles were removed in vacuo, the residue
redissolved in CHCl (15 mL) and washed with brine (2 x 15 mL). The aqueous phase was
3
extracted with CHCl (3 x 10 mL) and the combined organic layers dried over MgSO before
3
4
the volatiles were removed in vacuo. The residue was subjected to silica gel column
chromatography (15% ethyl acetate in hexane) to afford the title compound (91 mg, 56%) as
a yellow oil. R = 0.32 (10% ethyl acetate in hexane).
f
Method 2:
A mixture of ethyl 4,6-dimethyl-2-(((trifluoromethyl)sulfonyl)oxy)pyrimidine-5-carboxylate
(
21) (0.50 g, 1.52 mmol), Na CO (0.48 g, 4.57 mmol), o-tolylboronic acid (0.25 g, 1.83
2 3
mmol) and tetrakis(triphenylphosphine) palladium (88 mg, 76 µmol) in dioxane/H O (3:1, 15
2
mL) was subjected to microwave irradiation at 130 °C for 25 min. The volatiles were
removed in vacuo, the residue redissolved in CHCl (15 mL) and washed with brine (2 x 15
3
mL). The aqueous phase was extracted with CHCl (3 x 10 mL) and the combined organic
3
layers dried over MgSO before the volatiles were removed in vacuo. The residue was
4
subjected to silica gel column chromatography (15% ethyl acetate in hexane) to afford the
1
title compound (344 mg, 84%) as a yellow oil. H NMR (CDCl ) δ: 7.77 (m, 1 H), 7.36 –
3
7
.27 (m, 3 H), 4.47 (q, J = 7.2 Hz, 2 H), 2.62 (s, 6 H), 2.52 (s, 3 H), 1.44 (t, J = 7.2 Hz, 3 H).
1
3
C NMR (CDCl ) δ: 168.0, 167.0, 164.3, 138.0, 137.3, 131.4, 130.4, 129.7, 126.1, 123.8,
3
+
·+
·+
6
1.9, 23.0, 27.0, 14.2. LRMS (EI ): m/z 270.1 [M] (100%), 241.1 [M−C H ] (81%).
2 5
+
·+
HRMS (EI ): m/z calculated for C H N O [M] : 270.1363, found: 270.1358.
1
6
18
2
2
4
.3.5 Ethyl 4,6-dimethyl-2-(4-methylpyridin-3-yl)pyrimidine-5-carboxylate (23).
A mixture of ethyl 4,6-dimethyl-2-(((trifluoromethyl)sulfonyl)oxy)pyrimidine-5-carboxylate
(21) (0.25 g, 0.76 mmol), Na CO (0.16 g, 1.52 mmol), (4-methylpyridin-3-yl)boronic acid
2 3
(0.12 g, 0.84 mmol) and tetrakis(triphenylphosphine) palladium (44 mg, 38 µmol) in
dioxane/H O (3:1, 5 mL) was subjected to microwave irradiation at 130 °C for 20 min. The
2
volatiles were removed in vacuo, the residue redissolved in CHCl (10 mL) and washed with
3
H O (2 x 15 mL). The aqueous phase was extracted with CHCl (3 x 10 mL) and the
2
3
combined organic layers dried over MgSO before the volatiles were removed in vacuo. The
4
residue was subjected to silica gel column chromatography (2.5% methanol in DCM) to
afford an inseparable mixture of the title compound (89 mg, 43%; determined by NMR
spectroscopy) and triphenylphosphine oxide (10:1 ratio respectively). R = 0.29 (2.5%
f
methanol in DCM). Spectral data reported omits resonances attributed to triphenylphosphine
1
oxide. H NMR (CDCl ) δ: 9.02 (s, 1 H), 8.51 (d, J = 5.2 Hz, 1 H), 7.19 (d, J = 5.2 Hz, 1 H),
4
3
1
3
.47 (q, J = 7.2 Hz, 2 H), 2.62 (s, 6 H), 2.58 (s, 3 H), 1.44 (t, J = 7.2 Hz, 3 H). C NMR
(
1
[
CDCl ) δ: 167.7, 164.6, 164.5, 151.4, 150.3, 146.8, 133.8, 126.1, 124.4, 62.1, 23.1, 20.8,
3
+
·+
·+
4.3. LRMS (EI ) m/z 271.1 [M] (100%), 242.1 [M−C H ]
(74%), 226.1
2
5
+
M−C H O·] (16%).
2 5
4
.3.6 2-(4,6-Dimethyl-2-(o-tolyl)pyrimidin-5-yl)-5-methyl-1,3,4-oxadiazole (11).
A solution of ethyl 4,6-dimethyl-2-(o-tolyl)pyrimidine-5-carboxylate (22) (90 mg, 0.33 mmol)
in 1:1 ethanol/hydrazine monohydrate (4 mL) was stirred at reflux for 36 h. The reaction
mixture was concentrated in vacuo, diluted in H O (10 mL) and extracted with ethyl acetate
2
(
5
1
3 x 7 mL). The solvent was removed in vacuo to afford 4,6-dimethyl-2-(o-tolyl)pyrimidine-
1
-carbohydrazide (24) as a white solid (30 mg, 35%). H NMR (CDCl ) δ: 7.73 (d, J = 8.0 Hz,
3
H), 7.36 – 7.28 (m, 3 H), 7.04 (br s, 1 H), 4.20 (br s, 2 H), 2.56 (s, 6 H), 2.51 (s, 3 H).
10

ACCEPTED MANUSCRIPT
+
·+
+
+
LRMS (EI ): m/z 256.1 [M] (35%), 225.1 [M−N H ·] (100%). HRMS (EI ): m/z
2
3
·
+
calculated for C H N O [M] : 256.1319, found: 256.1321.
