2-Aminopyrimidines as dual adenosine A1/A2A antagonists

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Graphical abstract

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2

-Aminopyrimidines as dual adenosine A /A antagonists

1

2A

a,b

b

Sarel J. Robinson , Jacobus P. Petzer , Gisella Terre’Blanche , Anél Petzer , Mietha M. van der

b

a,b

a,b*

Walt , Jacobus J. Bergh , Anna C.U. Lourens

a

Department of Pharmaceutical Chemistry, School of Pharmacy, North-West University, Private Bag

X6001, Potchefstroom, 2520, South Africa

b

Centre of Excellence for Pharmaceutical Sciences, School of Pharmacy, North-West University,

Private Bag X6001, Potchefstroom, 2520, South Africa

a

*

Corresponding author. Department of Pharmaceutical Chemistry, School of Pharmacy, North-West

University, Private Bag X6001, Potchefstroom, 2520, South Africa. Tel: (+27 18 2992266). E-mail

address: Arina.Lourens@nwu.ac.za

Abstract

In this study thirteen 2-aminopyrimidine derivatives were synthesised and screened as potential

antagonists of adenosine A and A receptors in order to further investigate the structure activity

1

2A

relationships of this class of compounds. 4-(5-Methylfuran-2-yl)-6-[3-(piperidine-1-

carbonyl)phenyl]pyrimidin-2-amine (8m) was identified as a compound with high affinities for both

receptors, with an A Ki value of 6.34 nM and an A Ki value of 9.54 nM. The effect of selected

2A

1

compounds on the viability of cultured cells was assessed and preliminary results indicate low

cytotoxicity. In vivo efficacy at A receptors was illustrated for compounds 8k and 8m since these

2

A

compounds attenuated haloperidol-induced catalepsy in rats. A molecular docking study revealed that

the interactions between the synthesised compounds and the adenosine A binding site most likely

2

A

involve Phe168 and Asn253, interactions which are similar for structurally related adenosine A₂_A

receptor antagonists.

Keywords: Adenosine A antagonist, Adenosine A antagonist, 2-aminopyrimidine, Parkinson’s

2

A

1

disease

1

. Introduction

Parkinson’s disease is a complex, chronic neurodegenerative disorder, mainly characterised by a

decline in motor function, but also associated with non-motor manifestations such as cognitive

deficits (e.g. dementia) and neuropsychiatric symptoms, such as depression [1]. Management of the

non-motor symptoms in particular is challenging and represents an important unmet medical need

[

2,3,4]. The neuropathology of the disease is hallmarked by the selective degeneration of

dopaminergic neurons of the nigrostriatal pathway and the resulting deficiency of dopamine in the

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basal ganglia [5]. However, the importance of other neurotransmitter systems and organs in

Parkinson’s disease pathogenesis are increasingly recognised [6,7,8].

Nevertheless, current treatments are still focused on enhancing dopaminergic neurotransmission and

include the use of levodopa, (which is still the gold standard nearly 50 years since its introduction),

dopamine agonists, catechol-O-methyltransferase and monoamine oxidase inhibitors. Since the

chronic nature of the disease requires long-term use of medication, the occurrence of side-effects is

unavoidable, and levodopa especially is associated with the development of debilitating dyskinesias

[

9, 10]. Limited progress has been made in altering the progress of neurodegeneration, and to date, no

agent has been established as neuroprotective or disease modifying [11,12]. Non-dopaminergic targets

for the disease are thus particularly appealing; especially if these would also improve non-motor

symptoms and provide neuroprotection [13]. In recent years, antagonists of the adenosine A₂_A

receptor in particular has received attention as a promising non-dopaminergic alternative (for

examples of reviews see 3, 4, 14-17] and several of these agents such as istradefylline (KW-6002),

preladenant and tozadenant have been investigated clinically with promising results. Istradefylline for

example has been approved for use as adjunctive treatment for Parkinson’s disease in Japan [18-22].

Adenosine receptors are G-protein coupled receptors and consist of four subtypes, namely A , A , A

3

1

2A

and A [23,24]. Of particular importance to Parkinson’s disease are adenosine A receptors which

2B

2A

are concentrated in the indirect striatopallidal GABAergic pathway. This pathway also expresses the

D dopamine receptor and enkephalin [25-27]. Antagonism of A receptors potentiate dopamine

2

2A

mediated responses and partly restores the imbalance between the hypoactive direct striatonigral and

hyperactive indirect striatopallidal pathways that develops in Parkinson’s disease, thus relieving

motor symptoms [14,16,28,29]. Both epidemiological and experimental data have shown that

adenosine A antagonists exert a neuroprotective effect [16]. Furthermore, it has been reported that

2A

the adenosine A antagonist KW-6002, exhibit antidepressant properties in animal models of

2

A

depression [30,31] and that adenosine A antagonists have potential in the management of

2

A

dyskinesias [29,32]. This illustrates the promise of these agents as multifactorial non-dopaminergic

treatment of Parkinson’s disease.

Adenosine A receptors on the other hand, are expressed throughout the brain, including the cortex,

1

hippocampus and striatum [23]. Since A receptor antagonism also results in motor activation in

1

animals [33,34] it has been suggested that dual antagonism of A and A receptors would act

1

2A

synergistically in improving motor deficits in Parkinson’s disease [4]. Furthermore, since the A₁

receptor also occurs in systems that are important for cognitive function, adenosine A antagonism

1

may improve cognitive deficits experienced in Parkinson’s disease, as illustrated in animals [35-37].

Several dual adenosine A /A antagonists have in fact been investigated in animals and has not only

1

2A

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shown effectiveness in improving motor disabilities [4,38-43], but has also illustrated effectiveness in

enhancing cognition [38]. In summary, dual adenosine A and A antagonists would thus not only

1

2A

treat the motor symptoms of Parkinson’s disease and potentially be neuroprotective, but may also

improve non-motor symptoms.

The 2-aminopyrimidine motif frequently occurs in compounds (e.g. 1- 6) that exhibit potent adenosine

A and/or adenosine A affinity (Figure 1) and indicates that this scaffold is privileged for

2

A

1

antagonism of these receptors [4,15,17,40-48]. Of particular relevance to this paper are the findings of

Van Veldhoven, Matasi and Shook and co-workers [40-42,46,47], who synthesised a number of 2-

aminopyrimidines (e.g. 3), indenopyrimidones (e.g. 4, 5) and indenopyrimidines (e.g. 6), respectively.

Our research group has been interested in the design, synthesis and evaluation of heterocycles as

antagonists of adenosine receptors. Based on the aforementioned findings, the aim of the present

study was to explore the necessity of the methylene bridge as present in the indenopyrimidine or

indenopyrimidone scaffolds previously synthesised [40-42,46] and to further investigate the structure-

activity relationships of the 2-aminopyrimidine scaffold for dual antagonism of adenosine A and A₂_A

1

receptors. We thus set out to synthesise firstly, a set of 2-aminopyrimidines substituted with simple

electron withdrawing and donating groups (8a - h), and secondly an amide substituted series (8j - n),

related to the indenopyrimidones (e.g. 5) as synthesised by Shook and co-workers [41]. We report

herein their affinities for adenosine A and A receptors, the results of docking selected compounds

2A

1

into the adenosine A receptor’s binding site, as well as the in vivo activities of selected compounds.

2A

2

. Chemistry

As shown in scheme 1, the 2-aminopyrimidines were readily synthesised in two or three steps, albeit

in low yields. Firstly, a Claisen-Schmidt condensation, using commercially available ketones and

aldehydes under basic conditions [49] yielded the desired intermediate chalcones (7a - i). For the

amide derivatives (8j - n), the condensation reaction was followed by an amide coupling reaction

mediated by 1,1′-carbonyldiimidazole (CDI) resulting in chalcones 7j - n. All chalcones were cyclised

with guanidine hydrochloride in the presence of sodium hydride [50] to yield the desired 2-

aminopyrimidines (8a - n), since the use of sodium hydroxide in ethanol resulted in complicated

mixtures of products.

3

. Results and discussion

The affinities of the 2-aminopyrimidines for the adenosine A₂_Aand A receptors were determined by

1

radioligand binding and are expressed as the receptor-ligand dissociation constants (Ki, nM) (Tables 1

and 2). Adenosine A₂_Areceptor affinity was determined using the non-selective adenosine antagonist,

3

6

[

H]5’-N-ethylcarboxamide-adenosine ([ H]NECA) in the presence of N -cyclopentyladenosine

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3

(

CPA), and A receptor affinity was determined using 1,3-[ H]-dipropyl-8-cyclopentylxanthine

1

3

[ H]DPCPX) [51-53].

