The discovery of novel antitrypanosomal 4-phenyl-6-(pyridin-3-yl)pyrimidines

Article abstract of DOI:10.1016/j.ejmech.2020.112871

Human African trypanosomiasis, or sleeping sickness, is a neglected tropical disease caused by Trypanosoma brucei rhodesiense and Trypanosoma brucei gambiense which seriously affects human health in Africa. Current therapies present limitations in their application, parasite resistance, or require further clinical investigation for wider use. Our work herein describes the design and syntheses of novel antitrypanosomal 4-phenyl-6-(pyridin-3-yl)pyrimidines, with compound 13, the 4-(2-methoxyphenyl)-6-(pyridine-3-yl)pyrimidin-2-amine demonstrating an IC₅₀ value of 0.38 μM and a promising off-target ADME-Tox profile in vitro. In silico molecular target investigations showed rhodesain to be a putative candidate, supported by STD and WaterLOGSY NMR experiments, however, in vitro evaluation of compound 13 against rhodesain exhibited low experimental inhibition. Therefore, our reported library of drug-like pyrimidines present promising scaffolds for further antikinetoplastid drug development for both phenotypic and target-based drug discovery.

Full text of DOI:10.1016/j.ejmech.2020.112871

Journal Pre-proof
The Discovery of Novel Antitrypanosomal 4-phenyl-6-(pyridin-3-yl)pyrimidines
William J. Robinson, Annie E. Taylor, Solange Lauga-Cami, George W. Weaver,
Randolph R.J. Arroo, Marcel Kaiser, Sheraz Gul, Maria Kuzikov, Bernhard Ellinger,
Kuldip Singh, Tanja Schirmeister, Adolfo Botana, Chatchakorn Eurtivong, Avninder S.
Bhambra
PII:
S0223-5234(20)30843-6
DOI:
https://doi.org/10.1016/j.ejmech.2020.112871
EJMECH 112871
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 31 July 2020
Revised Date: 10 September 2020
Accepted Date: 19 September 2020
Please cite this article as: W.J. Robinson, A.E. Taylor, S. Lauga-Cami, G.W. Weaver, R.R.J. Arroo,
M. Kaiser, S. Gul, M. Kuzikov, B. Ellinger, K. Singh, T. Schirmeister, A. Botana, C. Eurtivong, A.S.
Bhambra, The Discovery of Novel Antitrypanosomal 4-phenyl-6-(pyridin-3-yl)pyrimidines, European
Journal of Medicinal Chemistry, https://doi.org/10.1016/j.ejmech.2020.112871.
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The Discovery of Novel Antitrypanosomal 4-phenyl-6-(pyridin-
3-yl)pyrimidines
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a
a
a
b
William J. Robinson , Annie E. Taylor , Solange Lauga-Cami , George W. Weaver ,
Randolph R. J. Arroo c, Marcel Kaiser d, e, Sheraz Gul f, g, Maria Kuzikov f, g, Bernhard Ellinger f, g
Kuldip Singh h, Tanja Schirmeister i, Adolfo Botana j, Chatchakorn Eurtivong k and Avninder S.
Bhambra a, *
,
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a Leicester School of Allied Health Sciences, De Montfort University, The Gateway, Leicester, LE1 9BH,
UK.
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b Department of Chemistry, Loughborough University, Loughborough, LE11 3TU, UK
c Leicester School of Pharmacy, De Montfort University, The Gateway, Leicester, LE1 9BH, UK.
d Swiss Tropical and Public Health Institute, Socinstrasse 57, 4051 Basel, Switzerland
e University of Basel, Petersplatz 1, 4003 Basel, Switzerland
f Fraunhofer Institute for Molecular Biology and Applied Ecology Screening Port, Hamburg, Germany
g
Fraunhofer Cluster of Excellence Immune-Mediated Diseases CIMD, Hamburg Site, Hamburg,
Germany.
h Department of Chemistry, University of Leicester, Leicester LE1 7RH, UK.
i
Institute of Pharmaceutical and Biomedical Sciences, Johannes Gutenberg University of Mainz,
Staudingerweg 5, D-55128 Mainz, Germany
j JEOL UK, JEOL House, Silvert Court, Watchmead Welwyn Garden City, Herts AL7 1LT, UK.
k
Program in Chemical Sciences, Chulabhorn Graduate Institute, Chulabhorn Royal Academy, Bangkok
10210, Thailand
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* corresponding author, email address: abhambra@dmu.ac.uk.
Abstract
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Human African trypanosomiasis, or sleeping sickness, is a neglected tropical disease caused
by Trypanosoma brucei rhodesiense and Trypanosoma brucei gambiense which seriously
affects human health in Africa. Current therapies present limitations in their application,
parasite resistance, or require further clinical investigation for wider use. Our work herein
describes the design and syntheses of novel antitrypanosomal 4-phenyl-6-(pyridin-3-
yl)pyrimidines, with compound 13, the 4-(2-methoxyphenyl)-6-(pyridine-3-yl)pyrimidin-2-
amine demonstrating an IC50 value of 0.38 µM and a promising off-target ADME-Tox profile
in vitro. In silico molecular target investigations showed rhodesain to be a putative
candidate, supported by STD and WaterLOGSY NMR experiments, however, in vitro
evaluation of compound 13 against rhodesain exhibited low experimental inhibition.
Therefore, our reported library of drug-like pyrimidines present promising scaffolds for
further antikinetoplastid drug development for both phenotypic and target-based drug
discovery.
KEYWORDS: antitrypanosomal, sleeping sickness, Human African Trypanosomiasis,
Trypanosoma brucei rhodesiense, pyrimidines, rhodesain, docking, ADME-Tox
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1.0 Introduction
Human African trypanosomiasis (HAT), also known as sleeping sickness, is a vector-borne
parasitic disease caused by Trypanosoma brucei rhodesiense (T.b.r) and Trypanosoma brucei
gambiense (T.b.g), two haemoflagellate subspecies of Trypanosoma brucei (Baker and
Welburn, 2018). The disease is endemic in 36 sub-Saharan Africa countries and parasites are
typically transmitted to humans by the tsetse fly. Infection with T.b.r manifests into the
acute form of the disease whilst T.b.g causes the chronic form of illness. HAT has two
notable stages where the first, known as the haemolymphatic stage occurs when
trypomastigotes circulate in the bloodstream and lymphatic fluids. If untreated, the second
stage, also known as the neurological stage ensues where parasites cross the blood brain
barrier and patient recovery is unlikely leading to death.
No vaccines exist to treat sleeping sickness and chemotherapeutic treatments including
pentamidine, melarsoprol, suramin and NECT have been associated with undesirable dosing
regimens and unwanted side-effects including patient death (Fairlamb and Horn, 2018;
Mesu et al., 2018). Recently, fexinidazole (figure 1) was approved to treat infections caused
Figure 1. Fexinidazole
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by T.b.g specifically in the Democratic Republic of Congo (DRC) which is host to around 85%
of cases recorded. However, factors affecting the success of fexinidazole will include the
further success of drugs under evaluation for clinical and veterinary use, drug resistance and
reservoir control of causative pathogens (Wyllie et al., 2016; Wall et al., 2018; Hulpia et al.,
2020).
The use of privileged structures or scaffolds as building blocks in medicinal chemistry
provide opportunities for developing new compound structures from an already active
framework (Zhuang et al., 2017). This strategy, combined with concepts such as scaffold
hopping can support the generation of detailed structure activity relationships in a bid to
develop suitable compounds for preclinical evaluation. For example, chalcones which
contain the α, β-unsaturated carbonyl moiety form the core chemical scaffold for a range of
natural products found within fruits, vegetables and other plants. This structural motif also
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presents in medications, including sofalcone which is used as a gastric mucosa protective
agent (Gomes et al., 2017; Kim et al., 2019).
Another important class of compounds accessible from chalcones are pyrimidines, which
have been reported for a wide range of biological activities and are incorporated in clinically
used drugs such as 5-fluorouracil (Maring et al., 2005). Moreover, antitrypanosomal
activities have been reported for compounds bearing the pyrimidine functional group
demonstrating the significance of therapeutic potential (Monti et al., 2018; Volkov et al.,
2018). Within this context, our previous work reported the syntheses of novel
antitrypanosomal pyridylchalcones showing sub-micromolar IC50 values against T.b.r
(Bhambra et al., 2017). Using this chemical scaffold as a suitable template for modification,
we investigated the syntheses and antitrypanosomal activities of substituted 4-phenyl-6-
(pyridin-3-yl)pyrimidines in a continued interest in antikinetoplastid drug development
(Bhambra et al., 2016).