1
4
16
4
A mixture of 4,6-dimethyl-2-(o-tolyl)pyrimidine-5-carbohydrazide (24) (30 mg, 0.12 mmol)
in triethylorthoacetate (2 mL) was heated at reflux for 6 h. The reaction was then allowed to
cool to room temperature, diluted in saturated K CO3(aq) (30 mL) and stirred for an additional
2
2
h. The volatiles were removed in vacuo before the reaction mixture was extracted with
ethyl acetate (3 x 10 mL). The organic layers were concentrated in vacuo and the residue
purified via silica gel column chromatography (3:7 ethyl acetate/hexane containing 0.5%
triethylamine) to afford the product as a white solid (16 mg, 49%), which was crystallised
from methanol to afford clear needle like crystals. R = 0.4 (3:7 ethyl acetate/hexane
f
1
containing 0.5% triethylamine). H NMR (CDCl ) δ: 7.86 (dd, J = 8.0, 2.0 Hz, 1 H), 7.39 –
3
1
3
7
1
.29 (m, 3 H), 2.68 (s, 3 H), 2.63 (s, 6 H), 2.58 (s, 3 H). C NMR (CDCl ) δ: 167.9, 166.8,
3
+
64.8, 162.2, 137.7, 137.6, 131.6, 130.6, 130.0, 126.2, 115.2, 23.8, 21.3, 11.3. LRMS (EI ):
m/z 280.2 [M] (100%). HRMS (EI ): m/z calculated for C H N O [M] : 280.1319, found:
·+
+
·+
1
6
16
4
2
80.1316. Microanalysis: calculated for C H N O: C, 68.55%; H, 5.75%; N, 19.99%, found:
16 16 4
C, 68.54%; H, 5.81%; N, 20.04%.
4
.3.7 2-(4,6-Dimethyl-2-(4-methylpyridin-3-yl)pyrimidin-5-yl)-5-methyl-1,3,4-oxadiazole
(12).
A solution of ethyl 4,6-dimethyl-2-(4-methylpyridin-3-yl)pyrimidine-5-carboxylate (23) (35
mg, 147 µmol) in hydrazine monohydrate (1 mL) was stirred at reflux for 14 h. The reaction
mixture was allowed to cool, diluted in H O (10 mL) and washed with DCM (3 x 5 mL). The
2
aqueous layer was freeze dried to afford 4,6-dimethyl-2-(4-methylpyridin-3-yl)pyrimidine-5-
1
carbohydrazide (25) as an orange-brown solid (30 mg, 88%). H NMR (MeOD) δ: 8.79 (s, 1
1
3
H), 8.46 (d, J = 4.8 Hz, 1 H), 7.40 (d, J = 4.8 Hz, 1 H), 2.61 (s, 6 H), 2.55 (s, 3 H). C NMR
(
MeOD) δ: 168.3, 165.7, 164.9, 151.2, 150.2, 149.4, 135.5, 128.0, 127.7, 22.1, 20.8. LRMS
+
+
−
−
(ESI ): m/z 258.0 [M+H] (100%); (ESI ): m/z 255.9 [M−H] (100%).
A mixture of 4,6-dimethyl-2-(4-methylpyridin-3-yl)pyrimidine-5-carbohydrazide (25) (7 mg,
7.2 µmol) in triethylorthoacetate (2 mL) was heated at reflux for 6 h. The reaction was then
allowed to cool to room temperature, diluted in saturated K CO (10 mL) and stirred at
2
2
3(aq)
7
0 °C for 2 h. Volatiles were removed in vacuo before the reaction mixture was extracted
with DCM (3 x 10 mL). The organic layers were concentrated in vacuo and the residue
purified via silica gel column chromatography (2% methanol in DCM) to afford the product
1
as a white solid (4 mg, 52%). R = 0.2 (2% methanol in DCM). H NMR (CDCl ) δ: 9.11 (s, 1
f
3
H), 8.54 (d, J = 4.8 Hz, 1 H), 7.23 (d, J = 4.8 Hz, 1 H), 2.69 (s, 3 H), 2.65 (s, 6 H), 2.64 (s, 3
1
3
H). C NMR (CDCl ) δ: 167.1, 165.3, 164.9, 161.9, 151.6, 150.5, 147.2, 133.4, 126.3, 115.8,
3
+
·+
+
2
3.8, 21.0, 11.3. LRMS (EI ): m/z 281.2 [M] (100%). HRMS (EI ): m/z calculated for
C H N O [M] : 281.1277, found: 281.1273.
·+
15
15
5
4
.3.8 4,6-Dimethyl-2-(o-tolyl)pyrimidine-5-carboxylic acid (26).
A solution of ethyl 4,6-dimethyl-2-(o-tolyl)pyrimidine-5-carboxylate (22) (0.20 0.78 mmol)
in ethanol/2 M NaOH(aq) (2:1, 15 mL) was heated at reflux for 3 h. The reaction mixture was
allowed to cool to room temperature, acidified with 2 M HCl , and cooled on ice. The
(aq)
resulting precipitate was isolated by filtration to afford the desired product as a white solid
1
(
8
1
0.18 g, quant.). R = baseline (1:1 ethyl acetate/hexane). H NMR (CDCl ) δ: 7.74 (dd, J =
f
3
1
3
.0, 1.6 Hz, 1 H), 7.38 – 7.27 (m, 3 H), 2.68 (s, 6 H), 2.52 (s, 3 H). C NMR (CDCl ) δ:
71.1, 167.1, 165.4, 137.5, 137.4, 131.4, 130.4, 130.0, 126.1, 123.1, 23.4, 20.9. LRMS (ESI ):
m/z 241.0 [M−H] (100%), m/z 197.0 [M−CO H] (81%); (ESI ): m/z 243.1 [M+H] (100%).
3
−
−
−
+
+
2
11

ACCEPTED MANUSCRIPT
+
·+
HRMS (EI ): m/z calculated for C H N O [M] : 242.1050, found: 242.1039; calculated for
1
4
14
2
2
·+
C H N O [M−H] : 241.0972, found: 241.0968.
14
13
2
2
4
.3.9 4,6-Dimethyl-2-(4-methylpyridin-3-yl)pyrimidine-5-carboxylic acid (27).
4
.3.10 (4,6-Dimethyl-2-(o-tolyl)pyrimidin-5-yl)(3-methylpiperazin-1-yl)methanone (13).