The 2-aminopyrimidines of series 1 (8a - h) exhibited moderate to weak affinities for the adenosine

A₂_Areceptor and moderate to good affinities for A receptors with A K values ranging from

1

2A

i

approximately 3 µM (8f, 8g) to approximately 250 nM (8b, 8d, 8h) and A Ki values ranging from 23

1

nM (8a) to 650 nM (8f). These compounds all have higher affinities for the adenosine A receptor

1

than for the adenosine A₂_Areceptor, with compound 8a being the most selective (selectivity index of

3

4

2). For adenosine A₂_Areceptor affinity, the replacement of the phenyl substituent (R ) with either a

furan or methyl furan group, resulted in improved affinity (compound 8a vs. compounds 8b and 8c),

whereas the opposite was true for A affinity, where methyl furan substitution in particular proved to

1

be detrimental. When the affinities of compounds 8b and 8c, and compounds 8d and 8e are compared,

it is clear that methyl substitution of the furan ring results in decreased affinity, especially for the

3

adenosine A receptor. Based on these results, it thus appears that for R , furan substitution is

1

preferable for A₂_Aaffinity, while phenyl substitution is optimal for A receptor affinity. These results

1

are in agreement with literature since the preference of the adenosine A₂_Areceptor for furan

substitution is well documented [e.g. 46]. The affinity is apparently not significantly affected by the

2

electronic effects of the substituent on the phenyl ring (R ) as compounds 8b and 8d (A K values

2A

i

approx. 250 nM, A K values 40 - 60 nM) as well as compounds 8c and 8e (A K values approx. 400

1

i

2A

i

nM, A K values 130- 140 nM) had similar affinities for A and A receptors, respectively.

1

i

2A

1

On the other hand, the position of the substituent on the phenyl ring seems to have a significant effect

1

2

on both A and A affinity, as substitution on the meta position (8c, R = H, R = Cl) is superior to

1

2A

1

2

substitution on the para position (8g, R = Cl, R = H). A similar observation is made when the

affinities of compounds 8e and 8f are compared, where addition of a second methoxy group, in the

para position, results in much weaker A and A affinity. However, since only a limited number of

1

2A

derivatives have been synthesised, these structure-activity relationships should be seen as preliminary.

3

For compounds 8j - n, the 5-methyl-2-furanyl group was selected as the R substituent, since it was

synthetically easier to work with than furan and is also less likely to present with metabolic liabilities

[

40]. Gratifyingly, this amide series showed improved adenosine A₂_Aaffinity, while still retaining

good A affinity. The most promising candidate was compound 8m, with dual affinity for both A

1

2A

and A receptors with K values of 6.34 nM and 9.54 nM, respectively. For adenosine A affinity, the

1

i

2A

amine groups yielded the following order of affinity: piperidine > methyl piperazine > morpholine =

ethyl piperazine > pyrrolidine. Piperidine substitution was also most advantageous for A affinity,

1

while methyl piperazine substitution was least favourable. The affinities of this series of compounds

are quite similar to those reported for related arylindenopyrimidones [41], and indicate that the

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presence of the five-membered indenyl ring (e.g as present in 5 and 6) is not an absolute requirement

for dual affinity.

To gain an indication of potential cytotoxicity of the amide derivatives, the effect of these compounds

on the viability of cultured HeLa cells were measured. For this purpose, the 3-(4,5-dimethylthiazol-2-

yl)-2,5-diphenyltetrazolium bromide (MTT) cell viability assay was used [54]. Cell viability was

generally above 60% when exposed for 24 h to either 1 µM or 10 µM of test compound (Table 3).

These concentrations are almost 1000-fold higher than the reported Ki values of these compounds.

Cytotoxicity at the doses required to obtain adenosine A and A affinity should thus not be

1

2A

problematic.

Selected representatives 8m (the compound with best dual affinity, A K = 6.34 nM, A K = 9.54 nM)

2

A

i

1

i

and 8k (A K = 16.3 nM), were subjected to in vivo studies to determine the effectiveness of these

2A

i

compounds as dual adenosine A /A antagonists. For these studies the effect of the test compounds

1

2A

on haloperidol-induced catalepsy in rats was investigated [33].

As depicted in figure 2, a significant reduction in catalepsy time was observed for 8m at all

intraperitoneal (ip) administered doses while a significant reduction in catalepsy time was only

observed for the highest two doses (1 and 2 mg/kg) for compound 8k (Figures 2a and b).

The results obtained with compounds 8m and 8k are thus similar to those obtained with other

adenosine A₂_Aantagonists where catalepsy is reversed in the presence of A₂_Aantagonists or dual

adenosine A /A antagonists [33,34,43], and provides evidence of in vivo efficacy of these

2

A

1

compounds as antagonists.

In order to rationalise the results obtained in the radioligand binding studies, a docking study was

performed using CDOCKER (Discovery Studio 3.1). The synthesised compounds were docked into a

model of the binding site of the adenosine A₂_Areceptor (PDB code: 3PWH). Visual inspection of the

docked poses with most favourable CDOCKER interaction energy firstly revealed that the three-

membered ring system of all derivatives (8a - n), undergo π-π interactions with Phe168. Hydrogen

bonding interactions also occur between the exocyclic amino-group and Asn253 for most derivatives.

These interactions most likely anchor the aminopyrimidine in the binding site and are also important

binding interactions predicted for other 2-aminopyrimidine antagonists [55] (Figure 3 and 4).

Additionally, hydrogen bonding interactions were also observed with His250, Ser67, Glu169, Phe168

and Ala63 for some compounds. Interestingly, for compounds 8a - h there were two different

orientations, one where the C-6 substituent was orientated towards His250 and another where the C-4

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substituent was orientated towards His250 (Figure 3). The CDOCKER interaction energies of these

two poses were in all cases very similar. The docking results gave no clear explanation for the

observed higher affinity of the amide derivatives (8j - n) compared to the series 1 compounds (8a - h).

However, when ranked according to CDOCKER interaction energy, most of the series 2 compounds

ranked above the series 1 compounds, except for compound 8f, which ranked above compound 8n. It

would thus appear that the interaction between the amide derivatives (8j - n) and the receptor is

generally more favourable than for the “smaller” derivatives and provides some explanation for the

superior affinity of compounds 8j - n.

4

. Conclusion

This series of 2-aminopyrimidines, particularly the amide derivatives (8j - n), which are related to

previously synthesised arylindenopyrimidines, retain affinity for both adenosine A and A receptors.

1

2A

The 2-aminopyrimidine scaffold can thus be optimised and the presence of a five-membered “linker

ring” is not an absolute requirement for affinity. In vivo activity is indicative of adenosine antagonism

rather than agonism and has been illustrated for compounds 8k and 8m with A Ki values of 16.3 nM

2

A

and 6.34 nM and A Ki values of 136 nM and 9.54 nM, respectively. Docking results indicate that

1

these compounds are predicted to bind in a fashion similar to that illustrated for other compounds of

the 2-aminopyrimidine class.

5

. Experimental section

5

.1 Chemistry

Chemical reagents were purchased from Sigma-Aldrich, and used without further purification.

Reactions were routinely monitored on TLC using precoated Kieselgel 60 F254 sheets with ethyl

acetate: petroleum ether (1:4) as mobile phase for series 1 (8a - h) and dichloromethane: methanol

(

9:1) as mobile phase for series 2 compounds (8j - n). Melting points were determined using a Buchi

B-545 apparatus, and are uncorrected. Mass spectra were obtained on a dual focusing DFS magnetic

sector mass spectrometer in EI+ mode. The mass spectrum for compound 7n was obtained with a

Bruker micrOTOF-QII mass spectrometer in atmospheric-pressure chemical ionisation (APCI) mode.

1

13

Proton ( H) and carbon ( C) NMR spectra were recorded on a Bruker Avance III 600 spectrometer at

frequencies of 600 MHz and 150 MHz, respectively. Samples were dissolved in either

1

deuterochloroform (CDCl ) or deuterated dimethylsulfoxide (DMSO-d6). H NMR data are reported

3

in parts per million (ppm) and the following abbreviations are used: s (singlet), br s (broad singlet), d

(

doublet), br d (broad doublet), dd (doublet of doublets), t (triplet), br t (broad triplet), q (quartet), p

pentet/quintet) or m (multiplet). Chemical shifts are referenced to the residual solvent signal (CDCl₃

(

1

13

1

13

7

.26 and 77.0 ppm for H and C respectively; DMSO-d6: 2.5 and 39.5 ppm for H and C,

1

13

respectively). Assignments were based on data obtained from 1D ( H, C, DEPT) and 2D (HSQC,

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HMBC, COSY) NMR experiments. HPLC analyses were conducted with an Agilent 1100 HPLC

system equipped with a quaternary pump and an Agilent 1100 series diode array detector. A Venusil

XBP C18 column (4.60 × 150 mm, 5 µm) was used with a solvent gradient program (30% acetonitrile

and 70% MilliQ water initially) at a flow rate of 1 ml/min. The concentration of acetonitrile in the

mobile phase was linearly increased up to 85% over a period of 5 min. Each HPLC run lasted 15 min

and a time period of 5 min was allowed for equilibration between runs. The test compound was

injected (20 µl, 1 mM) into the HPLC system and the eluent was monitored at a wavelength of 254

nm.