2.0 Results and Discussion
2.1 Syntheses
Chalcones were synthesised as previously described by Bhambra et al. (2017) and
subsequently converted to corresponding 4-phenyl-6-(pyridin-3-yl)pyrimidines (scheme 1) as
reported by Varga et al. (2005). The synthesis was achieved in a one pot procedure by
treating chalcones (1 mmol) with either formamidine or guanidine (1.5 mmol) and sodium
ethoxide (4 mmol). Compounds were purified by recrystallisation or column
chromatography using either ethyl acetate/hexane or dichloromethane/methanol as the
eluent. The structures of new compounds were fully in accord with their analytical and
Scheme 1. Reagents and conditions: a) sodium ethoxide, formamidine, reflux b) sodium ethoxide, guanidine
hydrochloride, reflux
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spectroscopic properties.
The structure of 13 was confirmed by single crystal X-ray diffraction analysis, with the
Figure 2. Crystal structure of compound 13
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molecular structure shown in figure 2.
2.2 Antitrypanosomal and Antiproliferative Activity
The antitrypanosomal activities (table 1) of our 4-phenyl-6-(pyridin-3-yl)pyrimidines were
determined against Trypanosoma brucei rhodesiense (STIB900) and cytotoxicity was
assessed using L6 cells (rat skeletal myoblasts) as described by Bernal et al. (2019).
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Table 1. Antitrypanosomal and cytotoxic evaluation of synthesised novel 4-phenyl-6-(pyridin-3-yl)pyrimidines.
SD – standard deviation.
IC50 (µM)
CC50 (µM)
L6 cells
>100
>100
>100
>100
>100
>65
Compound
Ar (A-ring)
Ar’ (B-ring)
R1
±SD
±SD
SI
T. b. rhodesiense
1
2
3-Py-
3-Py-
3-Py-
3-Py-
3-Py-
3-Py-
3-Py-
3-Py-
3-Py-
3-Py-
3-Py-
3-Py-
3-Py-
3-Py-
Ph-
3-Pyridyl-
2-OMe-Ph-
2-Br-Ph-
H
H
4.8
34.7
19.6
13.5
8.0
0.21
1.81
2.82
2.58
1.13
0.22
1.07
0.54
4.36
2.52
9.06
3.40
0.18
4.71
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
3
H
4
H
5
3-OMe-Ph-
3-Br-Ph-
H
6
H
2.4
7
3-F-Ph-
H
12.4
6.8
>100
>100
>100
>100
>100
>100
23
8
4-OMe-Ph-
3,4-OMe-
3,4-O2CH2-Ph-
Ph-
H
9
H
12.0
6.7
10
11
12
13
14
H
NH2
NH2
NH2
NH2
13.8
57.4
0.38
20.2
3-Pyridyl-
2-OMe-Ph-
2-Br-Ph-
7.38 61
>100
-
-
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3-Py-
3-Py-
3-Py-
3-Py-
3-Py-
3-Py-
3-Py-
2-Py-
2-Py-
4-Py-
4-Py-
3-OMe-Ph-
3-Br-Ph-
NH2
NH2
NH2
NH2
NH2
NH2
12.0
10.0
12.2
14.6
7.9
0.36
2.38
3.51
1.98
3.20
6.24
0.63
0.19
1.96
5.00
0.69
0.005
>100
>65
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
16
17
3-F-Ph-
>100
>100
>100
>100
>100
>15
18
4-OMe-Ph-
4-Br-Ph-
19
20
3,4-OMe-
20.3
12.9
8.7
21
3,4-O2CH2-Ph- NH2
22
2-OMe-Ph-
2-OMe-Ph-
2-OMe-Ph-
2-OMe-Ph-
H
23
24
NH2
H
13.9
13.0
24.2
0.011
>100
>100
>70
25
NH2
Melarsoprol
Podophyllotoxin
0.019
0.007
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2.2.1 4-Phenyl-6-(pyridin-3-yl)pyrimidine Derivatives
Derivative 1, 4-phenyl-6-(pyridin-3-yl)pyrimidine,
exhibited
low
micromolar
antitrypanosomal activity with an IC50 value of 4.8 µM whilst showing negligible toxicity
towards L6 cells (> 100 µM). In comparison, the di-pyridyl substituted analogue 2
demonstrated less antitrypanosomal activity but similarly to 1 remained non-toxic to L6
cells. Compounds 3 and 4, the 2-methoxyphenyl and 2-bromophenyl analogues both
demonstrated antitrypanosomal IC50 values of 19.6 µM and 13.5 µM with no toxicity
determined against L6 cells. The 3-methoxypheny analogue 5 showed a lower
antitrypanosomal IC50 value of 8.0 µM, however, 6, the 3-bromophenyl derivative exhibited
an increased antitrypanosomal effect with an IC50 value of 2.0 µM. Subsequently, derivative
7, bearing the 3-fluorophenyl substituent had reduced activity in comparison to both the 3-
bromo and 3-methoxyphenyl analogues with an IC50 value of 12.4 µM but again no toxicity
was observed against L6 cells. In comparison, 8 bearing a 4-methoxyphenyl substituent
showed better antitrypanosomal activity than the other methoxyphenyl substituted
analogues 3 and 5 with an IC50 value of 6.8 µM. The 3,4-dimethoxyphenyl substituted
analogue 9 showed less activity than both 5 and 8 with similar cytotoxicity values observed
and 10, bearing a methylenedioxyphenyl substituent demonstrated an IC50 value of 6.7 µM.
2.2.2 4-Phenyl-6-(pyridin-3-yl)pyrimidin-2-amine Derivatives
In relation to 1, derivative 11, the 4-phenyl-6-(pyridin-3-yl)pyrimidin-2-amine, exhibited
greater than 3-fold higher antitrypanosomal activity with an IC50 value of 13.8 µM and 12
also demonstrated less antitrypanosomal activity than its counterpart 2. Compound 13,
bearing a 2-methoxyphenyl substituent, showed submicromolar antitrypanosomal activity
with an IC50 value of 0.38 µM which was greater than 50-fold lower than that observed for 3.
Although 13 exhibited a CC50 value of 23 µM against L6 cells, this compound was over 60
times more selective against trypanosomes and provided a compound of interest from the
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4-phenyl-6-(pyridin-3-yl)pyrimidin-2-amine library. Replacing the methoxy group with Br
resulted in a reduction of antitrypanosomal activity as observed with 14 but did not exhibit
cytotoxic results against L6 cells. By viewing 13 as a pharmacophore of interest, moving the
methoxy group to the 3-position to give 15 showed less antitrypanosomal activity. The 3-
bromophenyl analogue 16 demonstrated similar activity to 15 although its 4-phenyl-6-
(pyridin-3-yl)pyrimidine counterpart 6 showed low micromolar antitrypanosomal activity.
Replacing Br with F as shown with 17 did not increase antitrypanosomal potency and
demonstrated a similar result to 7. Derivative 18, designed with a 4-methoxyphenyl
substituent also showed comparable activity to 17 but the 4-bromophenyl substituted
compound 19 exhibited nearly two times more activity than 18. Incorporating a 3,4-
dimethoxyphenyl group into the framework as shown with 20 raised the IC50 value to 20.3
µM and in line with 17, 18 and 19 did not demonstrate cytotoxic activity. In comparison, 21
showed nearly two times less activity than 10 but again didn’t demonstrate cytotoxic
activity. Furthermore, to add to our findings 22, 23 and 24 were prepared bearing the 2-
methoxyphenyl group for the B-ring but with either a 2- or 4- positioned N in the pyridyl A-
ring. However, the results obtained did not improve on 13, the 4-(2-methoxyphenyl)-6-
(pyridin-3-yl)pyrimidin-2-amine, as the antitrypanosomal values were 8.7, 13.9, 13.0 and
24.2 µM for 22, 23, 24 and 25 respectively with no notable cytotoxicity recorded apart from
compound 22 which exhibited a CC50 value of 17.0 µM.
With the overall data observed, it can be seen that antitrypanosomal activity is favoured
with a 3-pyridyl A-ring, and 2-methoxyphenyl B-ring and a 2-aminopyrimidine core (figure 3).