A solution of 4,6-dimethyl-2-(o-tolyl)pyrimidine-5-carboxylic acid (26) (40 mg, 0.17 mmol),
45
(
±)-tert-butyl 2-methylpiperazine-1-carboxylate (50 mg, 0.25 mmol), HOBt (25 mg, 0.18
mmol), EDCI (28 mg, 0.18 mmol) and DIPEA (0.06 mL, 0.33 mmol) in DCM (10 mL) was
stirred at room temperature for 18 h. The solution was then concentrated in vacuo before the
residue was purified via silica gel chromatography (1:1 ethyl acetate/hexane) to afford
compound 28, a clear oil (45 mg, 65%), as a mixture of conformers. R = 0.44 (1:1 ethyl
f
1
acetate/hexane). H NMR (CDCl ) δ: 7.77 – 7.74 (m, 1 H), 7.34 – 7.25 (m, 3 H), 4.65 (m, 0.5
3
H), 4.51 (m, 0.5 H), 4.47 (m, 0.5 H), 4.27 (m, 0.5 H), 4.02 (m, 0.5 H), 3.90 (m, 0.5 H), 3.40 –
3
.32 (m, 1 H), 3.21 – 2.95 (m, 3 H), 2.59 (s, 1.5H), 2.57 (s, 1.5 H), 2.52 (s, 3 H), 2.45 (s, 1.5
1
3
H), 2.43 (s, 1.5 H), 1.46 (s, 9 H), 1.27 (d, J = 6.8 Hz, 1.5 H), 1.12 (d, J = 6.8 Hz, 1.5 H). C
NMR (CDCl ) δ: 168.1, 167.8, 166.8, 166.8, 163.4, 162.8, 162.4, 154.41, 154.37, 137.9,
3
137.8, 137.4, 137.3, 131.4, 131.43, 131.41, 130.38, 130.36, 129.64, 129.60, 126.0, 125.8,
125.4, 80.6, 50.3, 46.9, 46.3, 46.2, 45.5, 41.5, 38.8, 38.0, 28.5, 22.55, 22.50, 22.20, 22.15,
21.10, 21.08, 15.6, 15.5.
To
a
solution of tert-butyl 4-(4,6-dimethyl-2-(o-tolyl)pyrimidine-5-carbonyl)-2-
methylpiperazine-1-carboxylate (28) (45 mg, 0.11 mmol) in DCM (3 mL) was added 10
drops of TFA and allowed to stir at room temperature for 18 h. The volatiles were evaporated
1
to afford the product, a colourless oil (35 mg, quant.) as a mixture of conformers. H NMR
(
CDCl ) δ: 9.64 (br s, 0.5 H), 9.25 (br s, 0.5 H), 7.64 – 7.58 (m, 1 H), 7.44 (m, 1 H), 7.35 –
3
7
.26 (m, 2 H), 4.80 (m, 1 H), 3.63 – 3.06 (m, 6 H), 2.67 (s, 1.5 H), 2.66 (s, 1.5 H), 2.62 (s, 1.5
H), 2.60 (s, 1.5 H), 2.49 (s, 1.5 H), 2.47 (s, 1.5 H), 1.43 (d, J = 6.0 Hz, 1.5 H), 1.25 (d, J = 6.8
1
3
Hz, 1.5 H). C NMR (CDCl ) δ: 165.1, 164.8, 164.6, 164.4, 164.3, 164.2, 138.20, 138.15,
3
1
4
33.0, 132.8, 131.9, 131.8, 130.5, 130.4, 126.3, 126.2, 126.0, 52.1, 51.8, 48.8, 44.4, 43.6,
+
3.2, 42.4, 37.9, 21.6, 21.5 , 21.11, 21.05, 20.5, 20.3, 15.7, 15.3. HRMS (APCI ): m/z
+
−
calculated for C H N O [M+H] : 325.2028, found: 325.2022; (APCI ): m/z calculated for
C H N O [M−H] : 323.1872, found: 323.1886.
1
9
25
4
−
19
23
4
4
.3.11
(4,6-Dimethyl-2-(4-methylpyridin-3-yl)pyrimidin-5-yl)(3-methylpiperazin-1-
yl)methanone (14).
A mixture of ethyl 4,6-dimethyl-2-(4-methylpyridin-3-yl)pyrimidine-5-carboxylate (23) (23
mg, 85 µmol) and lithium hydroxide (50 mg, 2.09 mmol) in ethanol (3 mL) was heated at
reflux for 3 h. The solvent was removed in vacuo before the residue was purified via reverse
phase (C ) HPLC with a gradient elution of H O/MeOH (100/0% to 10/90%) to afford
1
8
2
1
compound 27 as an orange-brown solid (18 mg, 87%). H NMR (D O+MeOD) δ: 8.77 (s, 1
H), 8.43 (d, J = 4.8 Hz, 1 H), 7.39 (d, J = 4.8 Hz, 1 H), 2.59 (s, 6 H), 2.53 (s, 3 H). C NMR
D O+MeOD) δ: 170.5, 163.2, 162.6, 150.8, 149.8, 149.0, 136.3, 133.8, 127.5, 22.3, 20.4.
2
1
3
(
2
+
+
−
−
LRMS (ESI ): m/z 244.1 [M+H] (100%); (ESI ): m/z 242.0 [M−H] (100%).
A solution of 4,6-dimethyl-2-(4-methylpyridin-3-yl)pyrimidine-5-carboxylic acid (27) (40
4
5
mg, 0.16 mmol), (±)-tert-butyl 2-methylpiperazine-1-carboxylate (36 mg, 0.18 mmol),
HOBt (24 mg, 0.18 mmol), EDCI (36 mg, 0.18 mmol) and DIPEA (0.08 mL, 0.33 mmol) in
DCM (10 mL) was stirred at room temperature for 18 h. The reaction mixture was diluted in
12

ACCEPTED MANUSCRIPT
H O, extracted with DCM (3 x 10 mL), and the combined organic layers concentrated to 3
2
mL. To the solution was then added 10 drops of TFA before stirring for 3 h. The reaction
mixture was then extracted with H O (3 x 3 mL) and the combined aqueous layers freeze
2
dried. The residue was purified via reverse phase (C ) HPLC with a gradient elution of
18
H O/MeOH (100/0% to 10/90%), to afford the desired product to afford the product as a pale
2
1
yellow solid (4 mg, 7%) as a mixture of conformers. H NMR (MeOD) δ: 8.44 (br s, 1 H),
7
–
1
3
.64 – 7.58 (m, 1 H), 7.45 (m, 1 H), 7.15 – 7.07 (m, 1 H), 4.59 (m, 2 H), 3.95 (br s, 2H), 3.63
3.06 (complex, 6 H), 2.39 (bs, 3 H), 1.32 (m, 3 H). C NMR (MeOD) δ: 157.0, 148.9,
13
ǂ
47.8, 139.2, 129.7, 129.4, 127.4, 127.0, 125.2, 123.4, 123.1, 113.6, 113.3, 52.0, 51.9, 47.1,
+
+
0.7, 19.9, 17.1, 16.9. HRMS (APCI ): m/z calculated for C H N O [M+H] : 326.1981,
1
8
24
5
−
−
found: 326.1978; (APCI ): m/z calculated for C H N O [M−H] : 324.1824, found:
1
9
22
4
3
24.1829.