5

.1.1 Procedure for the synthesis of 3-[(1E)-3-(5-methylfuran-2-yl)-3-oxoprop-1-en-1-yl]benzoic acid

(

7i)

A solution of 4% (w/v) sodium hydroxide (34 mmol) was added to a suspension of 3-formylbenzoic

acid (17 mmol) and 1-(5-methyl-2-furyl)ethanone (17 mmol) in methanol (100 ml). The mixture was

stirred at room temperature for 24 h and acidified with concentrated hydrochloric acid to a pH of 1-2.

The precipitate that formed was filtered, rinsed with water and recrystallised from methanol to afford

7

i.

5

.1.1.1 3-[(1E)-3-(5-methylfuran-2-yl)-3-oxoprop-1-en-1-yl]benzoic acid (7i)

1

Yield 68%; Pale yellow crystals; mp 191.7-194.1 °C (methanol); H NMR (600 MHz, DMSO-d6) δ

13.20 (br s, 1H, OH), 8.33 (br s, 1H, H-2′), 8.07 (br d, J = 7.8 Hz, 1H, H-6′), 7.98 (dt, J = 1.4, 7.7 Hz,

1H, H-4′), 7.82 (d, J = 3.5 Hz, 1H, H-3′′), 7.75 (d, J = 16.0 Hz, 1H, H-7 or H-8), 7.71 (d, J = 16.0 Hz,

1H, H-7 or H-8), 7.57 (t, J = 7.7 Hz, 1H, H-5′), 6.42 (dd, J = 1.1, 3.5 Hz, 1H, H-4′′), 2.40 (s, 3H,

13

CH ). C NMR (151 MHz, DMSO-d6) δ 175.6 (C-1), 167.0 (acid C=O), 158.8 (C-5′′), 151.8 (C-2′′),

3

1

2

41.2 (C-3), 135.0 (C-1′), 132.8 (C-6′), 131.6 (C-3′), 131.0 (C-4′), 129.2 (C-2′, C-5′), 123.1 (C-2),

21.9 (C-3′′), 109.6 (C-4′′), 13.8 (CH ). EI-HRMS m/z: calcd for C H O , 256.07356, found

3

15 12

4

56.07292; Purity (HPLC): 100%.

5

.1.2 General procedure for the synthesis of chalcones (7j – 7n)

,1'-Carbonyldiimidazole (CDI) (7.0 mmol) was added to a suspension of the acid (7i) (5.8 mmol) in

1

dichloromethane (70 ml). The reaction mixture was stirred under nitrogen at room temperature for 2 h

and the appropriate amine (7.0 mmol) was added. The mixture was then stirred for a further 3 h. The

reaction was quenched by the addition of brine and the aqueous phase extracted with dichloromethane

(

2 × 20 ml). The combined organic fractions were washed once with saturated sodium hydrogen

carbonate and twice with brine. The organic fraction was concentrated (in vacuo), purified with

column chromatography [dichloromethane: methanol (98:2)] and recrystallised from methanol.

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5

.1.2.1 (2E)-1-(5-methylfuran-2-yl)-3-[3-(morpholine-4-carbonyl)phenyl]prop-2-en-1-one (7j)

The title compound was prepared from (1E)-1-(5-methylfuran-2-yl)-3-[3-oxoprop-1-en-1-yl]benzoic

1

acid (7i) and morpholine in a yield of 54%: mp 149.1-149.9 °C (methanol), pale yellow crystals. H

NMR (600 MHz, CDCl ) δ 7.80 (d, J = 15.8 Hz, 1H, H-3), 7.69 (br t, J = 1.6 Hz, 1H, H-2′), 7.65 (dt, J

3

=

1.6, 7.7 Hz, 1H, H-6′), 7.47 – 7.36 (m, 3H, H-2, H-5′, H-4′), 7.25 (br d, J = 3.4 Hz, 1H, H-3′′), 6.21

13

(

dd, J = 1.0, 3.4 Hz, 1H, H-4′′), 3.93 – 3.31 (m, 8H, 4 x morpholine CH ), 2.43 (s, 3H, CH ). C

2 3

NMR (151 MHz, CDCl ) δ 176.8 (C-1), 169.6 (amide C=O), 158.4 (C-5′′), 152.3 (C-2′′), 141.8 (C-3),

3

1

36.0 (C-1′ or C-3′), 135.4 (C-1′ or C-3′), 129.9 (C-6′), 129.1, 128.5 (C-4′, C-5′)*, 126.6 (C-2′), 122.4

(

C-2), 119.8 (C-3′′), 109.4 (C-4′′), 66.8, 48.2, 42.5 (morpholine CH ). EI-HRMS m/z: calcd for

2

C H NO , 325.13141, found 325.13031; Purity (HPLC): 100%. * In no particular order.

19

4

5

.1.2.2 (2E)-1-(5-methylfuran-2-yl)-3-[3-(4-methylpiperazine-1-carbonyl)phenyl]prop-2-en-1-one

(

7k)

The title compound was prepared from (1E)-1-(5-methylfuran-2-yl)-3-[3-oxoprop-1-en-1-yl]benzoic

acid (7i) and 1-methylpiperazine in a yield of 67 %: mp 131.0-132.2 °C (methanol), yellow crystals.

1

H NMR (600 MHz, CDCl ) δ 7.79 (d, J = 15.8 Hz, 1H, H-3), 7.68 (br t, J = 1.6 Hz, 1H, H-2′), 7.63

3

(

dt, J = 1.6, 7.7 Hz, 1H, H-6′), 7.45 – 7.35 (m, 3H, H-4′, H-5′, H-2), 7.25 (d, J = 3.5 Hz, 1H, H-3′′),

6

1

.20 (d, J = 3.5 Hz, 1H, H-4′′), 3.81 (br s, 2H, CONCH ), 3.44 (br s, 2H, CONCH ), 2.50-2.27 (m,

2 2

1

3

0H, 2 x CH NCH , 2 x CH ). C NMR (151 MHz, CDCl ) δ 176.8 (C-1), 169.5 (amide C=O), 158.3

2

3

(

C-5′′), 152.3 (C-2′′), 141.9 (C-3), 136.5 (C-1′ or C-3′), 135.3 (C-1′ or C-3′), 129.7 (C-6′), 129.0 (C-4′

or C-5′), 128.5 (C-4′ or C-5′), 126.6 (C-2′), 122.3 (C-2), 119.8 (C-3′′), 109.4 (C-4′′), 55.2

CH NCH ), 54.6 (CH NCH ), 47.6 (CONCH ), 45.9 (piperazine CH ,), 42.0 (CONCH ), 14.1 (furan

(

2

3

2

3

2

3

2

CH ,). EI-HRMS m/z: calcd for C H N O , 338.16304, found 338.16270; Purity (HPLC): 97%.