Figure 3. SAR observations for antitrypanosomal pyrimidines explored
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2.3 In Vitro ADME-Tox and In Silico ADME Assessment
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Compound 13 was selected for further in vitro ADME-Tox studies based on the sub-
micromolar antitrypanosomal activity observed and selectivity over L6 cells. This included
screening Compound 13 at 10 µM against a panel of cytochrome P450s (CYP1A2, CYP2C9,
CYP2C19, CYP2D6 and CYP3A4), histone deacetylases (HDAC1, HDAC3 and HDAC6) and hERG
(figure 4). Cytotoxicity was also determined against 786-O and A549 cells.
ADME-Tox
Figure 4. Data generated from the in vitro ADME-Tox assessment of compound 13. The results were reported as
percentage inhibition where 0-50 % inhibition is indicated as a green tile, 51-70 % as amber and 71-100 % as a red tile.
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Compound 13 showed favourable results against CYP3A4 and CYP2D6, some inhibition
against CYP2C9 and CYP2C19 and inhibitory activity against CYP1A2. No inhibition was
observed against HDAC 1, 3 and 6 and 13 was not cytotoxic towards 786-O and A549 cells.
This dataset shows 13 to be a promising early stage pre-clinical compound for further
development to increase on-target activity and decrease off-target toxicity. Compound 13
was further evaluated in silico using the ADME prediction program QikProp and the findings
are reported below in table 2 (Schrödinger Release 2020-3: Maestro; LigPrep; QikProp,
Schrödinger, LLC, New York, NY, 2020). All properties calculated are satisfactory physico-
chemical properties for compound 13.
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Table 2. QikProp ADME Predictions with ranges as described for the program: CNS, Predicted central nervous
system activity on a −2 (inacꢀve) to +2 (active) scale; QPlogPo/w, predicted octanol/water partition coefficient
(−2.0 – 6.5); QPPCaco, Predicted apparent Caco-2 cell permeability in nm/sec (<25 poor, >500 great); QPlogBB,
predicted brain/blood partition coefficient (−3.0 – 1.2); HOralAbs, predicted qualitative human oral absorption (1,
2, or 3 for low, medium, or high); Ro5, number of violations of Lipinski’s rule of five; and Ro3, number of
violations of Jorgensen’s rule of three.
Predicted ADME Properties
Compound CNS QPlogPo/w QPPCaco QPlogBB HOralAbs Ro5 Ro3
13
-1
2.602
906.463
-0.722
3
0
0
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2.4 In Silico Target Exploration
Previous findings have shown pyrimidines to putatively inhibit cruzain, a cysteine protease
expressed in Trypanosoma cruzi (T. cruzi) which is essential for parasite survival (de Melo,
2018). Based on this, we performed docking analyses of our pyrimidine library against
rhodesain, an analogous cysteine protease essential for parasite survival since it is involved
in several pathological processes in the host, including the crossing of the parasite through
the blood brain barrier, turnover of variant surface glycoproteins (VSGs) and degradation of
host immunoglobulins (Ettari et al., 2016; Ferreira and Andricopulo, 2017).
The 4-phenyl-6-(pyridine-3-yl)pyrimidines docked into the binding site with similar binding
modes and were predicted to occupy the S2 binding pocket of the catalytic site. Both the A
and B rings of compounds can occupy the hydrophobic pocket deeply buried into the S2 site
and can form favourable hydrophobic interactions with nearby hydrophobic residues:
Ala136; Ile137; Ala138; Ala208; and Leu67. The importance of hydrophobic interactions
between the mentioned residues with bioactive ligands has been documented (de Melo,
2018; Ogungbe and Setzer, 2009; Zhang et al., 2018). The pyrimidine core can interact with
nearby hydrophobic Leu160 and Leu67 residues and hydrogen bonds with His162. In this
case, when the A ring is oriented towards the catalytic triad, the 3-pyridine nitrogen atom
can form hydrogen bonds with Trp26 and catalytic Cys25 residues as seen for derivative 1
(figure 5, A and B). In the hydrophobic pocket, the 3-pyridine nitrogen was able to form
hydrogen bonds with the backbones of Met68 and Asp69 as seen for derivative 6 (figure 5, C
Figure 5. Docking of compounds 1, 6 and 13 with rhodesain. Red and grey surfaces indicate partially negative and neutral
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charges, respectively. The protein folds were shown as blue ribbons. Hydrogen bonding amino acid residues were depicted
as: Cys25 (red), Trp26 (turquoise), Met68 (yellow), Asp69 (brown), Asp161 (green) and His162 (orange).

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and D). The role of catalytic Cys25 and His162 residues are renowned for their catalytic
function in cysteine proteases and intermolecular interactions with the residues implicates
cysteine protease inhibitory effect (Kerr, 2009; Rogers, 2012). The amino group at R1 was
seen frequently to form a hydrogen bond with Asp161 residue as in the case of derivative 13
(figure 5, E and F).
2.5 Nuclear Magnetic Resonance
2.5.1 Saturation Transfer Difference (STD) and WaterLOGSY
In order to support the outcome observed from docking studies, protein-ligand interactions
were probed via Saturation Transfer Difference (STD) and WaterLOGSY experiments (Dalvit
Figure 6. STD and WaterLOGSY spectra of compound 13 in the presence of rhodesain.
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et al., 2000; Mayer and Meyer, 1999). STD is an NMR technique where the protein is
selectively saturated and, if the ligand is binding to the protein in the timescale of the
experiment, the saturation gets transferred to the ligand via intermolecular NOE and only
then it is possible to observe an NMR signal. WaterLOGSY relies on the detection of the
intermolecular NOE between water (which is selectively irradiated) and the ligand. The sign
of the NOE depends on the rotational correlation time of the molecule, thus large molecules
such as proteins, have an opposite NOE sign than small molecules such as the pyrimidines
investigated. If the pyrimidines bind to the protein, their rotational correlation time changes
and the sign of the signals in the WaterLOGSY experiment will be opposite than in the
absence of a binding protein. NMR signals for 13 were detected by STD, confirming binding
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to rhodesain (figure 6). The WaterLOGSY results, which as expected is generally a more
sensitive technique, further corroborate our findings (Antanasijevic, Ramirez and Caffrey,
2014). However, proton signals at 8.5 ppm and 9.0 ppm present in the STD results are
almost absent or have the opposite sign in the WaterLOGSY spectra. The STD indicates these
protons are binding to rhodesain, while the WaterLOGSY, as it is solvent-mediated, indicates
that these protons have weak solvent accessibility meaning they are buried in the protein
(Ludwig et al., 2008; Raingeval et al., 2019).
2.6 In Vitro Rhodesain Activity
Based on the in silico docking studies and outcomes of the NMR experiments performed,
compound 13 was selected for evaluation of its potential inhibition against rhodesain in
vitro. Compound 13, at a concentration of 50 µM was assayed with varying concentrations
of the Cbz-Phe-Arg-AMC substrate (10, 5 and 1.25 µM) and a maximum of 15% inhibition of
rhodesain enzymatic activity was detected. Although in silico docking and NMR experiments
showed favourable protein-ligand interactions, in vitro enzymatic screening data indicates
that the weak experimental inhibition observed signifies rhodesain is not the proposed
target for compound 13. This result warrants further investigations into the
antitrypanosomal mechanism of action of compound 13 but also establishes a new chemical
scaffold for further development with the potential of inhibiting rhodesain.
3.0 Conclusion
Our work has described the discovery of novel 4-phenyl-6-(pyridine-3-yl)pyrimidines
demonstrating promising antitrypanosomal activities in vitro. In particular, compound 13
exhibited potent antitrypanosomal activity against T.b.r with an IC50 value of 0.38 µM.
Limited off-target liabilities were recorded against CYP450s and HDACs and there was no
notable hERG or cell cytotoxicity. To our knowledge, this is the first report detailing such
findings encompassing this compound class in relation to T.b.r. In silico docking and in vitro
NMR experiments indicated the cysteine protease rhodesain as a putative target for our
novel compound library and subsequent in vitro enzymatic studies indicated compound 13
had a weak inhibitory effect. These findings suggest the antitrypanosomal effects observed
for our pyrimidines are not linked to rhodesain inhibition, prompting further investigations
to elucidate the mechanism of action. In vivo evaluation and further chemical development
of the presented chemical scaffold from both a phenotypic and target-based drug design
approach could exploit the antikinetoplastid potential of this compound class and we shall
report further findings in due course.