4
.3.12 (±)-3-Methylpentanal (31).
4
2
In a procedure adapted from Kumaraswamy and Markondaiah, a mixture of 3-
methylpentan-1-ol (30) (2.00 g, 19.6 mmol) and PCC (4.22 g, 19.6 mmol) in DCM (30 mL)
was stirred at room temperature for 1 h. The DCM was then removed under reduced pressure
before distillation of the residue afforded the product as a clear oil (1.84 g, 94%) as a 1:1
4
2
mixture of stereoisomers, with spectral data in accordance with the literature. R = 0.47
f
1
(
DCM). H NMR (CDCl ) δ: 9.72 (s, 0.5 H), 9.71 (s, 0.5 H), 2.38 – 2.33 (m, 1 H), 2.20 – 2.14
3
(
m, 1 H), 1.98 - 1.89 (m, 1 H), 1.37 – 1.28 (m, 1 H), 1.26 – 1.19 (m, 1 H), 0.92 – 0.90 (m, 1.5
1
3
H), 0.87 – 0.83 (m, 1.5 H). C NMR (CDCl ) δ: 203.1, 50.7, 29.8, 29.6, 19.5, 11.3.
3
4
.3.13
Ethyl
6-methyl-4-(2-methylbutyl)-2-oxo-1,2,3,4-tetrahydropyrimidine-5-
carboxylate (32).
A solution of urea (16) (2.36 g, 39.2 mmol) in ethanol (15 mL) was stirred at 60 °C until
completely dissolved. The reaction mixture was then allowed to cool to room temperature
before the addition of ethylacetoacetate (17) (2.5 mL, 19.6 mmol), 3-methylpentanal (31)
(2.36 g, 19.6 mmol) and concentrated HCl(aq) (1 mL). The mixture was stirred at 70 °C for 72
h before the volatiles were removed in vacuo and the residue washed with cold H O (3 x 15
2
mL) to afford the product as a white solid (2.79 g, 56%). R = 0.32 (5% methanol in DCM).
f
1
m.p.: 161.2 °C (decomp.). H NMR (MeOD) δ: 4.29 (m, 1 H), 4.16 (m, 2 H), 2.26 (s, 3 H),
1
0
4
1
.64 – 1.46 (m, 2 H), 1.41 (m, 1 H), 1.28 (t, J = 7.2 Hz, 3 H), 1.25 – 1.10 (m, 2 H), 0.95 –
1
3
.86 (m, 6 H). C NMR (MeOD) δ: 167.6, 167.5, 156.1, 156.2, 149.1, 103.2, 61.0, 50.2, 50.1,
+
4.8, 44.6, 31.7, 31.0, 30.8, 29.2, 20.6, 18.9, 18.09, 18.05, 14.7, 11.7, 11.0. LRMS (EI ): m/z
+
+
+
83.1 [M−C H ] (100%), 155.1 [M−C H −C H ] (23%). HRMS (EI ): m/z calculated for
5
11
5
11
2
2
·
+
C H N O [M] 254.1606, found: 254.1625. IR (neat): ν 3235m, 3105m, 2979m, 2935m,
13
22
2
3
max
1
704s, 1646s, 1438m, 1288m, 1223s, 1177m, 1090s, 1020m, 777m, 758m, 721s, 694s, 668s
−
1
cm .
4
.3.14 Ethyl 2-hydroxy-4-methyl-6-(2-methylbutyl)pyrimidine-5-carboxylate (33).
To a solution of ethyl 6-methyl-4-(2-methylbutyl)-2-oxo-1,2,3,4-tetrahydropyrimidine-5-
carboxylate (32) (1.45 g, 5.70 mmol) in acetone (30 mL) was added a solution of cerium
ammonium nitrate (9.38 g, 17.1 mmol) in H O (20 mL) dropwise over 1 h. The reaction
2
mixture was allowed to stir for 24 h at room temperature before the acetone was removed
under reduced pressure. The reaction mixture was extracted with CHCl (40 mL) and washed
3
with H O (3 x 15 mL) before the organic layer was dried over MgSO and the volatiles
2
4
removed in vacuo. The residue was purified via silica gel column chromatography (5%
methanol in DCM) to afford the product as an amber oil (0.67 g, 47%). R = 0.38 (5%
f
1
methanol in DCM). H NMR (DMSO-d ) δ: 5.91 (br s, 1 H), 4.32 (q, J = 7.2 Hz, 2 H), 2.83
6
13

ACCEPTED MANUSCRIPT
(
dd, J = 12.8, 6.4 Hz, 1 H), 2.55 (dd, J = 12.8, 8.8 Hz, 1 H), 2.47 (s, 3 H), 1.74 (m, 1 H), 1.36
1
–
4
2
1.28 (m, 1 H), 1.28 (t, 3 H), 1.25 – 1.16 (m, 1 H), 0.86 – 0.83 (m, 6 H). H NMR (CDCl ) δ:
3
.34 (q, J = 7.2 Hz, 2 H), 2.86 (dd, J = 13.2, 6.4 Hz, 1 H), 2.59 (dd, J = 13.2, 8.4 Hz, 1 H),
.50 (s, 3 H), 1.84 (m, 1 H), 1.43 – 1.30 (m, 4 H), 1.29 – 1.18 (m, 1 H), 0.94 – 0.84 (m, 6 H).