3

20 22

2

3

5

.1.2.3 (2E)-3-[3-(4-ethylpiperazine-1-carbonyl)phenyl]-1-(5-methylfuran-2-yl)prop-2-en-1-one (7l)

The title compound was prepared from (1E)-1-(5-methylfuran-2-yl)-3-[3-oxoprop-1-en-1-yl]benzoic

1

acid (7i) and 1-ethylpiperazine in a yield of 63%: mp 98.5-98.8 °C (methanol), orange solid. H NMR

(

600 MHz, CDCl ) δ 7.80 (d, J = 15.9 Hz, 1H, H-3), 7.68 (br s, 1H, H-2′), 7.63 (br d, J = 7.6 Hz, 1H,

3

H-6′), 7.45 – 7.36 (m, 3H, H-4′, H-5′, H-2), 7.25 (d, J = 3.5 Hz, 1H, H-3′′), 6.21 (d, J = 3.5 Hz, 1H,

H-4′′), 3.81 (br s, 2H, CONCH ), 3.44 (br s, 2H, CONCH ), 2.55 – 2.33 (m, 9H, 2 x CH NCH ,

2

3

1

3

NCH CH , furan CH ), 1.08 (t, J = 7.2 Hz, 3H, NCH CH ). C NMR (151 MHz, CDCl ) δ 176.8 (C-

2

3

2

3

1

), 169.4 (amide C=O), 158.3 (C-5′′), 152.3 (C-2′′), 141.9 (C-3), 136.5 (C-1′ or C-3′), 135.3 (C-1′ or

C-3′), 129.8 (C-6′), 129.0 (C-4′ or C-5′), 128.5 (C-4′ or C-5′), 126.6 (C-2′), 122.3 (C-2), 119.8 (C-3′′),

09.4 (C-4′′), 53.1 (CH NCH ), 52.3 (CH NCH ), 52.2 (NCH CH ), 47.7 (CONCH ), 42.1

1

2

3

2

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(

CONCH ), 14.1 (furan CH ), 11.8 (piperazine CH ). EI-HRMS m/z: calcd for C H N O ,

2

3

21 24

2

3

52.17869, found 352.17790; Purity (HPLC): 98%.

5

.1.2.4 (2E)-1-(5-methylfuran-2-yl)-3-[3-(piperidine-1-carbonyl)phenyl]prop-2-en-1-one (7m)

The title compound was prepared from (1E)-1-(5-methylfuran-2-yl)-3-[3-oxoprop-1-en-1-yl]benzoic

acid (7i) and piperidine in a yield of 78%: mp 144.7 – 145.1 144.2-144.4 °C (methanol), pale yellow

1

solid. H NMR (600 MHz, CDCl ) δ 7.81 (d, J = 15.8 Hz, 1H, H-3), 7.68 (br s, 1H, H-2′), 7.63 (br d, J

3

=

7.8 Hz, 1H, H-6′), 7.45 – 7.35 (m, 3H, H-4′, H-5′, H-2), 7.25 (d, 1H, J = 3.4 Hz, H-3′′), 6.21 (d, J =

3

6

5

.4 Hz, 1H, H-4′′), 3.71 (s, 2H, CONCH ), 3.33 (s, 2H, CONCH ), 2.43 (s, 3H, CH ), 1.71 – 1.42 (m,

2

3

1

3

H, 3 x piperidine CH ). C NMR (151 MHz, CDCl ) δ 176.9 (C-9), 169.5 (amide C=O), 158.3 (C-

2

3

′′), 152.3 (C-2′′), 142.1 (C-3), 137.2 (C-3′), 135.2 (C-1′), 129.5 (C-6′), 128.9 (C-4′ or C-5′), 128.3

(

C-4′ or C-5′), 126.4 (C-2′), 122.2 (C-2), 119.8 (C-3′′), 109.4 (C-4′′), 48.7 (CONCH ), 43.1

2

(

CONCH ), 26.5, 25.5, 24.5 (3 x piperidine CH ), 14.2 (CH ). EI-HRMS m/z: calcd for C H NO ,

2

3

20 21

3

23.15214, found 323.15116; Purity (HPLC): 96%.

5

.1.2.5 (2E)-1-(5-methylfuran-2-yl)-3-[3-(pyrrolidine-1-carbonyl)phenyl]prop-2-en-1-one (7n)

The title compound was prepared from (1E)-1-(5-methylfuran-2-yl)-3-[3-oxoprop-1-en-1-yl]benzoic

1

acid (7i) and pyrrolidine in a yield of 74%: mp 120.8-122.6 °C (methanol), yellow crystals. H NMR

(

600 MHz, CDCl ) δ 7.82 – 7.76 (m, 2H, H-2′, H-3), 7.62 (br d, J = 7.7 Hz, 1H, H-6′), 7.49 (br d, J =

3

7

1

.7 Hz, 1H, H-4′), 7.44 – 7.37 (m, 2H, H-5′, H-2), 7.24 (d, J = 3.5 Hz, 1H, H-3′′), 6.2 (d, J = 3.5 Hz,

H, H-4′′), 3.63 (t, J = 7.1 Hz, 2H, CONCH ), 3.41 (t, J = 6.6 Hz, 2H, CONCH ), 2.41 (s, 3H, CH ),

2

3

1

3

.95 (p, J = 6.9 Hz, 2H, CH CH CH ), 1.86 (p, J = 6.7 Hz, 2H, CH CH CH ). C NMR (151 MHz,

2

CDCl ) δ 176.9 (C-1), 168.9 (amide C=O), 158.3 (C-5′′), 152.3 (C-2′′), 142.1 (C-3), 137.8 (C-3′),

3

1

35.0 (C-1′), 129.9 (C-6′), 128.8 (C-4′ or C-5′), 128.6 (C-4′ or C-5′), 126.6 (C-2′), 122.1 (C-2), 119.8

(

C-3′′), 109.4 (C-4′′), 49.5 (CONCH ), 46.2 (CONCH ), 26.3 (CH CH CH ), 24.3 (CH CH CH ),

2

+

1

4.1 (CH ). APCI-HRMS m/z: calcd for C H NO (M + H) , 310.1438, found 310.1448; Purity

3 19 19 3

(

HPLC): 93%.

5

.1.3 General procedure for the synthesis of 2-aminopyrimidines

Guanidine hydrochloride (4.6 mmol) was dissolved in a small amount of DMF (15 ml), and the

appropriate chalcone (3.1 mmol) and sodium hydride (9.2 mmol) were added while stirring. The

reaction mixture was heated (110 ˚C) for 24 h under nitrogen, allowed to cool to room temperature

and then diluted with equal volumes of ethyl acetate and water. The aqueous phase was extracted with

ethyl acetate (twice) and the organic layers were combined. All traces of DMF were removed by

washing the combined organic layers with water (4 times). The organic layer was concentrated in

ACCEPTED MANUSCRIPT

vacuo and the crude product was purified with column chromatography [petroleum ether: ethyl

acetate (4:1)] and recrystallised from ethanol.

5

.1.3.1 4-(3-chlorophenyl)-6-phenylpyrimidin-2-amine (8a)

The title compound was prepared from (2E)-3-(3-chlorophenyl)-1-phenylprop-2-en-1-one (7a) in a

1

yield of 15%: mp 128.6-131.4 °C (ethanol), (lit. 132 – 133 °C) [56] white solid. H NMR (600 MHz,

CDCl ) δ 8.05 (m, 3H, Ar-H), 7.92 (dt, J = 1.5, 7.6 Hz, 1H, H-4'/6'), 7.53 – 7.39 (m, 6H, Ar-H, H-5),

3

1

3

5

1

9

.42 (s, 2H, NH₂). C NMR (151 MHz, CDCl ) δ 166.5, 164.64, 163.6 (C-2, C-4, C-6)*, 139.5,

3

37.4, 134.8 (C-1′, C-1′′, C-3′′)*, 130.6, 130.3, 130.0, 128.8 (2C), 127.2, 127.1 (2C), 125.1 (Ar-C),

04.1 (C-5). EI-HRMS m/z: calcd for C H ClN , 281.07198, found 281.07167; Purity (HPLC):

1

6

12

3

9%.* In no particular order.

5

.1.3.2 4-(3-chlorophenyl)-6-(furan-2-yl)pyrimidin-2-amine (8b)

The title compound was prepared from (2E)-3-(3-chlorophenyl)-1-(furan-2-yl)prop-2-en-1-one (7b) in

1

a yield of 22%: mp 144.1-144.4 °C (ethanol), light yellow solid. H NMR (600 MHz, CDCl ) δ 8.05

3

(

br t, J = 1.9 Hz, 1H, H-2'), 7.91 (dt, J = 1.45, 7.6 Hz, 1H, H-6'), 7.59 (dd, J = 0.8, 1.8 Hz, 1H, H-5''),

7

.42 (ddd, J = 8.0, 2.1, 1.2 Hz, 1H, H-4'), 7.38 (br t, J = 7.8 Hz, 1H, H-5'), 7.34 (s, 1H, H-5), 7.20 (dd,

1

3

J = 0.8, 3.5 Hz, 1H, H-3''), 6.56 (dd, J = 1.8, 3.5 Hz, 1H, H-4''), 5.41 (s, 2H, NH ). C NMR (151

2

MHz, CDCl ) δ 164.6 (C-2 or C-4), 163.4 (C-2 or C-4), 157.24 (C-6), 152.0 (C-2''), 144.6 (C-5'' ),

3

1

39.3 (C-1'), 134.8 (C-3'), 130.4 (C-4' or C-5'), 129.9 (C-4' or C-5'), 127.2 (C-2'), 125.1 (C-6'), 112.3

(

C-3'' or C-4''), 111.80 (C-3'' or C-4′′), 101.94 (C-5). EI-HRMS m/z: calcd for C H ClN O,

1

4

10

3

2

71.05124, found 271.05049; Purity (HPLC): 98%.