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4.0 Experimental
4.1 General
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All solvents and chemicals were used as purchased without further purification. The H and
13C-NMR spectra were recorded on a JEOL ECZ-R 600 MHz or Bruker Avance AV400 NMR
spectrometer. Chemical shifts are reported in δ units (ppm) relative to either TMS or the
residual solvent signal. IR spectra were recorded on a Bruker Alpha ATR IR instrument. HRMS
was performed using a Thermo Scientific LTQ Orbitrap XL at the EPSRC UK National Mass
Spectrometry Facility at Swansea University. Melting points were determined on
Gallenkamp melting point apparatus. TLC was performed on Merck Silica Gel 60F254 coated
plates and visualised under UV light (254/366 nm). Fisher silica gel 60 (20-45 or 35-70 µm)
was used for flash chromatography.
4.2 Chemistry
4.2.1 General Method for Synthesis of Chalcones
Chalcones were synthesised using the methods described by our previous work (Bhambra et
al., 2017).
4.2.2 General Method for the Synthesis of 4-phenyl-6-(pyridine-3-yl)pyrimidines
In an inert atmosphere sodium ethoxide was generated in situ by reacting sodium metal (4
mmol) with dry EtOH (50 mL) under reflux. After the sodium metal had dissolved, either
guandine hydrochloride (1.5 mmol) or formamidine hydrochloride (1.5 mmol) was added
depending on the target pyrimidine. Subsequently, the corresponding chalcone (1.0 mmol)
was added and the solution was left to stir (18 hr) under reflux. The completion of the
reaction was indicated by TLC analyses and the reaction was quenched with water (50 mL).
The crude mixture was extracted with ethyl acetate (3 x 50 mL), washed with brine (50 mL)
and dried over MgSO4. The crude product was purified by recrystallisation in ethanol or flash
column chromatography (ethyl acetate/hexane or dichloromethane/methanol as the
eluent).
4-Phenyl-6-(pyridin-3-yl)pyrimidine (1)
Yield: 18%, mp: 116-118 ˚C. 1H NMR (400 MHz, CDCl ): δ 9.31 (1H, s), 8.73 (1H, d, J = 3), 8.71
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(1H,s), 8.67 (1H, d, J = 5), 8.38-8.33 (2H, m), 7.61-7.54 (4H, m).13C NMR (101 MHz, CDCl3): δ
165.08, 162.32, 159.42, 151.72, 148.49, 136.61, 134.71, 132.73, 131.27, 129.15, 127.27,
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123.83, 112.82. IR, ꢀmax/cm-1: 3038, 1677, 1412, 1369, 1020. HRMS: m/z found 234.1028,
C15H12N3 (M+H+) requires 234.1026.
4,6-Bis(Pyridin-3-yl)pyrimidine (2)
Yield: 63%, mp: 206-207 ˚C. 1H NMR (400 MHz, DMSO-d6): δ 9.54 (2H, s), 9.40 (1H, s), 8.84
(1H, s), 8.78 (2H, d), 8.71 (2H, dd, J = 5, 1), 7.61-7.65 (2H, m). 13C NMR (101 MHz, DMSO-d6):
δ 162.07, 159.13, 151.88, 148.56, 134.79, 131.73, 123.96, 113.48. IR, ꢀmax/cm-1: 3038, 1591,
1571, 1414, 1371, 1259. HRMS: m/z found 235.0981, C14H11N4 (M+H+) requires 235.0978.
4-(2-Methoxyphenyl)-6-(pyridin-3-yl)pyrimidine (3)
1
Yield: 21%, mp: 102-104 ˚C. H NMR (400 MHz, CDCl3): δ 9.37 (1H, s), 9.36 (1H, s), 8.77 (1H,
bs), 8.57 (1H, bs), 8.56 (1H, s), 7.94 (1H, d, J = 5), 7.63-7.61 (1H, m), 7.55 (1H, t, J = 5), 7.23
(1H, d, J = 5), 7.13 (1H, t, J = 6), 3.90 (3H, s). 13C NMR (101 MHz, CDCl3): δ 163.76, 161.13,
159.08, 157.93, 151.42, 148.60, 134.72, 132.03, 131.10, 125.96, 123.77, 121.28, 117.71,
111.59, 55.73. IR, ꢀmax/cm-1: 2922, 1589, 1574, 1460, 1251. HRMS: m/z found: 264.1131
C16H14N3O (M+H+) requires 264.1131.
4-(2-Bromophenyl)-6-(pyridin-3-yl)pyrimidine (4)
1
Yield: 12%, Yellow oil. H NMR (400 MHz, CDCl ): δ 9.40 (1H, d, J = 2), 9.34 (1H, d, J = 1),
3
8.77 (1H, dd, J = 5, 1), 8.63 (1H, dt, J = 8, 2), 8.45 (1H, d, J = 1), 7.83 (1H, dd, J = 8, 1), 7.70
(1H, dd, J = 8, 2), 7.57-7.63 (2H, m), 7.48 (1H, td, J = 8, 2). 13C NMR (100 MHz, DMSO-d6): δ
166.18, 161.06, 158.70, 151.88, 149.70, 148.39, 147.26, 138.67, 134.73, 133.31, 131.45,
128.05, 124.03, 120.86, 117.67. IR, ꢀmax/cm-1: 3054, 2924, 1497, 1451. HRMS: m/z found
312.0133, C15H1179BrN3 (M+H+) requires 312.0131.
4-(3-Methoxyphenyl)-6-(pyridin-3-yl)pyrimidine (5)
1
Yield: 8%, mp: 124-125 ˚C. H NMR (400 MHz, DMSO-d6): δ 9.53 (1H, d), 9.34 (1H, s), 8.79-
8.77 (1H, m), 8.74 (1H, s), 8.72 (1H, dd, J = 5, 1),7.98 (1H, d, J = 6), 7.92 (1H, s), 7.66-7.61 (1H,
m), 7.52, (1H, td, J = 5, 1), 7.16 (1H, dt, J = 5, 1), 3.89 (3H, s). 13C NMR (101 MHz, DMSO-d6) δ
163.72, 161.88, 159.82, 158.93, 151.71, 148.54, 137.52, 134.76, 131.90, 130.08, 123.89,
119.70, 117.20, 113.11, 112.31, 55.35. IR, ꢀmax/cm-1: 1594, 1571, 1523, 1495, 1296, 1268.
HRMS: m/z found: 264.1134 C16H14N3O(M+H+) requires 264.1131.
4-(3-Bromophenyl)-6-(pyridin-3-yl)pyrimidine (6)
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Yield: 22 %, mp: 162-163 ˚C. NMR H (400 MHz, DMSO-d6): δ 9.55 (1H, s), 9.37 (1H, s), 8.81
(1H, s), 8.79-8.77 (1H, m), 8.72 (1H, d, J = 2), 8.61 (1H, s), 8.41 (1H, d, J = 2), 7.81 (1H, d, J =
2), 7.60-7.57 (1H, m), 7.52 (1H, td, J = 6, 2). 13C NMR (101 MHz, DMSO-d6): δ 162.32, 162.14,
158.98, 151.82, 148.58, 138.38, 134.78, 133.93, 131.75, 131.09, 129.87, 126.30, 123.89,
122.52, 113.29. IR, ꢀmax/cm-1: 3056, 2919, 1583, 1561, 1518, 1460, 1240. HRMS: m/z found:
312.0314 C15H11Br79N3 (M+H+) requires 312.0131.
4-(3-Fluorophenyl)-6-(pyridin-3-yl)pyrimidine (7)
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Yield: 14%, mp: 193-195 ˚C. NMR H (400 MHz, DMSO-d6): δ 9.54 (1H, s), 9.37 (1H, s), 8.80
(1H, s), 8.78 (1H, d, J = 3), 8.71 (1H, d, J = 5), 8.32 (1H, d, J = 1), 8.27 (1H, d, J = 6), 8.24 (1H. d,
J = 7), 7.68-7.61 (1H, m), 7.45 (1H, t, J = 5). 13C NMR (100 MHz, DMSO-d6): δ 163.52 (d, J =
241), 163.14, 162.71, 159.59, 152.36, 149.13, 139.14 (d, J = 8), 135.36, 132.32, 131.62 (d, J =
8), 124,58, 123, 92, 118.69 (d, J = 23), 114.55 (d, J = 28), 113.92. IR, ꢀmax/cm-1 : 3051, 2921,
2852, 1585, 1571, 1448, 1261. HRMS: m/z found 252.0934, C15H11FN3 (M+H+) requires
252.0932.