1
3
ǂ
C NMR (CDCl ) δ: 165.8, 161.7, 158.2, 151.9, 112.2, 61.8, 41.4 , 35.1, 29.6, 22.3ǂ, 18.9,
3
+
·+
+
·+
·+
1
4.3, 11.4. LRMS (EI ): m/z 252.2 [M] (14%), 237.1 [M−CH ] (39%), 223.1 [M−C H ]
3 2 5
+
+
(100%), 207.1 [M−C H O·] (61%). HRMS (EI ): m/z calculated for C H N O [M]
2 7 13 20 2 3
2
1
52.1468, found: 252.1461. IR (neat): νmax 3120bw, 2962m, 2932m, 1717m, 1648s, 1597s,
551m, 1438m, 1368m, 1253s, 1174w, 1099s, 1014m, 955w, 910m, 864m, 798m, 727s, 671s
−
1
cm .
4
.3.15 Ethyl 4,6-dimethyl-2-(((trifluoromethyl)sulfonyl)oxy)pyrimidine-5-carboxylate
(34). A solution of ethyl 2-hydroxy-4,6-dimethylpyrimidine-5-carboxylate (33) (244 mg, 1.25
mmol) in dry DCM (15 mL) was cooled to −5 °C before dry pyridine (0.20 mL, 2.50 mmol)
was added and the mixture stirred for 5 min. To the reaction mixture was added
trifluoromethanesulfonic anhydride (0.32 mL, 1.88 mmol) dropwise, before the mixture was
allowed to warm to room temperature and stirred for 16 h. The mixture was then washed with
1
M HCl(aq) (3 x 15 mL), the organic phase dried over MgSO and the volatiles removed in
4
vacuo. The residue was then purified via silica gel column chromatography (10% ethyl
acetate in hexane) to afford the desired product as a yellow oil (322 mg, 78%). R = 0.49 (20%
f
1
ethyl acetate in hexane). H NMR (CDCl ) δ: 4.45 (q, J = 7.2 Hz, 2 H), 2.83 (dd, J = 14.0, 6.0
3
Hz, 1 H), 2.58 (dd, J = 14.0, 8.0, 1 H), 2.57 (s, 3 H), 1.97 (m, 1 H), 1.41 (t, J = 7.2 Hz, 3 H),
1
3
1
1
1
[
(
.39 – 1.31 (m, 1 H), 1.28 – 1.17 (m, 1 H), 0.91 – 0.86 (m, 6 H). C NMR (CDCl ) δ: 172.7,
3
69.7, 166.3, 157.3, 127.3, 118.6 (q, J = 478.2 Hz), 62.6, 42.3, 34.7, 29.5, 22.9, 19.0, 14.3,
1.3. F NMR (CDCl ) δ: −73.4. LRMS (APCI ): m/z 385.1 [M+H] (100%), 237.1
M−OTf+H] (22%); (APCI ): m/z 383.1 [M−H] (4%), 251.1 [M−Tf] (100%). HRMS
APCI ): m/z calculated for C H N O F S [M+H] : 385.1045, found: 385.1040; (APCI ):
C-F
19
+
+
3
+
−
−
−
+
32
+
−
1
4
20
2
5 3
3
2
−
m/z calculated for C H N O F S [M−H] : 383.0889, found: 383.0904 IR (neat): ν
1
4
20
2
5
3
max
2
1
965m, 2934w, 2879w, 1731s, 1576s, 1545m, 1424s, 1327w, 1262m, 1208s, 1133s, 1099s,
038m, 986s, 866s, 823s, 764m cm .
−
1
4
.3.16 1-Bromo-2-(2-methylprop-1-en-1-yl)benzene (36).
4
3
In a procedure adapted from Liu et al., to a solution of isopropyltriphenylphosphonium
iodide (3.86 g, 8.92 mmol) in THF (50 mL) at 0 °C was added n-BuLi (2.5 M in hexanes,
3
.73 mL, 9.32 mmol) dropwise. The reaction mixture was maintained at 0 °C and 2-
bromobenzaldehyde (35) (0.95 mL, 8.11 mmol) was added dropwise. The solution was
allowed to warm to room temperature over 2 h, before the reaction mixture was diluted with
hexane (15 mL). The reaction mixture was then filtered through a silica plug and the solvent
removed in vacuo to afford the product as a clear oil (1.71 g, quant.), with spectral data in
4
4
1
accordance with the literature. R = 0.55 (hexane). H NMR (CDCl ) δ: 7.56 (d, J = 7.6 Hz, 1
f
3
H), 7.28 – 7.21 (m, 2 H), 7.06 (m, 1 H), 6.24 (m, 1 H), 1.93 (d, J = 1.2 Hz, 3 H), 1.75 (d, J =
+
·+ 81
·+ 79
1
.2 Hz, 3 H). LRMS (EI ): m/z 212.0 [M] ( Br, 13%), 210.0 [M] ( Br, 13%), 169.0
·+
[
M−C H ] (100%).
3 5
4
.3.17 1-Bromo-2-isobutylbenzene (37).
A solution of 1-bromo-2-(2-methylprop-1-en-1-yl)benzene (36) (0.25 g, 1.20 mmol) and PtO2
13 mg, 55 µmol) in ethanol (10 mL) was stirred under 1 atm. of H at room temperature for
(
2
3
h. The reaction mixture was then filtered and the solids washed with hexane. The filtrate
was concentrated in vacuo before being redissolved in CHCl (15 mL), washed with H O (3 x
3
2
1
0 mL) and dried over MgSO . Evaporation of the volatiles afforded the product as a clear oil
4
14

ACCEPTED MANUSCRIPT
1
(
0.22 g, 87%). R = 0.70 (hexane). H NMR (CDCl ) δ: 7.53 (dd, J = 8.0, 1.2 Hz, 1 H), 7.26 –
f
3
7
.15 (m, 2 H), 7.05 (m, 1 H), 2.61 (d, J = 7.2 Hz, 2 H), 1.99 (m, 1 H), 0.94 (d, J = 6.6 Hz, 6
1
3
H). C NMR (CDCl ) δ: 141.2, 132.9, 131.5, 127.5, 127.1, 125.0, 45.3, 28.9, 22.5. LRMS
3
+
·+ 81
·+ 79
·+
(EI ): m/z 214.0 [M] ( Br, 16%), 212.0 [M] ( Br, 16%), 169.0 [M−C H ] (100%).