5

.1.3.3 4-(3-chlorophenyl)-6-(5-methylfuran-2-yl)pyrimidin-2-amine (8c)

The title compound was prepared from (2E)-3-(3-chlorophenyl)-1-(5-methylfuran-2-yl)prop-2-en-1-

1

one (7c) in a yield of 45%: mp 170.5-172.1°C (ethanol), yellow solid. H NMR (600 MHz, CDCl ) δ

3

8

7

.05 (br s, 1H, H-2'), 7.92 (d, J = 7.6 Hz, 1H, H-6'), 7.46 – 7.37 (m, 2H, H-4', H-5'), 7.31 (s, 1H, H-5),

1

3

.11 (br s, 1H, H-3''), 6.17 (br s, 1H, H-4''), 5.26 (s, 2H, NH ), 2.43 (s, 3H, CH ). C NMR (151 MHz,

2

3

CDCl ) δ 164.3 (C-2 or C-4), 163.3 (C-2 or C-4), 157.3 (C-6), 155.4 (C-5''), 150.5 (C-2''), 139.4 (C-

3

1

2

'), 134.8 (C-3'), 130.3 (C-4' or C-5'), 129.9 (C-4' or C-5'), 127.2 (C-2'), 125.1 (C-6'), 113.3 (C-3''),

08.8 (C-4''), 101.6 (C-5), 14.0 (CH ). EI-HRMS m/z: calcd for C H ON Cl, 285.06690, found

3

15 12

3

85.06556; Purity (HPLC): 100%.

5

.1.3.4 4-(3-methoxyphenyl)-6-(furan-2-yl)pyrimidin-2-amine (8d)

The title compound was prepared from (2E)-1-(furan-2-yl)-3-(3-methoxyphenyl)prop-2-en-1-one (7d)

1

in a yield of 19%: mp 113.8-116.8 °C (ethanol), dark brown crystals. H NMR (600 MHz, CDCl ) δ

3

ACCEPTED MANUSCRIPT

7

.64 – 7.57 (m, 3H, H-2',H-4',H-5''), 7.40 – 7.37 (m, 2H, H-5, H-5'), 7.19 (dd, J = 0.82, 3.4 Hz, 1H,

H-3''), 7.03 (ddd, J = 0.96, 2.59, 8.1 Hz, 1H, H-6'), 6.55 (dd, J = 1.7, 3.4 Hz, 1H, H-4''), 5.41 (s, 2H,

1

3

NH ), 3.89 (s, 3H, OCH ). C NMR (151 MHz, CDCl ) δ 165.92 (C-4), 163.35 (C-2), 159.96 (C-3'),

2

3

1

56.43 (C-6), 152.15 (C-2''), 144.51 (C-5''), 138.89 (C-1'), 129.68 (C-5'), 119.47 (C-4'), 116.54 (C-6'),

12.21 (C-2'/C-3''/C-4''), 112.21 (C-2'/C-3''/C-4''), 112.07 (C-2'/C-3''/C-4''), 102.18 (C-5), 55.37

1

(

OCH ). EI-HRMS m/z: calcd for C H N O , 267.10078, found 267.09949; Purity (HPLC): 92%.

3 15 1 3 2

5

.1.3.5 4-(3-methoxyphenyl)-6-(5-methylfuran-2-yl)pyrimidin-2-amine (8e)

The title compound was prepared from (2E)-3-(3-methoxyphenyl)-1-(5-methylfuran-2-yl)prop-2-en-

1

-one (7e) in a yield of 43%: mp 149.0 -150.6 °C (ethanol), light orange crystals. H NMR (600 MHz,

CDCl ) δ 7.64 – 7.58 (m, 2H, H-2', H-4'), 7.38 (br t, J = 7.9 Hz, 1H, H-5'), 7.33 (s, 1H, H-5), 7.09 (d,

3

J = 3.3 Hz, 1H, H-3''), 7.02 (dd, J = 2.6, 8.2 Hz, 1H, H-6'), 6.15 (dd, J = 0.9, 3.3 Hz, 1H, H-4''), 5.44

1

3

(

s, 2H, NH ), 3.88 (s, 3H, OCH ), 2.44 (s, 3H, CH ). C NMR (151 MHz, CDCl ) δ 165.6 (C-4),

2 3 3 3

1

63.4 (C-2), 159.9 (C-3'), 157.0 (C-6), 155.1 (C-5''), 150.6 (C-2''), 139.1 (C-1'), 129.6 (C-5'), 119.4

(

C-4'), 116.3 (C-6'), 113.0 (C-3''), 112.1 (C-2'), 108.7 (C-4''), 101.7 (C-5), 55.4 (OCH ), 14.0 (CH ).

3 3

EI-HRMS m/z: calcd for C H N O , 281.11643, found 281.11556 ; Purity (HPLC): 100%.

16

15

3

2

5

.1.3.6 4-(3,4-dimethoxyphenyl)-6-(5-methylfuran-2-yl)pyrimidin-2-amine (8f)

The title compound was prepared from (2E)-3-(3,4-dimethoxyphenyl)-1-(5-methylfuran-2-yl)prop-2-

1

en-1-one (7f) in a yield of 27%: mp 167.4-168.1 °C (ethanol), dark yellow crystals. H NMR (600

MHz, CDCl ) δ 7.68 (d, J = 2.0 Hz, 1H, H-2′), 7.61 (dd, J = 2.1, 8.4 Hz, 1H, H-6'), 7.29 (s, 1H, H-5),

3

7

5

.08 (d, J = 3.3 Hz, 1H, H-3''), 6.93 (d, J = 8.4 Hz, 1H, H-5'), 6.15 (dd, J = 0.8, 3.3 Hz, 1H, H-4''),

13

.35 (s, 2H, NH ), 3.98 (s, 3H, OCH ), 3.93 (s, 3H, OCH ), 2.42 (s, 3H, CH ). C NMR (151 MHz,

2

3

CDCl ) δ 165.2 (C-4), 163.3 (C-2), 156.7 (C-6), 155.0 (C-5''), 151.1, 150.6 (C-4' or C-3' and C-2′′)*,

3

1

3

49.0 (C-3′ or C-4′), 130.2 (C-1'), 120.1 (C-6'), 112.8 (C-3''), 110.7 (C-5'), 109.8 (C-2'), 108.7 (C-4''),

00.8 (C-5), 56.0 (OCH ), 55.9 (OCH ), 14.00 (CH ). EI-HRMS m/z: calcd for C H N O ,

3

17 17

3

11.12699, found 311.12618; Purity (HPLC): 100%* In no particular order.

5

.1.3.7 4-(4-chlorophenyl)-6-(5-methylfuran-2-yl)pyrimidin-2-amine (8g)

The title compound was prepared from (2E)-3-(4-chlorophenyl)-1-(5-methylfuran-2-yl)prop-2-en-1-

1

one (7g) in a yield of 30%: mp 204.6 – 205.8 205-205.1°C (ethanol), dark yellow powder. H NMR

(

600 MHz, DMSO-d6) δ 8.14 (d, J = 8.6 Hz, 2H, H-2', H-6'), 7.56 (d, J = 8.6 Hz, 2H, H-3', H-5'), 7.39

(

s, 1H, H-5), 7.21 (d, J = 3.3 Hz, 1H, H-3''), 6.76 (s, 2H, NH ), 6.32 (dd, J = 1.0, 3.3 Hz, 1H, H-4''),

2

1

3

2

.37 (s, 3H, CH ). C NMR (151 MHz, DMSO-d6) δ 163.9 (C-2 or C-4), 163.1 (C-2 or C-4), 156.7

3

(

C-6), 154.7 (C-5''), 150.4 (C-2''), 136.0 (C-1'), 135.2 (C-4'), 128.8 (C-2', C-6' or C-3', C-5'), 128.6 (C-

ACCEPTED MANUSCRIPT

2

', C-6' or C-3', C-5'), 113.3 (C-3''), 108.9 (C-4''), 99.4 (C-5), 13.7 (CH ). EI-HRMS m/z: calcd for

3

C H ClN O, 285.06689, found 285.06633; Purity (HPLC): 99%.