4-(4-Methoxyphenyl)-6-(pyridin-3-yl)pyrimidine (8)
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Yield: 21%, mp: 165-167 ˚C. H NMR (400 MHz, DMSO-d6): δ 9.52 (1H, s), 9.27 (1H, s), 8.77-
8.75 (1H, m), 8.69 (1H, dd, J = 6, 1), 8.64 (1H, s), 8.37 (2H, mAA’), 7.64-7.60 (1H, m), 7.13 (2H,
mBB’), 3.87 (3H, s). 13C NMR (101 MHz, DMSO-d6): δ 163.54, 161.95, 161.45, 158.89, 151.59,
148.44, 134.65, 132.05, 129.03, 128.35, 123.87, 114.34, 111.96, 55.42. IR, ꢀmax/cm-1: 1590,
1512. 1467, 1372, 1247, 1174, 1022. HRMS: m/z found 264.1134, C16H14N3O (M+H+) requires
264.1131.
4-(3,4-Dimethoxyphenyl)-6-(pyridin-3-yl)pyrimidine (9)
1
Yield: 18%, mp: 131-134 ˚C. H NMR (400 MHz, CDCl ): δ 9.53 (1H, s), 9.28 (1H, s), 8.77 (1H,
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d, J = 3), 8.69 (1H, dd, J = 5, 1), 8.67 (1H, s), 8.05 (1H, dt, , J = 6, 1), 7.94 (1H, s), 7.64-7.61 (1H,
m), 7.15 (1H, dd, J = 6, 1), 3.91 (3H, s), 3.87 (3H, s). 13C NMR (101 MHz, DMSO-d6): δ 163.34,
161.21, 158.62, 151.45, 151.39, 149.04, 148.27, 134.46, 131.82, 128.15, 123.67, 120.50,
111.83, 111.40, 109.89, 55,72, 55.69. IR, ꢀmax/cm-1: 2967, 2915, 2838, 1674, 1587, 1511,
1372, 1249. HRMS: m/z found 294.1236, C17H16N3O2 (M+H+) requires 294.1237.
4-(2H-1,3-Benzodioxol-5-yl)-6-(pyridin-3-yl)pyrimidine (10)
1
Yield: 15%, mp: 173-175 ˚C. H NMR (400 MHz, DMSO-d6): δ 9.51 (1H, s), 9.26 (1H, s), 8.75
(1H, d, J = 3), 8.68 (1H, d, J = 5), 8.63 (1H, s), 8.02 (1H, d, J = 6), 7.96 (1H, s), 7.61 (1H, dd, J =
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6, 5), 7.12 (1H, dd, J = 6, 1), 6.15 (2H, s). 13C NMR (101 MHz, DMSO-d6) δ 163.29, 161.57,
158.81, 151.63, 150.07, 148.47, 148.18, 134.67, 131.97, 130.16, 123.86, 122.37, 112.20,
108.62, 107.10, 101.78. IR, ꢀmax/cm-1: 2920, 2852, 1678, 1585, 1524, 1502, 1425, 1252.
HRMS: m/z found 278.0926, C16H12O2N3 (M+H+) requires 278.0924.
4-Phenyl-6-(pyridin-3-yl)pyrimidin-2-amine (11)
1
Yield: 18%, mp: 172-175 ˚C. H NMR (400 MHz, DMSO-d6): δ 9.40 (1H, s), 8.71 (1H, d, J = 3),
8.56 (1H, dd, J = 3), 8.28-8.23 (2H, m), 7.82 (1H, d, J = 2), 7.59-7.55 (1H, m), 7.55-7.51 (3H,
m), 6.90 (2H, s). 13C NMR (101 MHz, DMSO-d6): δ 165.13, 164.00, 162.78, 151.08, 148.24,
137.08, 134.36, 132.79, 130.58, 128.60, 127.02, 123.66, 102.08. IR, ꢀmax/cm-1: 3320 (NH),
3155(NH), 1650, 1590, 1544, 1357. HRMS: m/z found 249.1137, C15H13N4 (M+H+) requires
249.1135.
4,6-Bis(Pyridin-3-yl)pyrimidin-2-amine (12)
1
Yield: 20%, mp: 229-230 ˚C. H NMR (400 MHz, CDCl ): δ 9.41 (2H, s), 8.72 (2H, dt, J = 3, 1),
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8.57 (2H, dd, J = 5, 2), 7.93 (2H, d, J = 1), 7.58 (2H, dd, J = 5, 3), 7.00 (2H, s). 13C NMR (101
MHz, CDCl3): δ 164.16, 163.69, 151.52, 148.57, 134.57, 132.98, 123,64, 103.93. IR, ꢀmax/cm-1 :
3340 (NH), 3220 (NH), 3064, 1645, 1583, 1549, 1365. HRMS: m/z found 250.1090, C14H12N5
(M+H+) requires 250.1087.
4-(2-Methoxyphenyl)-6-(pyridin-3-yl)pyrimidin-2-amine (13)
Yield: 16%. mp: 108-110 ˚C. 1H H NMR (400 MHz, CDCl3): δ 9.22 (1H, d, J = 1), 8.69 (1H, dd, J
= 5, 1), 8.34 (1H, dt, J = 8, 2), 7.86 (1H, dd, J = 8, 1), 7.64 (1H, s), 7.39-7.45 (2H, m), 7.09 (1H,
td, J = 8, 1), 7.02 (1H, d, J = 8), 5.25 (2H, s), 3.90 (3H, s). 13C NMR (101 MHz, CDCl3): δ 165.41,
163.56, 162.49, 157.64, 151.03, 148.63, 134.67, 133.59, 131.42, 130.68, 126.84, 123.60,
121.05, 111.51, 108.92, 55.70. IR, ꢀmax/cm-1: 3400-3200 (NH), 2927 2837, 1677, 1597, 1534,
1279 (C-H). HRMS: m/z found 279.1244, C16H15N4O (M+H+) requires 279.1240.
4-(2-Bromophenyl)-6-(pyridin-3-yl)pyrimidin-2-amine (14)
1
Yield: 25%, mp: 149-152 ˚C. H NMR (400 MHz, DMSO-d6): δ 9.28 (1H, s), 8.69 (1H, d, J = 3),
8.45 (1H, d, J = 5), 7.76 (1H, d, J = 5) 7.48-7.58 (3H, m), 7.35-7.44 (2H, m), 6.98 (2H, s). 13C
NMR (400 MHz, DMSO-d6): δ 167.51, 163.68, 161.63, 150.43, 147.37, 139.72, 135.22,
133.01, 130.80, 130.60, 128.62, 127.75, 124.13, 120.57, 106.30. IR, ꢀmax/cm-1: 3294 (NH),
3069, 2922, 2852, 1657, 1593, 1543, 1351. HRMS: m/z found 327.0243, C15H12Br79N4 (M+H+)
requires 327.0240.
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4-(3-Methoxyphenyl)-6-(pyridin-3-yl)pyrimidin-2-amine (15)
Yield: 26%, mp: 162-164 ˚C. NMR: 1H NMR (400 MHz, DMSO-d6): δ 9.40 (1H, s), 8.71 (1H, d, J
= 3), 8.56 (1H, dd, J = 5, 1), 7.83 (1H, d, J = 5), 7.81 (1H, d, J = 1), 7.79 (1H, bs), 7.56 (1H, dd, J
= 5, 3), 7.44 (1H, td, J = 5, 1), 7.10 (1H, d, J = 6), 6.88 (2H, s), 3.86 (3H, s). 13C NMR (101 MHz,
DMSO-d6): δ 164.93, 163.93, 162.78, 159.57, 151.07, 148.27, 138.59, 134.41, 132.77, 129.68,
123.67, 119.43, 116.35, 112.13, 102.22, 55.27. IR, ꢀmax/cm-1: 3431 (NH), 3304 (NH), 3178,
.2921, 2852, 1634, 1564, 1538, 1451. HRMS: m/z found 279.1243, C16H15N4O (M+H+) requires
279.1240.