3 7
4
.3.18 2-(2-Isobutylphenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (38).
A solution of 1-bromo-2-isobutylbenzene (37) (0.20 g, 0.94 mmol), bis(pinacolato)diboron
0.26 g, 1.03 mmol), [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (38 mg, 50
µmol) and potassium acetate (0.28 g, 2.82 mmol) in dioxane (20 mL) was heated at 85 °C for
(
1
2
8 h. The reaction mixture was then diluted in ethyl acetate (20 mL), washed with brine (3 x
5 mL) and the combined aqueous layers extracted with ethyl acetate (2 x 20 mL). The
combined organic layers were dried over MgSO and concentrated in vacuo before
4
purification via silica gel column chromatography (3% ethyl acetate in hexane) to afford the
1
product as a clear oil (0.16 g, 65%). H NMR (CDCl ) δ: 7.78 (d, J = 7.6 Hz, 1 H), 7.33 (t, J
3
=
7.6 Hz, 1 H), 7.18 (t, J = 7.6 Hz, 1 H), 7.13 (d, J = 7.6 Hz, 1 H), 2.77 (d, J = 6.8 Hz, 2 H),
1
.77 (m, 1 H), 1.35 (s, 12 H), 0.91 (d, J = 6.4 Hz, 6 H). R = 0.36 (3% ethyl acetate in hexane).
f
1
3
C NMR (CDCl ) δ: 148.9, 136.1, 130.5, 130.2, 125.0, 83.5, 44.9, 32.0, 25.0, 22.5. A signal
3
5
1 11
for the carbon directly attached to the boron atom (C-1) was not observed.
B NMR
(16%), 217.2
M−C H ] (25%), 203.1 [M−C H ] (63%). HRMS (EI ): m/z calculated for C H BO
−
·−
·−
(
[
[
CDCl ) δ: 31.1.GCMS (EI ): m/z 260.1 [M] (51%), 245.2 [M−CH ]
3
3
·−
·−
+
3
7
4
9
16 25
2
·+
M] : 260.1948, found: 260.1954. IR (neat): ν
2954m, 2929w, 2866w, 1599m, 1569w,
max
1
1
486w, 1466w, 1440m, 1380m, 1344s, 1309s, 1273m, 1259m, 1214w, 1144s, 1111w, 1085s,
062m, 1036m, 963m, 862m, 843w, 761m, 742s, 674m, 661s cm .
−
1
4
.3.19 2-(2-Isobutylphenyl)-4-methyl-6-(2-methylbutyl)pyrimidine-5-carboxylic acid
(39).
A mixture of ethyl 4-methyl-6-(2-methylbutyl)-2-(((trifluoromethyl)sulfonyl)oxy)pyrimidine-
5
4
-carboxylate (34) (66 mg, 0.17 mmol), Na CO (55 mg, 0.52 mmol), 2-(2-isobutylphenyl)-
2 3
,4,5,5-tetramethyl-1,3,2-dioxaborolane
(38)
(45
mg,
0.17
mmol)
and
tetrakis(triphenylphosphine) palladium (10 mg, 9 µmol) in a mixture of dioxane/H O (3:1, 5
2
mL) was microwave irradiated at 130 °C for 35 min. The reaction mixture was then diluted in
ethyl acetate (20 mL) and washed with H O (20 mL). The aqueous phase was extracted with
2
ethyl acetate (2 x 10 mL) and the combined organic layers dried over MgSO before the
4
volatiles were removed in vacuo. The residue was purified via silica gel column
chromatography (5% ethyl acetate in hexane) to afford ethyl 2-(2-isobutylphenyl)-4-methyl-
6
-(2-methylbutyl)pyrimidine-5-carboxylate (39a) as a clear oil (45 mg, 71%). R = 0.33 (5%
f
1
ethyl acetate in hexane). H NMR (CDCl ) δ: 7.73 (dd, J = 7.2, 1.6 Hz, 1 H), 7.36 – 7.23 (m,
3
3
H), 4.46 (q, J= 7.2 Hz, 2 H), 2.94 – 2.83 (m, 3 H), 2.62 (dd, J= 13.2, 8.0 Hz, 1 H), 2.59 (s, 3
H), 2.07 (m, 1 H), 1.69 (m, 1 H), 1.44 (t, J = 7.2 Hz, 3 H), 1.42 – 1.30 (m, 1 H), 1.31 – 1.12
1
3
(
m, 1 H), 0.93 – 0.88 (m, 6 H), 0.78 (d, J = 6.4 Hz, 6 H). C NMR (CDCl ) δ: 168.3, 167.2,
3
1
2
3
67.0, 164.0, 140.9, 138.3, 131.5, 130.9, 129.3, 126.2, 124.6, 62.0, 42.8, 42.7, 35.0, 30.0,
+
·+
·+
9.7, 23.0, 22.6, 19.2, 14.4, 11.5. LRMS (EI ): m/z 368 [M] (18%), 353 [M−CH ] (20%),
3
·
+
·+
+
25 [M−C H ] (17%), 311 [M−C H ] (100%). HRMS (EI ): m/z calculated for
3
7
4
9
·+
C H N O [M] : 368.2463, found: 368.2439. IR (neat): ν 2957s, 2929m, 2868m, 1725s,
23
32
2
2
max
1
9
A
541s, 1459m, 1448m, 1402m, 1382m, 1364m, 1251s, 1147m, 1097s, 1071s, 1015m, 964w,
18w, 863w, 841w, 752s, 737s, 696m, 660w cm .