15

12

3

5

.1.3.8 4-(4-fluorophenyl)-6-(furan-2-yl)pyrimidin-2-amine (8h)

The title compound was prepared from (2E)-1-(4-fluorophenyl)-3-(furan-2-yl)prop-2-en-1-one (7h) in

1

a yield of 41%: mp 162.3-162.5 °C (lit. 170 °C ) [57] (ethanol), faded yellow crystals. H NMR (600

MHz, CDCl ) δ 8.09 – 8.03 (m, 2H, H-2',H-6'), 7.59 (dd, J = 0.8, 1.8 Hz, 1H, H-5''), 7.37 (s, 1H, H-5),

3

13

7

.19 – 7.13 (m, 3H, H-3'', H-3', H-5'), 6.57 (dd, J = 1.8, 3.4 Hz, 1H, H-4''), 5.28 (s, 2H, NH ). C

2

NMR (151 MHz, CDCl ) δ 165.0 (C-2 or C-4), 164.4 (d, J = 250.5 Hz, C-4′), 163.3 (C-2 or C-4),

3

C-F

1

57.1 (C-6), 152.2 (C-2''), 144.6 (C-5''), 133.5 (d, J_C_-_F= 3.5 Hz, C-1'), 129.1 (d, J_C_-_F= 8.5 Hz C-2', C-

'), 115.7 (d, J_C_-_F= 21.3 Hz, C-3′, C-5′), 112.3 (C-3''), 111.6 (C-4''), 101.7 (C-5). EI-HRMS m/z:

6

calcd for C H FN O, 255.08079, found 255.07982; Purity (HPLC): 100%.

14

10

3

5

.1.3.9 4-(5-methylfuran-2-yl)-6-[3-(morpholine-4-carbonyl)phenyl]pyrimidin-2-amine (8j)

The title compound was prepared from (2E)-1-(5-methylfuran-2-yl)-3-[3-(morpholine-4-

carbonyl)phenyl]prop-2-en-1-one (7j) in a yield of 20%: mp 205.9 – 206.4 205-205.1 °C (methanol),

1

yellow solid. H NMR (600 MHz, CDCl ) δ 8.11 (m, 2H, H-2', H-6'), 7.55 – 7.46 (m, 2H, H-4', H-5'),

3

7

3

1

.35 (s, 1H, H-5), 7.09 (d, J = 3.4 Hz, 1H, H-3''), 6.16 (br d, J = 3.4 Hz, 1H, H-4''), 5.32 (s, 2H, NH₂),

1

3

.87 – 3.43 (m, 8H, morpholine CH ), 2.42 (s, 3H, CH ). C NMR (151 MHz, CDCl ) δ 170.0 (C=O),

2

3

64.6 (C-2 or C-6), 163.4 (C-2 or C-6), 157.3 (C-4), 155.3 (C-5''), 150.4 (C-2''), 138.1 (C-1'), 135.7

(

C-3'), 128.8, 128.7, 128.4, 125.9 (C-2′, C-4′, C-5', C-6')*, 113.2 (C-3''), 108.7 (C-4''), 101.5 (C-5),

6.8, 48.2, 42.6 (morpholine CH ) 14.0 (CH ). EI-HRMS m/z: calcd for C H N O , 364.15354,

6

2

3

20 20

4

3

found 364.15292; Purity (HPLC): 92%. * In no particular order.

5

.1.3.10 4-(5-methylfuran-2-yl)-6-[3-(4-methylpiperazine-1-carbonyl)phenyl]pyrimidin-2-amine (8k)

The title compound was prepared from (2E)-1-(5-methylfuran-2-yl)-3-[3-(4-methylpiperazine-1-

1

carbonyl)phenyl]prop-2-en-1-one (7k) in a yield of 57%: 194.3-194.4 °C (methanol), cream solid. H

NMR (600 MHz, CDCl ) δ 8.13 – 8.07 (m, 2H, H-2', H-6'), 7.53 – 7.45 (m, 2H, H-4', H-5'), 7.35 (s,

3

1

H, H-5), 7.09 (d, J = 3.4 Hz, 1H, H-3''), 6.15 (br d, J = 3.4 Hz, 1H, H-4''), 5.30 (s, 2H, NH ), 3.83 (br

2

1

3

s, 2H, CONCH ), 3.46 (br s, 2H, CONCH ), 2.60 – 2.19 (m, 10H, 2 x CH NCH , 2 x CH ). C NMR

2

3

(

151 MHz, CDCl ) δ 169.8 (C=O), 164.7 (C-2 or C-6), 163.4 (C-2 or C-6), 157.3 (C-4), 155.3 (C-5''),

3

1

50.5 (C-2''), 138.1 (C-1'), 136.2 (C-3'), 128.8, 128.7, 128.2, 125.9 (C-2', C-4′, C-5′, C-6′)*, 113.2 (C-

3

''), 108.8 (C-4''), 101.6 (C-5), 55.2 (CH NCH ), 54.6 (CH NCH ), 47.7 (CONCH ), 46.0 (piperazine

2

3

2

3

2

CH ), 42.1 (CONCH ), 14.0 (furan CH ). EI-HRMS m/z: calcd for C H N O , 377.18518, found

3

2

3

21 23

5

2

3

77.18422; Purity (HPLC): 85%. *In no particular order.

ACCEPTED MANUSCRIPT

5

.1.3.11 4-(5-methylfuran-2-yl)-6-[3-(4-ethylpiperazine-1-carbonyl)phenyl]pyrimidin-2-amine (8l)

The title compound was prepared from (2E)-3-[3-(4-ethylpiperazine-1-carbonyl)phenyl]-1-(5-

methylfuran-2-yl)prop-2-en-1-one (7l) in a yield of 16%: 178.1-178.4 °C (methanol), light orange

1

solid. H NMR (600 MHz, CDCl ) δ 8.12 – 8.07 (m, 2H, H-2', H-6'), 7.52 – 7.44 (m, 2H, H-4', H-5'),

3

7

.34 (s, 1H, H-5), 7.08 (d, J = 3.4 Hz, 1H, H-3''), 6.14 (dd, J = 1.0, 3.4 Hz, 1H, H-4''), 5.38 (s, 2H,

NH ), 3.83 (br s, 2H, CONCH ), 3.46 (s, 2H, CONCH ), 2.62 – 2.21 (m, 9H, 2 x CH NCH ,

2

3

1

3

NCH CH , furan CH ), 1.07 (t, J = 7.4 Hz, 3H, NCH CH ). C NMR (151 MHz, CDCl ) δ 169.7

2

3

2

3

(

C=O), 164.7 (C-2 or C-6), 163.4 (C-2 or C-6), 157.2 (C-4), 155.2 (C-5''), 150.4 (C-2''), 138.0 (C-1'),

1

36.2 (C-3'), 128.7, 128.7, 128.2, 125.9 (C-2', C-4', C-5', C-6')*, 113.1 (C-3''), 108.7 (C-4''), 101.5 (C-

), 53.0 (CH NCH ), 52.3 (CH NCH ), 52.2 (NCH CH ), 47.7 (CONCH ), 42.1 (CONCH ), 14.0

5

2

3

2

(

furan CH ), 11.8 (NCH CH ). EI-HRMS m/z: calcd for C H N O , 391.20083, found 391.20052;

3 2 3 22 25 5 2

Purity (HPLC): 100%. *In no particular order.

5

.1.3.12 4-(5-methylfuran-2-yl)-6-[3-(piperidine-1-carbonyl)phenyl]pyrimidin-2-amine (8m)

The title compound was prepared from (2E)-1-(5-methylfuran-2-yl)-3-[3-(piperidine-1-

carbonyl)phenyl]prop-2-en-1-one (7m) in a yield of 56%: 179.2-180.5 °C (methanol), orange crystals.

1

H NMR (600 MHz, CDCl ) δ 8.12 – 8.05 (m, 2H, H-2', H-6'), 7.52 – 7.44 (m, 2H, H-4', H-5'), 7.35

3

(

s, 1H, H-5), 7.09 (d, J = 3.4 Hz, 1H, H-3''), 6.15 (dd, J = 1.1, 3.4 Hz, 1H, H-4''), 5.35 (s, 2H, NH₂),

.73 (br s, 2H, CONCH ), 3.36 (br s, 2H, CONCH ), 2.42 (s, 3H, CH ), 1.77 – 1.39 (m, 6H, 3 x

3

2

3

1

3

piperidine CH ). C NMR (151 MHz, CDCl ) δ 169.8 (C=O), 164.9 (C-2 or C-6), 163.4 (C-2 or C-6),

2

3

1

4

2

3

57.2 (C-4), 155.2 (C-5''), 150.4 (C-2''), 137.9 (C-1'), 136.8 (C-3'), 128.7, 128.5, 127.9 125.7 (C-2', C-

', C-5′, C-6′), 113.1 (C-3''), 108.7 (C-4''), 101.6 (C-5), 48.8 (CONCH ), 43.1 (CONCH ), 26.5, 25.5,

2

4.5 (3 x piperidine CH ), 14.00 (CH ). EI-HRMS m/z: calcd for C H N O , 362.17428, found

2

3

21 22

4

2

62.17292; Purity (HPLC): 97%.