4-(3-Bromophenyl)-6-(pyridin-3-yl)pyrimidin-2-amine (16)
Yield: 45%, mp: 169-171˚C NMR: 1H NMR (400 MHz, DMSO-d6): δ 9.41 (1H, s), 8.71 (1H, d, J =
3), 8.56 (1H, dd, J = 6, 1), 8.47 (1H, d, J = 1), 8.27 (1H, d, J = 5), 7.90 (1H, d, J = 1), 7.73 (1H, d,
J = 5), 7.57 (1H, dd, J = 5, 3), 7.50 (1H, td, J = 6, 1) 6.97 (2H, s). 13C NMR (101 MHz, DMSO-d6):
δ 163.95, 163.39, 163.18, 151.20, 148.34, 139.39, 134.41, 133.22, 132.61, 130.78, 129.56,
126.00, 123.64, 122.22, 102.17. IR, ꢀmax/cm-1: 3482 (NH), 3316 (NH), 3181, 2922, 2851, 1630,
1580, 1561. 1461, 1248. HRMS: m/z found 327.0243, C15H12Br79N4 (M+H+) requires 327.0240.
4-(3-Fluorophenyl)-6-(pyridin-3-yl)pyrimidin-2-amine (17)
1
Yield: 15%, mp: 196-198 ˚C. H NMR (400 MHz, DMSO-d6): δ 9.41 (1H, s), 8.72 (1H, d, J = 3),
8.56 (1H, dd, J = 5, 2), 8.12 (1H, d, J = 6), 8.08 (1H, d, J = 7), 7.89 (1H, s), 7.62-7.54 (2H, m),
7.37 (1H, t, J = 6), 6.90 (2H, s). 13C NMR (101 MHz, DMSO-d6): δ 165.24, 164.04, 163.63,
163.23 (d, J = 245), 151.43, 148.56, 139.69 (d, J = 8), 134.39, 133.13, 130.38 (d, J = 8), 123.60,
122.70 (d, J = 3), 117.62 (d, J = 22), 114.16 (d, J = 23), 104.04. IR, ꢀmax/cm-1: 3483 (NH), 3319
(NH), 2921, 2852, 1634, 1584, 1569, 1447, 1351, 1261. HRMS: m/z found 267.1043,
C15H12FN4 (M+H+) requires 267.1041.
4-(4-Methoxyphenyl)-6-(pyridin-3-yl)pyrimidin-2-amine (18)
1
Yield: 27%, mp: 163-165 ˚C. H NMR (400 MHz, DMSO-d6): δ 9.38 (1H, s), 8.70 (1H, d, J = 3),
8.53 (1H, dd, J = 6, 1), 8.23 (2H, d, J = 5), 7.76 (1H, s), 7.55 (1H, dd, J = 5, 3), 7.07 (2H, d, J = 5),
6.79 (2H, s), 3.84 (3H, s). 13C NMR (101 MHz, DMSO-d6): δ 164.65, 163.89, 162.41, 161.34,
150.97, 148.19, 134.32, 132.91, 129.33, 128.63, 123.65, 113.93, 101.27, 55.31. IR, ꢀmax/cm-1
3326 (NH), 3186 (NH(, 2921, 1642, 1580, 1451, 1359. HRMS: m/z found 279.1242, C16H15N4O
(M+H+) requires 279.1240.
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4-(4-Bromophenyl)-6-(pyridin-3-yl)pyrimidin-2-amine (19)
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Yield: 32%, mp: 210-211 ˚C. H NMR (400 MHz, DMSO-d6): δ 9.38 (1H, s), 8.72-8.71 (1H, m),
8.54 (1H, d, J = 5), 8.20 (2H, d, J = 6), 7.84 (1H, s), 7.74 (2H, d, J = 6), 7.57-7.53 (1H, m), 6.92
(2H, s). 13C NMR (101 MHz, DMSO-d6): δ 163.97, 163.92, 163.06, 151.17, 148.28, 136.27,
134.36, 132.66, 131.60, 129.04, 124.26, 123.66, 101.94. IR, ꢀmax/cm-1: 34854 (NH), 3323
(NH), 3072, 2921, 1627, 1592, 1536, 1455, 1360. HRMS: m/z found 327.0243, C15H12Br79N4
(M+H+) requires 327.0240.
4-(3,4-Dimethoxyphenyl)-6-(pyridin-3-yl)pyrimidin-2-amine (20)
1
Yield: 24%, mp: 165-167 ˚C. H NMR (400 MHz, DMSO-d6): δ 9.40 (1H, s), 8.70 (1H, d, J = 3),
8.54 (1H, dd, J = 5, 1), 7.88 (1H, dt, J = 6, 1), 7.81 (1H, s), 7.79 (1H, s), 7.56 (1H, dd, J = 5, 3),
7.09 (1H, dd, J = 6, 1), 6.80 (2H, s), 3.88 (3H, s), 3.84 (3H, s). 13C NMR (101 MHz, DMSO-d6): δ
165.30, 164.78, 163.84, 162.40, 151.07, 148.72, 134.20, 134.32, 132.92, 129.53, 123.62,
120.31, 116.90, 112.75, 102.75, 55.66, 55.57. IR, ꢀmax/cm-1: 3508 (NH), 3372 (NH), 2937,
1607, 1564, 1511, 1448, 1362. HRMS: m/z found 309.1347, C17H17N4O2 (M+H+) requires
309.1346.
4-(2H-1,3-Benzodioxol-5-yl)-6-(pyridin-3-yl)pyrimidin-2-amine (21)
1
Yield: 16%, mp: 236-238 ˚C. H NMR (400 MHz, DMSO-d6): δ 9.38 (1H, s), 8.69 (1H, d, J = 3)
8.53 (1H, dd, J = 5, 1) 7.87 (1H, dt, J = 6, 1) 7.82 (1H, s) 7.75 (1H, s) 7.54 (1H, dd, J = 5, 3) 7.06
(1H, d, J = 5) 6.80 (2H, s), 6.12 (2H, s). 13C NMR (101 MHz, DMSO-d6): δ 164.38, 163.82,
162.57, 151.01, 149.39, 148.25, 147.84, 134.31, 132.83, 131.23, 123.61, 121.73, 108.26,
106.95, 102.10, 101.54. IR, ꢀmax/cm-1: 3492 (NH), 3309 (NH), 3195, 2900, 1633, 1571, 1504,
1370, 1253. HRMS: m/z found 293.1035, C16H13O2N4 (M+H+) requires 293.1033.
4-(2-Methoxyphenyl)-6-(pyridin-2-yl)pyrimidine (22)
Yield: 12%, mp: 94-96 ˚C 1H NMR (400 MHz, CDCl3): δ 8.74 (1H, dd, J = 4, 1), 8.33 (1H, s), 8.31
(1H, s), 8.18 (1H, dt, J = 8, 1), 7.86 (1H, td, J = 8, 2), 7.79 (1H, dd, , J = 8, 2), 7.47 (1H, ddd, J =
6, 5, 1), 7.37 (1H, ddd, J = 8, 7, 2), 6.99 (1H, td, J = 7, 1), 6.93 (1H, dd J = 8, 1), 3.92 (3H, s). 13C
NMR (101 MHz, CDCl3): δ 158.92, 157.37, 154.61, 153.37, 148.84, 148.42, 139.97, 131.88,
128.84, 126.66, 124.25, 122.91, 121.16, 120.67, 111.20, 55.89. IR, ꢀmax/cm-1: 2937, 2835,
1687. 1600, 1584, 1492, 1435. HRMS: m/z found: 264.1134, C16H14N3O (M+H+) requires
264.1131.
4-(2-Methoxyphenyl)-6-(pyridin-2-yl)pyrimidin-2-amine (23)
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Yield: 19%, mp: 178-180 ˚C 1H NMR (400 MHz, CDCl3): δ 8.72 (1H, d, J = 3), 8.34 (1H, d, J = 6),
8.01 (1H, s), 7.99 (1H, td, J = 5, 1), 7.81 (1H, dd, J = 5, 1), 7.52 (1H, ddd, J = 5, 4, 1), 7.48-7.43
(1H, m), 7.18 (1H, d, J = 6), 7.08 (1H, t, J = 5), 6.73 (2H, s), 3.87 (3H, s). 13C NMR (101 MHz,
CDCl3): δ 165.96, 163.72, 163.36, 157.64, 155.07, 149.48, 136.89, 131.09, 130.63, 127.37,
124.77, 121.75, 120.84, 111.39, 109.29, 55.73. IR, ꢀmax/cm-1: 3293 (NH), 3172 (NH), 3003,
2964, 2931, 1627. 1604, 1535, 1435. HRMS: m/z found: 279.1242 C16H15N4O (M+H+) requires
279.1240.