solution of ethyl 2-(2-isobutylphenyl)-4-methyl-6-(2-methylbutyl)pyrimidine-5-
−
1
carboxylate (39a) (26 mg, 72 µmol) in a mixture of ethanol/2 M NaOH(aq) (2:1, 10 mL) was
stirred at reflux for 3 h. The ethanol was then removed in vacuo before the remaining solution
was washed with ether (3 x 10 mL). The aqueous layer was acidified to pH 4 with 2 M
15

ACCEPTED MANUSCRIPT
HCl , and extracted with ethyl acetate (3 x 15 mL). The combined organic layers were dried
(aq)
over MgSO and the solvent removed in vacuo to afford the title compound as a white solid
4
1
(
24 mg, quant.). H NMR (CDCl ) δ: 11.12 (br s, 1 H), 7.67 (d, J = 7.6 Hz, 1 H), 7.38 (dt, J =
3
7
6
.6, 1.2 Hz, 1 H), 7.32 (dt, J = 7.6, 1.2 Hz, 1 H),7.28 (d, J = 7.6 Hz, 1 H), 2.95 (dd, J= 13.2,
.4 Hz, 1 H), 2.89 – 2.77 (m, 2 H), 2.73 (dd, J= 13.2, 8.4 Hz, 1 H), 2.61 (s, 3 H), 2.09 (m, 1
H), 1.67 (m, 1 H), 1.42 (m, 1 H), 1.26 (m, 1 H), 0.92 (t, J = 7.6 Hz, 3 H), 0.90 (d, J = 6.4 Hz,
1
3
3
1
H), 0.78 (d, J = 6.4 Hz, 6 H). C NMR (CDCl ) δ: 170.8, 167.9, 167.0, 164.5, 140.9, 137.5,
3
31.4, 130.8, 129.6, 126.2, 124.7, 42.7, 42.5, 35.2, 30.0, 29.7, 22.64, 22.60, 22.55, 19.2, 11.6.
LRMS (ESI ): m/z 341.3 [M+H] (100%). HRMS (ESI ): m/z calculated for C H N O
M+H] : 341.2229, found: 341.2226. IR (CHCl ): ν
718s, 1543s, 1459m, 1382m, 1250s, 1152s, 1115m, 1075w, 906s, 726s cm .
+
+
+
2
1
29
2
2
+
[
1
2959m, 2927m, 2870w, 2458 bw,
3
max
−
1
4
.3.20 (2-(2-Isobutylphenyl)-4-methyl-6-(2-methylbutyl)pyrimidin-5-yl)((S)-3-
isobutylpiperazin-1-yl)methanone (15).
A solution of 2-(2-isobutylphenyl)-4-methyl-6-(2-methylbutyl)pyrimidine-5-carboxylic acid
4
5
(
39) (33 mg, 97 µmol), (S)-tert-butyl 2-isobutylpiperazine-1-carboxylate (40) (24 mg, 99
µmol), HOBt (20 mg, 148 µmol, EDCI (27 mg, 141 µmol) and DIPEA (50 µL, 287 µmol) in
DCM (10 mL) was stirred at room temperature for 16 h. The solution was then diluted with
H O (10 mL) and extracted with DCM (2 x 10 mL). The combined organic layers were dried
2
over MgSO and concentrated in vacuo. The residue was purified via silica gel
4
chromatography (4:1 hexane/ethyl acetate) to afford the coupled product (2S)-tert-butyl 2-
isobutyl-4-(2-(2-isobutylphenyl)-4-methyl-6-(2-methylbutyl)pyrimidine-5-
carbonyl)piperazine-1-carboxylate, a yellow oil (28 mg, 51%), as a mixture of conformers. R
f
1
=
0.21 (20% ethyl acetate in hexane). H NMR (CDCl ) δ: 7.75 (dd, J = 7.6, 1.6 Hz, 1 H),
3
7
4
.35 (m, 1 H), 7.30 (m, 1 H), 7.25 (m, 1 H), 4.68 (m, 1 H), 4.38 (m, 0.6 H), 4.19 (m, 0.4 H),
.10 (m, 0.4 H), 3.95 (m, 0.6 H), 3.33 – 3.22 (m, 1 H), 3.18 – 3.05 (m, 1 H), 3.03 – 2.92 (m, 2
H), 2.92 – 2.86 (m, 1 H), 2.86 – 2.78 (m, 1 H), 2.78 – 2.68 (m, 0.5H), 2.66 – 2.31 (m, 4.5H),
2
–
.31 – 2.08 (m, 1 H), 1.73 – 1.58 (m, 2 H), 1.57 – 1.45 (m, 11 H), 1.45 – 1.31 (m, 1 H), 1.30
1.15 (m, 1H ), 1.03 – 0.96 (m, 4 H), 0.95 – 0.80 (m, 8 H), 0.79 – 0.76 (m, 6 H). C NMR
ʹ
13
(
CDCl ) δ: 168.0, 167.7, 167.61, 167.60, 167.0, 166.1, 165.9, 162.8, 162.7, 141.0, 138.1,
3
ǂ
ǂ
1
4
3
2
31.5, 130.9, 129.3, 126.2, 126.2 , 126.0 ,80.7, 49.5, 49.4, 46.7, 46.6, 46.2, 46.1, 44.42, 44.37,
4.1, 42.93, 42.88, 42.74, 42.71, 42.6, 42.4, 41.6, 41.5, 38.7, 34.3, 34.1, 34.0, 33.8, 30.13,
0.10, 30.03, 30.01, 29.6, 29.5, 28.5, 24.9, 23.13, 23.12, 23.01, 22.98, 22.88, 22.84, 22.67,
2.64, 22.60, 22.55, 22.28, 22.26, 19.8, 19.6, 19.2, 19.1, 11.75, 11.69, 11.5, 11.4. LRMS
+
+
+
+
(ESI ): m/z 1152.0 [2M+Na] (18%), 587.6 [M+Na] (100%), 565.6 [M+H] (33%), 531.5
+
+
+
[
M−C H +Na] (18%). HRMS (ESI ): m/z calculated for C H N O [M+H] : 565.4118,
4
8
34 53
4
3
found: 565.4119. IR (neat): νmax 2957m, 2927m, 2867w, 1694s, 1638s, 1540s, 1458m, 1437m,
1
7
411s, 1391s, 1364s, 1320m, 1281m, 1254m, 1228m, 1163s, 1126m, 1104m, 1055m, 1018m,
7m, 920w, 858w, 811w, 749s, 697w cm .