5

.1.3.13 4-(5-methylfuran-2-yl)-6-[3-(pyrrolidine-1-carbonyl)phenyl]pyrimidin-2-amine (8n)

The title compound was prepared from (2E)-1-(5-methylfuran-2-yl)-3-[3-(pyrrolidine-1-

carbonyl)phenyl]prop-2-en-1-one (7n) in a yield of 27%: 212.7 – 213.4 213.7-213.8 °C (methanol),

1

yellow crystals. H NMR (600 MHz, CDCl ) δ 8.20 (t, J = 1.8 Hz, 1H, H-2'), 8.10 (dt, J = 1.5, 7.8 Hz,

3

1

H, H-6'), 7.59 (dt, J = 1.4, 7.7 Hz, 1H, H-4'), 7.49 (t, J = 7.7 Hz, 1H, H-5'), 7.36 (s, 1H, H-5), 7.08

(

d, J = 3.4 Hz, 1H, H-3''), 6.14 (dd, J = 1.2, 3.3 Hz, 1H, H-4''), 5.37 (s, 2H, NH ), 3.66 (t, J = 7.0 Hz,

2

H, CONCH ), 3.44 (t, J = 6.7 Hz, 2H, CONCH ), 2.41 (s, 3H, CH ), 1.95 (p, J = 7.0 Hz, 2H,

2 2 3

1

3

CH CH CH ), 1.86 (p, J = 6.7 Hz, 2H, CH CH CH ). C NMR (151 MHz, CDCl ) δ 169.2 (C=O),

2

3

1

64.8 (C-2 or C-6), 163.4 (C-2 or C-6), 157.2 (C-4), 155.2 (C-4''), 150.5 (C-1''), 137.7 (C-1′ or C-3′),

37.6 (C-1' or C-3'), 128.8, 128.5, 128.3, (C-4', C-5', C-6')*, 125.9 (C-2'), 113.1 (C-3''), 108.7 (C-4''),

01.5 (C-5), 49.6 (CONCH ), 46.2 (CONCH ), 26.3 (CH CH CH ), 24.4 (CH CH CH ), 14.0 (CH ).

2

3

ACCEPTED MANUSCRIPT

EI-HRMS m/z: calcd for C H N O , 348.15863, found 348.15730; Purity (HPLC): 95%. *In no

20

4

2

particular order.

5

.2 Biological methods

.2.1.Materials

6

Adenosine deaminase (type X from calf spleen), N -cyclopentyladenosine (CPA), anhydrous

magnesium chloride, Trizma® Base, Trizma® Hydrochloride, silicone solution (Sigma-cote), 3-(4,5-

Dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), phosphate-buffered saline (PBS)

and isopropanol were obtained from Sigma Aldrich. Dimethyl sulfoxide (DMSO), Whatman® GF/B

2

5 mm diameter filters and formic acid were obtained from Merck. Filtercount scintillation fluid,

3

radioligands, [ H]5’-N-ethylcarboxamide-adenosine {[ H]NECA, 25 Ci/mmol (250 µCi)} and 1,3-

3

[

H]-dipropyl-8-cyclopentylxanthine ([ H]DPCPX, 120 Ci/mmol) were obtained from Separation

Scientific SA. Cell culture media (Dulbecco’s Modified Eagle Medium (DMEM)), fungizone,

trypsin/EDTA, streptomycin, fetal bovine serum and penicillin were obtained from Gibco and Merck.

Well-plates (24 and 96) and culture flasks were obtained from Corning. Sterile syringe filters (0.22

µM) were obtained from Pall Corporation Life Sciences.

5

.2.2 Radioligand binding studies

Tissue preparation for binding studies

Radioligand binding studies were carried out as reported in literature [51-53]. The Animal Research

Ethics Committee of the North-West University (NWU-0035-10-A5) approved the collection of

animal tissue required for these assays. Adult male Sprague Dawley rats were obtained from the

Vivarium of the North-West University, Potchefstroom campus. Striata (for the adenosine A assays)

2

A

and whole brains (for the adenosine A assay) were dissected on ice and immediately snap frozen with

1

liquid nitrogen and then stored at -70 ˚C until required. The frozen striata as well as the whole brain

tissue were suspended in ice-cold 50 mM Tris buffer (pH 7.7 at 25 °C) and homogenised using a

Polytron PT-10 homogeniser (Brinkman) to yield final suspensions of 1 g/5 ml, which were aliquoted

and stored at -70 ˚C until required. Test compounds were dissolved (10 mM) and further diluted in

DMSO, with the final concentration of DMSO in the incubations being 1%. All pipette tips as well as

the 4 ml polypropylene tubes used for the incubations were coated with Sigmacote®. The incubations

were prepared using 50 mM Tris buffer (pH 7.7 at 25 °C). For the adenosine A₂_Aassay, the final

volume of the incubations was 1 ml and each incubation contained test compound (0-100 µM),

membrane suspension yielding ~10 mg of original tissue weight of rat striata, 10 mM MgCl , 0.2 units

2

3

of adenosine deaminase, 50 nM CPA and 4 nM [ H]NECA. The MgCl (A assay) and adenosine

2

2A

deaminase (A₂_Aand A assays) were firstly added to the membrane suspension, and this mixture was

1

subsequently added to the incubations. The order of addition was test compound, membrane

ACCEPTED MANUSCRIPT

3

suspension, CPA and [ H]NECA. All incubations were carried out in duplicate. Non-specific binding

was determined in triplicate in the presence of CPA (100 µM replacing the test compound). For the

adenosine A assay, the final volume of the incubations was also 1 ml and each incubation contained

1

test compound (0-100 µM), membrane suspension yielding 5 mg of original tissue weight of rat whole

3

brain, 0.1 units of adenosine deaminase and 0.1 nM [ H]DPCPX. The order of addition was test

3

compound, membrane suspension and [ H]DPCPX. All incubations were carried out in duplicate.

Non-specific binding was determined in triplicate in the presence of CPA (100 µM replacing the test

compound). After incubation for 1 h (with vortexing after 30 minutes), the incubations were rapidly

filtered through Whatman® GF/B 25 filters (25 mm diameter) fitted on a Hoffeler vacuum system.

The damp filters were placed into scintillation vials and scintillation fluid (4 ml) was added. The vials

were shaken thoroughly and left for 2 h. A Packard Tri-CARB 2100 TR scintillation counter was used

to count the radioactivity retained on the filters. Specific binding was defined as total binding minus

nonspecific binding, and was expressed as counts per minute (CPM).

Data analysis

Using the one site competition model of the Prism 5 software package (GraphPad) the CPM values

were plotted against the logarithm of the ligand concentration to give a sigmoidal dose-response

curve, from which the IC₅₀values were determined. For adenosine A binding, K values were

1

i

calculated from the IC values by using the Cheng-Prusoff equation [58], as applicable to radioligand

5

0

3

binding assays [51]. K , the equilibrium dissociation constant for the radioligand [ H]DPCPX, was

d

taken as 0.36 nM [52]. Since adenosine A₂_Abinding was performed in the presence of CPA, an

3

adapted version of the Cheng-Prusoff equation was used. The K of the radioligand, [ H]NECA, was

d

taken as 15.3 nM and a K value of 685 nM was used for CPA [51]. The binding affinities of the

c

known adenosine A agonist, CPA, and the A antagonists, KW-6002 and ZM241385 were also

1

2A

determined as controls. The results of the radioligand binding studies are reported as the mean ±

standard error of the mean (SEM) of duplicate determinations.

5

.2.3 MTT cell viability assay

Cell culture

2

HeLa cells were maintained in 250 cm flasks in DMEM media (30 ml) containing 10% fetal bovine

serum, 1% penicillin (10 000 units/ml)/streptomycin (10 mg/ml), and fungizone (250 µg/ml). The

cells were incubated at 37 °C in an atmosphere of 10 % CO . The media was replaced once a week

2

and cells were allowed to reach confluency before use in assays.

Preparation of compounds

ACCEPTED MANUSCRIPT

Stock solutions of test compounds were prepared in DMSO (10 mM) and further diluted in DMSO to

concentrations of 1 µM and 10 µM. These solutions were filtered via a syringe filter before addition to

the cell cultures.