4-(2-Methoxyphenyl)-6-(pyridin-4-yl)pyrimidine (24)
Yield: 7%, mp: 124-126 ˚C. 1H NMR (400 MHz, CDCl3): δ 9.38 (1H, s), 8.81 (2H, bd, J = 2), 8.43
(1H, d, J = 1), 8.06 (1H, dd, J = 6, 1), 7.99 (2H, d, J = 4), 7.52-7.49 (1H, m), 7.15 (1H, td, J = 5,
1), 7.07 (1H, d, J = 6), 3.96 (3H, s). 13C NMR (101 MHz, CDCl3): δ 164.26, 161.07, 159.16,
157.90, 150.72, 144.71, 132.22, 131.13, 125.81, 121.36, 121.23, 118.15, 111.59. 55.79. IR,
ꢀ
max/cm-1: 2982, 1686, 1601, 1584, 1493, 1285. HRMS: m/z found: 264.1135 C16H14N3O
(M+H+) requires 264.1131.
4-(2-Methoxyphenyl)-6-(pyridin-4-yl)pyrimidin-2-amine (25)
Yield: 9%, mp: 179-180 ˚C 1H NMR (400 MHz, CDCl3): δ 8.74 (2H, d, J = 4), 7.99 (2H, d, J = 4),
7.81 (1H, dd, J = 6, 1), 7.63 (1H, s), 7.47 (1H, td, J = 6, 1), 7.18 (1H, d, J = 6), 7.08 (1H, t, J = 5),
6.84 (2H, s), 3.89 (3H, s). 13C NMR (101 MHz, CDCl3): δ 165.86, 163.55, 162.47, 157.66,
150.48, 145.32, 131.55, 130.70, 126.73, 121.25, 121.11, 111.54, 109.27, 55.74. IR, ꢀmax/cm-1:
3307 (NH), 3133 (NH), 1649, 1568, 1524, 1454, 1357. MS: m/z found: 279.1244 C16H15N4O
(M+H+) requires 279.1240.
4.3 Biological Assays
4.3.1 Antitrypanosomal and L6 Cytotoxicity Assays
Assays were performed as described by Bernal et al. (2019). In vitro antitrypanosomal
activities of compounds were determined against Trypanosoma brucei rhodesiense STIB900
(bloodstream trypomastigotes) where the parasitic stock was isolated in 1982 from a human
patient in Tanzania. Minimum Essential Medium (50 µl) supplemented with 25 mM HEPES, 1
g/l glucose, 1% MEM non-essential amino acids (100 x), 0.2 mM 2-mercaptoethanol, 1 mM
Na-pyruvate and 15 % heat inactivated horse serum was added to each well of a 96-well
microtiter plate. Serial drug dilutions from 100 to 0.002 μg/ml were prepared and 4 x 103
bloodstream forms of T. b. rhodesiense STIB 900 in 50 µl was added to each well and the
plate incubated at 37 °C under a 5 % CO2 atmosphere for 70 h. Then, 10 µl of Alamar Blue
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(resazurin, 12.5 mg in 100 ml double-distilled water) was added to each well and incubation
continued for a further 2–4 h. Plates were read with a Spectramax Gemini XS microplate
fluorometer (Molecular Devices Cooperation, Sunnyvale, CA, USA) using an excitation
wavelength of 536 nm and an emission wavelength of 588 nm. The data was analysed with
SoftmaxPro (Molecular Devices Cooperation, Sunnyvale, CA, USA), which calculated IC50
values by linear regression and 4-parameter logistic regression from the sigmoidal dose
inhibition curves.
In vitro cytotoxicity with L6 cells (a primary cell line derived from rat skeletal myoblasts)
were performed in 96-well microtiter plates with each well containing 100 µl of RPMI 1640
medium supplemented with 1 % L-glutamine (200mM) and 10 % fetal bovine serum and
4000 L6 cells. Serial drug dilutions from 100 to 0.002 μg/ml were prepared and after 70 h of
incubation the plates were microscopically inspected to ensure the validity of control wells
and sterile conditions. Then, 10 µl of Alamar Blue was added to each well and the plates
incubated for an additional 2 h. Plates were read with a Spectramax Gemini XS microplate
fluorometer (Molecular Devices Cooperation, Sunnyvale, CA, USA) using an excitation wave
length of 536 nm and an emission wave length of 588 nm. The IC50 values were calculated by
linear regression and 4-parameter logistic regression from the sigmoidal dose inhibition
curves using SoftmaxPro software (Molecular Devices Cooperation, Sunnyvale, CA, USA).
4.3.2 Pre-Clinical In Vitro ADME-Tox Assays
Experiments for determining pre-clinical in vitro ADME-Tox activities were performed as
described by Moraes et al. (2019) and also described in the following sections 4.3.2.1 to
4.3.2.4.
4.3.2.1 Cytochrome (CYP) P450 inhibition assay
The luminescence based P450-Glo™ (Promega Corp.) assay was used in 384-well assay
format.
The cytochrome P450 (CYP) panel included microsomal preparations of cytochromes P450
1A2, 2C9, 2C19, 2D6, and 3A4 (Corning) from baculovirus infected insect cells (BTI-TN-
5B1−4) which express cytochromes P450 and cytochrome c reductase (and cytochrome b5
for 3A4). Compounds were added (100 nL/well in 1 % DMSO v/v) using the Echo 550® Liquid
Handler followed by addition of 5 μL/well of CYP450/substrate mixture. Following
incubation for 30 min at 37°C, the reaction was initiated by the addition of 5 μL/well of the
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NADPH regeneration mixture. By the end of a further 30 min incubation (37 °C), the CYP450
reaction was stopped and the luciferase reaction was initiated by the addition of 10 μL/well
of the luciferin detection reagent, followed by an additional 30 min of incubation at 37 °C.
The luminescence signal was detected using an Infinite® M1000 PRO plate reader. The
negative controls yielded 0 % inhibition (1 % v/v DMSO) and standard CYP450 specific
inhibitors were used as positive controls with 100 % inhibition (CYP450 1A2, alpha-
naphthoflavone; CYP450 2C9, sulfaphenazole; CYP450 2C19, troglitazone; CYP450 2D6,
quinidine; CYP450 3A4, ketoconazole). The raw data were normalised relative to the positive
and negative controls yielding the % inhibition for each compound.
4.3.2.2 Histone Deacetylase (HDAC) Assay
Inhibition of histone deacetylase (HDAC) enzymes was determined using the bioluminogenic
HDAC-Glo™ I/II assay (Promega Corp.) in 384-well assay format. Human recombinant HDAC
enzymes were purchased from BPS Bioscience (San Diego, USA) and the standard inhibitor
trichostatin A (Sigma-Aldrich) was dissolved to a yield stock solution in 100 % v/v DMSO
(stored at –20 °C). Plate handling was performed using a Cell Explorer HTS platform
equipped with an Echo 550® Liquid Handler and Multidrop liquid handling system with
luminescence measurements taken using an EnVision® multilabel 2103 reader. Compounds
were added to plates (100 nL/well; 1% v/v DMSO) and the HDAC-Glo™ I/II assay reagent was
prepared by (i) rehydration of lyophilised HDAC-Glo™ I/II substrate (with an acetylated
peptide concentration of 100 μM) in 10 mL HDAC-Glo™ I/II assay buffer and (ii) addition of
10 μL of developer reagent (containing trypsin). The microtiter plates were mixed briefly by
orbital shaking (500 - 700 rpm) and luminescence was measured at steady-state
signal:background which was achieved after 20 min. The raw data were normalised relative
to the positive and negative controls yielding the % inhibition for each compound.