−
1
To
a
solution of (2S)-tert-butyl 2-isobutyl-4-(2-(2-isobutylphenyl)-4-methyl-6-(2-
methylbutyl)pyrimidine-5-carbonyl)piperazine-1-carboxylate (8 mg, 14 µmol) in DCM (4
mL) was added TFA (0.5 mL) and allowed to stirred at reflux for 3 h. The reaction mixture
was then cooled to room temperature and diluted with H O (10 mL) and extracted with DCM
2
(
3 x 5 mL). The combined organic layers were dried of MgSO before the volatiles were
4
removed in vacuo to afford the product 15, a yellow oil (6 mg, 91%), as a mixture of
1
conformers. H NMR (CDCl ) δ: 7.77 – 7.72 (m, 1 H), 7.40 – 7.35 (m, 1 H), 7.34 – 7.28 (m,
3
ʹ
1
3
2
H), 7.28 – 7.25 (m, 1 H), 4.96 – 4.84 (m, 1 H), 3.78 (br s, 1 H), 3.64 – 3.44 (m, 2 H), 3.44 –
.19 (m, 2.5 H), 3.18 – 3.05 (m, 1 H), 3.04 – 2.80 (m, 2.5 H), 2.80 – 2.60 (m, 1 H), 2.58 –
.31 (m, 4 H), 2.26 – 2.08 (m, 1 H), 1.94 – 1.81 (m, 0.5 H), 1.77 – 1.60 (m, 2 H), 1.60 – 1.51
16

ACCEPTED MANUSCRIPT
(
m, 0.5 H), 1.50 – 1.31 (m, 1 H), 1.31 – 1.17 (m, 1 H), 1.03 – 0.96 (m, 3 H), 0.97 – 0.81 (m, 8
13
H), 0.79 – 0.75 (m, 7 H). C NMR (CDCl ) δ: 167.51, 167.17, 166.51, 166.47, 163.0, 162.8,
3
1
4
3
2
41.1, 141.0, 137.5, 131.7, 130.9, 129.7, 126.33, 126.26, 124.9, 54.2, 54.0, 47.9, 43.0, 42.78,
2.75, 42.74, 42.70, 42.67, 42.62, 42.56, 42.5, 39.4, 38.0, 34.54, 34.45, 34.2, 33.3, 30.2, 30.1,
0.0, 29.5, 29.4, 24.3, 24.2, 22.9, 22.64, 22.60, 22.53, 22.4, 22.3, 22.08, 22.05, 21.9, 21.8,
+
1.7, 21.4, 19.70, 19.65, 19.1, 19.0, 11.6, 11.5, 11.44, 11.42 .LRMS (ESI ): m/z 465.5
+
+
+
[
M+H] (100%). HRMS (ESI ): m/z calculated for C H N O [M+H] : 465.3593, found:
29 45 4
4
1
8
65.3596. IR (neat): νmax 2956s, 2924m, 2868m, 1710w, 1636s, 1540s, 1491m, 1460s, 1435s,
384s, 1364s, 1339s, 1276m, 1254m, 1220w, 1160m, 1137m, 1158w, 1010w, 974w, 918w,
−
1
56w, 833w, 761s, 746s, 702w, 667w cm .
4
.4 AlphaScreen Assay.
4
6
The AlphaScreen assay was performed as previously described. The AlphaScreen GST
detection kits were purchased from PerkinElmer Life Sciences. Assays were performed in
white, 384-well low-volume assay plates and analysed on PerkinElmer Fusion alpha plate
reader (Ex680, Em520-620nM). The assay buffer was prepared fresh daily and contained 50
mM HEPES, 10 mM DTT, 100 mM NaCl, 0.05% Tween 20 and 0.1 mg/ml casein, pH 7.4.
Bead dilution buffer contained 50 mM Tris, pH 7.5, 0.01% Tween 20 and 0.1 mg/ml casein.
A solution of 4.75 µL of assay buffer and 0.25 µL of compound stock solution (20 mM in
DMSO) was titrated into a 384 well plate at 11 concentrations ranging from 300 to 0.03 µM
(
2.5 fold dilutions). A mixture of the GST tagged protein binding partner Bcl-x or Mcl-1
L
with anti-GST (antibody against GST coated) acceptor beads was prepared, whilst in another
tube a mixture of the biotinylated Bim BH3 26-mer peptide and streptavidin-coated donor
beads was prepared. The bead mixtures were pre-incubated at room temperature for 30 min.
To each well was added 10 µL of the acceptor bead-protein complex mixture. The 384 well
plate was sealed and incubated at room temperature for 30 min, before 10 µL of the donor
bead-BH3 complex mixture was added to each well. The 384 well plate was sealed with
aluminium foil and incubated at room temperature for 4 h. The final assay concentrations
were as follows: 1.0% v/v, DMSO; 0.6 nM Bcl-x /Mcl-1 protein; 5.0 nM biotinylated Bim
L
BH3 26-mer peptide; 15 µg/mL donor beads and 15 µg/mL acceptor beads. Regression
analysis was conducted to determine IC values.
5
0
Acknowledgements
The authors acknowledge the support of the School of Chemistry, Monash University, and
CSIRO Manufacturing; Biomedical Manufacturing Program. Z.L. gratefully acknowledges
receipt of a Faculty of Science Dean’s Postgraduate Research Scholarship and a top-up
scholarship from CSIRO. We are also thankful to Dr Craig Forsyth for X-ray crystal structure
determination and the Victorian Partnership for Advanced Computing for access to their
facilities for the molecular modelling component of this research. Part of this work was
supported by an NHMRC (GNT1025138), the WEHI de Burgh Fellowship to GL, the
Australian Cancer Research Foundation. Part of this work was made possible through
Victorian State Government Operational Infrastructure Support and Australian Government
NHMRC IRIISS (#361646).
Supplementary data
Supplementary data related to this article can be found at http:// . This includes the X-Ray
crystallography data for compound 11 (CCDC 993501) and the ab initio optimised low
energy conformers of compounds 11 and 13.
References
17

ACCEPTED MANUSCRIPT
1
.
Rual, J.-F.; Venkatesan, K.; Hao, T.; Hirozane-Kishikawa, T.; Dricot, A.; Li, N.;
Berriz, G. F.; Gibbons, F. D.; Dreze, M.; Ayivi-Guedehoussou, N.; Klitgord, N.; Simon, C.;
Boxem, M.; Milstein, S.; Rosenberg, J.; Goldberg, D. S.; Zhang, L. V.; Wong, S. L.; Franklin,
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