MTT Assay

Once confluent, cells were detached with 3 ml trypsin/EDTA (0.25%/0.02%) and seeded in 24-well

plates at 500 000 cells/ well. Plates were then incubated for 24 h and the wells subsequently rinsed

with 0.5 ml DMEM free from fetal bovine serum. A volume of 0.99 ml DMEM (free from fetal

bovine serum) was subsequently added to each well followed by 10 µl of the test compound. In each

2

4-well plate, wells were reserved as either positive control (100% cell death via lyses with 0.03%

formic acid) or negative controls (100% cell viability as a result of no drug treatment). The plates

were then incubated at 37 °C for a further 24 h where after media was aspirated from each well.

The wells were then washed twice with 0.5 ml/well PBS and 200 µl of 0.5% MTT (prepared in PBS)

was added to each well. The well plates were incubated at 37 °C for 2 h in the dark, where after the

residual MTT was aspirated and 250 µl isopropanol was added to dissolve the formed formazan

crystals. The well-plates were then incubated at room temperature for 5 min to allow dissolution of

the blue formazan crystals, where after 100 µl of the isopropanol solution of each well plate was

transferred to a corresponding well in a 96-well plate. The absorbance was measured

spectrophotometrically at 560 nM (using a Labsystems Multiscan RC UV/V spectrophotometer), with

the absorbance of the negative control signifying 100 % viability and the absorbance of the positive

control signifying 0% viability. The effects of the test compounds were evaluated in triplicate and the

residual cell viabilities reported as the mean ± SD of the percentage viable cells compared to the

negative control (100%).

5

.2.4 In vivo assays

Animals

Sprague-Dawley rats were given free access to standard laboratory food and water until the required

weight was obtained (240 g - 300 g). All efforts were made to minimise animal suffering as

experiments were carried out in accordance with the National Institute of Health's Guide for the Care

and Use of Laboratory Animals. The experimental protocol was approved by the North-West

University ethical committee (NWU-00035-10-A5).

Compounds

To induce catalepsy a dose of 5 mg/kg haloperidol (Serenace Injection; 5 mg/ml) was administered

intraperitoneally (ip) [33]. A vehicle solution was prepared by mixing a 1:1:4 solution of DMSO,

Tween 80 and saline. Compounds (8k and 8m) were dissolved in a required amount of vehicle

solution to yield concentrations of 0.1, 0.4, 1 and 2 mg/ml. A suitable volume of these solutions was

ACCEPTED MANUSCRIPT

injected depending on the weight of the rat, resulting in final dose concentrations of 0.1, 0.4, 1 and 2

mg/kg. All injections were administered ip.

Catalepsy test

The experiments were carried out between 8:00 and 15:30 in a lit room with a controlled temperature.

All the rats were drug naïve and were only used once. Haloperidol-induced catalepsy was measured

with the standard bar test, in a Perspex chamber (length, 23 cm; width, 10.5 cm; height, 9 cm) with a

horizontal plastic bar (diameter, 1 cm; length, 10.5 cm) fixed at 9 cm above the floor, and at 7 cm

from the back of the box.

Animals were divided into 5 groups, each group containing 6 rats. The 5 groups were treated with 0,

0

.1, 0.4, 1 and 2 mg/kg of the test compound, respectively. All rats received ip injections of

haloperidol (5.0 mg/kg) to induce catalepsy. 30 Min. later, the rats in each group received ip

injections of compound. The vehicle solution was administered to rats in the control group. Catalepsy

was measured 60 min. after the haloperidol injections by placing the rats in the Perspex box with their

front paws on the horizontal bar. Catalepsy was measured as the time the animal maintained its

position on the bar. Time was recorded until one or both of the rat’s front paws were removed from

the bar, or up to 120 seconds.

The results of the animal experiments are reported as the mean ± standard error of the mean (SEM).

Data were analysed by means of one-way analysis of variance (ANOVA) across all groups, and were

subsequently subjected to Dunnett’s post-test to determine if statistical differences exist between

mean values. A p value < 0.05 is judged as being statistical significant. These analyses were carried

out with the Prism software package.

5

.3 Molecular docking

®

Molecular docking studies were carried out with the Windows based Accelrys Discovery Studio 3.1

software. The crystal structure of the adenosine A₂_Areceptor [Protein Data Bank (PDB) code 3PWH]

co-crystalised with the known A₂_Aantagonist ZM241385 was used. This receptor was prepared with

the ‘Clean protein’ function to correct problems such as incomplete amino acid side chains and typed

with the CHARMm forcefield. A fixed atom constraint was applied to the backbone and a

minimisation was then carried out using the Generalised Born approximation with Molecular Volume

(

GBMV) as the solvent model to obtain a receptor at energetic minimum. A binding sphere with a

radius of 5 Å was defined using the existing ligand (ZM241385) before it was removed from the

receptor. Selected inhibitors were cleaned and prepared for docking with the 'Prepare ligand' protocol

to correct valences and remove duplicates whereafter ligands were visually inspected and remaining

errors corrected. The CDOCKER protocol was used for the docking of ligands. The orientations,

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CDOCKER and CDOCKER interaction energies of the ten different conformers of each ligand were

considered and the best conformation for each ligand selected. An in situ ligand minimisation was

then performed on the selected conformers and minimised conformers visually inspected and

compared.

Acknowledgements

We gratefully acknowledge the assistance of Dr. Johan Jordaan and Mr. André Joubert of the SASOL

Centre for Chemistry, North-West University for recording of NMR spectra and Prof. Jan du Preez for

HPLC data. The financial assistance of the North-West University, Medical Research Council (MRC)

and the National Research Foundation (UID 76308) towards this research is hereby acknowledged.

Opinions expressed and conclusions arrived at, are those of the authors and are not necessarily to be

attributed to the NRF.

Appendix A. Supplementary data

Supplementary data associated with this article can be found in the online version, at...... These data

include the NMR data of the intermediates (7a – 7h) and examples of sigmoidal dose response curves

obtained during the radioligand binding assays.

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NH₂

N

1

2

3

A Ki = 2.5 nM

A Ki = 0.6 nM

2A

A Ki = 405 nM

2A

2

A

A Ki = 133 nM

1

A Ki = 17.7 nM

1

4

5

6

A Ki 0.1 nM

A Ki = 8.2 nM

2A

A Ki = 0.8 nM

2A

2

A

A Ki = 1.1 nM

1

A Ki = 58.4 nM

1

Figure 1: Adenosine A antagonists containing the 2-aminopyrimidine moiety.

2

A

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8

m

150

100

*

**

50

**

0

Dose

Figure 2a. Reduction in haloperidol induced-catalepsy in male Sprague-Dawley rats by compound 8m.

Time to descend from the bar was measured in haloperidol (5 mg/kg, ip) treated rats after ip administration

of compound 8m (0.1, 0.4, 1, 2 mg/kg). Each bar represents average time (± SEM) of (n = 6) rats in the

cataleptic position (*,**indicate significant differences compared with the haloperidol + vehicle control

group as determined by one-way ANOVA [F (4, 25) = 5.893; P < 0.005) followed by Dunnet’s post test

with *P = 0.01 - 0.05 and **P = 0.001 – 0.01).

8

k

100

8

0

6

4

2

*

0

Dose

ACCEPTED MANUSCRIPT

Figure 2b. Reduction in haloperidol induced-catalepsy in male Sprague-Dawley rats by compound 8k.

Time to descend from the bar was measured in haloperidol (5 mg/kg, ip) treated rats after ip administration

of compound 8k (0.1, 0.4, 1, 2 mg/kg). Each bar represents average time (± SEM) of (n = 6) rats in the

cataleptic position (* indicates significant differences compared with the haloperidol + vehicle control

group as determined by one-way ANOVA [F (4, 25) = 3.103; P < 0.05) followed by Dunnet’s post test with

P = 0.01 – 0.05).

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A

B

Figure 3: Illustration of the different orientations of compound 8b in the binding site of the A₂_Areceptor.

Intermolecular hydrogen bond interactions are observed between the exocyclic amino group and Asn253, while

π=π stacking occurs between the three ring systems and Phe168 (A). In orientation B, an additional hydrogen

bonding interaction is observed between the furan oxygen and Asn253 (Figure generated using Pymol).

Article Doi

2-Aminopyrimidines as dual adenosine A1/A2A antagonists

DOI: 10.1016/j.ejmech.2015.09.035

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Article abstract of DOI:10.1016/j.ejmech.2015.09.035

Full text of DOI:10.1016/j.ejmech.2015.09.035

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