4.3.2.3 Cytotoxicity Assays
A549 and 786-O cells were grown on surface-modified T175 cell culture flasks in Dulbecco’s
Modified Eagle Medium with 10 % fetal calf serum, streptomycin (100 μg/mL), and 100
U/mL penicillin G. At about 80 % confluency, cells were washed, trypsinised, resuspended
and counted in RPMI-1640 medium and seeded into 384-well microtiter plates (20 μL) at 500
cells/well. After 24 h of incubating at 37 °C in the presence of 5 % CO2, test compounds
were added to cells using an Echo 550 Liquid Handler and read after 48 h of incubation, the
luminescence signal was read following addition of 20 μL using the CellTiter-GloCTG reagent.
The raw data were normalized to percentage of cell growth by using the baseline growth
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and the corresponding NC containing only 1 % v/v DMSO. The luminescence signal of each
sample (S) was converted into percentage of cell growth compared with the average signal
of the baseline control (BC). The raw data were normalised relative to the positive and
negative controls yielding the % cytotoxicity for each compound.
4.3.2.4 hERG Cardiotoxicity Assay
The Invitrogen Predictor™ hERG Fluorescence Polarisation Assay was used in 384-well assay
format. To each well, 100 nL of the test and control compounds, 5 µL of homogenised
membrane solution (undiluted) and 5 µL of the tracer (1 nM final concentration in assay)
were added. The plates were incubated for 2 h at 25 °C in a humidity-controlled incubator
and the fluorescence polarisation was measured using an EnVision® multilabel 2103 reader.
The negative controls (0 % inhibition) and positive controls with E-4031, a blocker of hERG-
type potassium channels (100 % inhibition) were used to normalise the raw data. The raw
data were normalised relative to the positive and negative controls yielding the %
cardiotoxicity for each compound.
4.3.2.5 In Silico ADME Predictions
In silico ADME predictions were calculated using Schrodinger software (Schrödinger Release
2020-3: Maestro; LigPrep; QikProp, Schrödinger, LLC, New York, NY, 2020). The ligand was
prepared using LigPrep and evaluated using QikProp in normal processing mode. Compound
ADME Prediction ranges are as described for the program: CNS, Predicted central nervous
system activity on a −2 (inacꢀve) to +2 (acꢀve) scale; QPlogPo/w, predicted octanol/water
partition coefficient (−2.0 – 6.5); QPPCaco, Predicted apparent Caco-2 cell permeability in
nm/sec (<25 poor, >500 great); QPlogBB, predicted brain/blood partition coefficient (−3.0 –
1.2); HOralAbs, predicted qualitative human oral absorption (1, 2, or 3 for low, medium, or
high); Ro5, number of violations of Lipinski’s rule of five; and Ro3, number of violations of
Jorgensen’s rule of three.
4.4 Docking Studies
The protein structure of T.b.r rhodesain was obtained from the Protein Data Bank (PDB) and
the PDB ID is 2P7U (Berman, 2000; Kerr et al., 2009). The crystal structure was viewed using
Discovery Studio visualizer version 3.5. All ligands including All ligands including water and
co-crystallised ligands were removed and the proteins were protonated using the add
hydrogen feature in Discovery Studio. The Genetic Optimisation for Ligand Docking (GOLD)
version 5.8.1 was used as the docking engine and the GoldScore (GS) scoring function was
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implemented to validate the predicted binding modes and relative energies of the ligands
(www.ccdc.cam.ac.uk; Jones et al., 1997). The ligand structures were prepared in 3D format
using the Scigress software package version 3.3.2 (Fujitsu Scigress, 2018). The ligands were
energy minimised using the MM2 force-field followed by the semi-empirical PM6 method
(Allinger, 1977; Stewart, 2007). The centre of the docking grid was defined from the N2
amide bond of the co-crystallised ligand (x = -7.134, y = 1.467, z = 9.526) with 10 Å radius.
Fifty docking runs were allowed for each ligand with default search efficiency (100%). The
basic amino acids lysine and arginine were defined as protonated. Furthermore, aspartic and
glutamic acids were assumed to be deprotonated. The co-crystallised ligand was removed
and re-docked into the binding site and the root-mean squared deviation (RMSD) of heavy
atoms measured by superimposing the top ranking re-docked conformation and the X-ray
co-crystallised ligand using the GS scoring function (see supplementary information). The
good overlays and low RMSD value of 2.45 Å was calculated suggesting the reliability and
reproducibility of the docking protocol.
4.5 NMR Experimental
NMR samples were prepared in a 50 % DMSO-d6 and 50 % buffer solution (50 mM sodium
acetate, 200 mM sodium chloride, 5 mM EDTA, 5 % DMSO and 2 mM DTT). The overall
concentration of rhodesain used was 50 µM and the final concentration of 13 was 5 mM.
All NMR studies were performed at 25°C on a JEOL ECZ-R 600 MHz spectrometer equipped
with a ROYAL room temperature probe. All experiments were run under automation, with
the centre of the spectrum being automatically set via a script looking for the frequency of
the tallest signal of a proton experiment preceding the actual experiment. The spectra were
processed and analysed using JEOL Delta software. Saturation Transfer Difference
experiments were performed with a saturation time of 6 seconds using REBURP pulses with
a field strength of 92.3 Hz, with an off-resonance frequency of -200 ppm and an on-
resonance frequency of 0.25 ppm in alternating 256 scans. A W5 Watergate solvent
suppression scheme was used before acquisition to suppress the solvent signal. WaterLOGSY
experiments were performed with a relaxation delay of 6 seconds and a mixing time of 1.6
seconds. 256 scans were acquired and excitation sculpting was used before acquisition to
supress the solvent signal. The resulting spectrum was phased using the same phase
correction used in the experiment run in the sample without protein.
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4.6 Rhodesain experimental
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Enzyme assays with rhodesain were performed as described previously (Ehmke et al., 2013;
Latorre et al., 2016; Schirmeister et al., 2016). The assay buffer consisted of 50 mM sodium
acetate (pH 5.5), 5 mM EDTA, 200 mM NaCl and 0.005 % Brij35. The enzyme buffer
consisted of 50 mM sodium acetate (pH 5.5), 5 mM EDTA, 200 mM NaCl and 2 mM DTT. The
substrate Cbz-Phe-Arg-AMC (Bachem, purchased as HCl salt) was diluted from a 1 mM stock
solution to reach concentrations of 10, 5 and 1.25 μM. The assay mixtures had a total
volume of 200 μL and consisted of 180 μL assay buffer, 5 μL enzyme, 10 μL Me2SO (as
negative control) or inhibitor and 5 μL of the substrate solution. Inhibition against rhodesain
was measured using Cbz-Phe-Arg-AMC as the substrate, which releases AMC (7-amino-4-
methylcoumarin) after amide bond cleavage by the enzyme. Proteolytic activity of the
enzyme was monitored spectrophotometrically by the increase of fluorescence intensity by
release of AMC (emission at 460 nm) upon hydrolysis.
4.7 X-Ray Crystallography
The crystal data was collected on a Bruker APEX 2000 CCD diffractometer using graphite
monochromated Mo-Ka radiation (l = 0.71073 Å). The data were corrected for Lorentz and
polarization effects, and empirical absorption corrections were applied. The structures were
solved by direct methods and refined by full-matrix least squares cycles on F2 for all data,
using SHELX 2018/3 (Sheldrick, 2015). All hydrogen atoms were included in calculated
positions (C-H = 0.95-0.98 Å) riding on the bonded atom with isotropic displacement
parameters set to 1.5 Ueq(C) for methyl H atoms and 1.2 Ueq(C) for all other H atoms. All
non-hydrogen atoms were refined with anisotropic displacement parameters.
Crystallographic data for the structure reported in this paper has been deposited with The
Cambridge Crystallographic Data Centre and allocated the deposition numbers CCDC:
2012793. Copy of the data can be obtained free of charge from The Cambridge
Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.
5.0 Author contributions
The manuscript was written through contributions of all authors a-k. All authors have given
approval to the final version of the manuscript and have contributed equally.
6.0 Acknowledgments
We thank the EPSRC UK National Mass Spectrometry Facility at Swansea University for
recording mass spectra. We thank the Wellcome Trust for support through the Biomedical
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Vacation Scholarship initiative and both De Montfort and Loughborough Universities for
financial support.
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Human African trypanosomiasis (sleeping sickness) is a neglected tropical disease.
Current drugs present limitations and severe side-effects in their application.
We describe the syntheses of antitrypanosomal 4-phenyl-6-(pyridin-3-yl)pyrimidines.
13 showed potent antitrypanosomal activity in vitro against T.b.r (0.38 µM).