Design, synthesis and evaluation of amino-3,5-dicyanopyridines and thieno[2,3-b]pyridines as ligands of adenosine A₁ receptors for the potential treatment of epilepsy

Abstract

Due to the implication of adenosine in seizure suppression, adenosine-based therapies such as adenosine receptor (AR) agonists have been investigated. This study aimed at investigating thieno[2,3-b]pyridine derivatives as non-nucleoside A₁ agonists that could be used in pharmaco-resistant epilepsy (PRE). Compound 7c (thieno[2,3-b]pyridine derivative), displayed good binding affinity to the rA₁ AR (K_i = 61.9 nM). This could be a breakthrough for further investigation of this heterocyclic scaffold as potential ligand. In silico evaluation of this compound raised bioavailability concerns but performed well on drug-likeness tests. The effect of intramolecular cyclisation that occurs during synthesis of thieno[2,3-b]pyridines from the lead compounds, amino-3,5-dicyanopyridine derivatives (6a-s) in relation to AR binding was also evaluated. A significant loss of activity against rA₁/rA_2A ARs with cyclisation was revealed. Amino-3,5-dicyanopyridines exhibited greater affinity towards rA₁ ARs (K_i < 10 nM) than rA_2A. Compound 6c had the best rA₁ affinity (K_i = 0.076 nM). Novel compounds (6d, 6k, 6l, 6m, 6n, 6o, 6p) were highly selective towards rA₁ AR (K_i between 0.179 and 21.0 nM). Based on their high selectivity for A₁ ARs, amino-3,5-dicyanopyridines may be investigated further as AR ligands in PRE with the right structural optimisations and formulations.

A decrease in rA₁ AR affinity is observed with intramolecular cyclisation that occurs during synthesis of thieno[2,3-b]pyridines (7a, 7d, 7c) from amino-3,5-dicyanopyridine derivatives (6a, 6f, 6g).

Introduction

Adenosine receptors (ARs) are a family of G protein-coupled receptors (GPCRs) with the nucleoside adenosine as endogenous agonist [1]. There are four known types of ARs, namely A₁, A_2A, A_2B and A₃ [2] which have been linked to both inhibition (A₁ and A₃) and activation (A_2A and A_2B) of adenylyl cyclase activity [3]. These receptors are widely expressed throughout all human body tissues and organs; such as the brain, heart, lung, liver, kidney, eye, joints, and blood cells [4]. ARs also play a role in various pathological conditions such as inflammatory diseases, ischaemia-reperfusion and neurodegenerative disorders [5], due to their broad spectrum of physiological and pathophysiological functions [6, 7]. All these physiological functions imply that ARs are potential drug targets for treatment of a variety of conditions such as asthma, neurodegenerative disorders, psychosis and anxiety, cardiac ischaemic diseases, sleep disorders, cancer and many other pathophysiological states that are believed to be associated with changes of adenosine levels [8].

The ARs are far more abundant in the brain than in any other cell type or organ in mammals [9], where it has a role in mechanisms of seizure susceptibility, sleep induction, pain perception, respiration and others [10]. Adenosine levels in the brain extracellular space increase dramatically during enhanced nerve activity conditions, such as ischaemia, seizures, or trauma to prevent neuronal injury [10]. The neuroprotective effects of adenosine may be due to stimulation of A₁ receptors and blockade of A_2A receptors [11]. Therefore, ARs are potential therapeutic targets for treatment of neurological [12] as well as neurodegenerative diseases including epilepsy [13].

Epilepsy is defined as a chronic neurological disorder characterised by recurrent, unprovoked seizures due to excessive discharge of cerebral neurons [14], which alter perception, consciousness, and motor activity. It affects about 50 million people worldwide, hence it is one of the most common neurological diseases globally [15]. Currently there is no available cure for epilepsy. The current treatment of epilepsy consists of antiepileptic drugs (AEDs) (also known as anticonvulsants). These therapies are employed to control symptoms of the disease (i.e. suppression of seizures) [16].

Approximately one-third of epileptic patients on treatment remain poorly controlled [17]. Pharmaco-resistance epilepsy can be defined as failure to control seizures after introduction of two or three anticonvulsants that are suitable for the type of epilepsy, prescribed and taken at maximum daily therapeutic doses [18]. A strategy that prevents seizures in drug-resistant epilepsy would be an important therapeutic advance and altering purinergic signalling may be a viable option [19].

Adenosine is a long-known endogenous anticonvulsant substance that effectively inhibits excitatory transmission in the brain [20] through activation of A₁ ARs [3]. Firstly, the released adenosine binds to presynaptic A₁ receptors, which blocks the influx of Ca²⁺ through voltage-dependent calcium channels leading to inhibition of glutamate release, and hence, decreased excitation of postsynaptic glutamate receptors [11, 21]. Secondly, postsynaptic activation of A₁ receptors by adenosine opens potassium channels leading to K⁺ efflux which results in resting membrane potential hyperpolarization rendering both ionotropic glutamate receptors (NMDA & AMPA) less responsive [22–24]. Both decreased neurotransmitter release and membrane potential hyperpolarization lead to decreased excitatory synaptic transmission and lower probability of seizure generation onset and propagation [21].

Therefore, adenosine receptor-based therapy—especially through A₁ AR activation—may provide therapeutic potential for patients who do not gain satisfactory seizure control with currently available AEDs [19, 21, 25].

Attempts have been made over the years to develop selective A₁ AR agonists that may be useful as antiepileptic agents. Initially the approach for discovering AR agonists as antiepileptics has been restricted to modification of the physiological agonist adenosine [26], and justly, these adenosine derivatives represent the great majority of molecules developed and reported to date [27]. The development of these agonists has been limited by the essential requirement of the retention of the ribose moiety of adenosine for agonist activity [26, 28, 29]. Examples of adenosine derivatives include non-selective AR agonists such as 2-chloroadenosine (2-CADO) and A₁ AR selective agonists such as 2-chloro-N6-cyclopentyladenosine (CCPA) [30].

However, the development of adenosine-based AR agonists as novel therapeutic agents has been limited by their pronounced peripheral side effects (mainly cardiovascular effects such as bradycardia and hypotension) and central side effects (like sedation) [3, 6, 13] at doses that have relatively weak anticonvulsant and neuroprotective effects [3]. In addition, they exhibited low blood brain barrier permeability, and hence, limited use in the central nervous system (CNS) [6, 31]. Therefore, these drugs have not been pursued clinically [14].

The said limitations led to development of new strategies to produce potent and selective AR agonists with dominant CNS activity [14]. Non-nucleoside agonists provide an alternative set of compounds which are highly potent and selective for specific AR subtypes [28]. In this study thieno[2,3-b]pyridine derivatives were explored as alternative non-nucleoside A₁ AR agonists for the potential management of seizure disorders.

Thienopyridines as a class of heterocyclic compounds have attracted considerable interest due to their broad spectrum of biological activities [32]. The pharmacological potential of thienopyridine derivatives made these compounds a privileged scaffold in medicinal chemistry [33]. There are six isomeric thienopyridine structures, one of them being thieno[2,3-b]pyridine (Fig. 1) and its derivatives which have since attracted attention due to their antitumor, antibacterial [34], antiviral [35, 36], vasodilator and antihypertensive [37], antidiabetic [38], anti-inflammatory [39], antidermatophytic [40], antimalarial activities [41] in addition to treatment of CNS disorders [42].

Fig. 1.

Chemical structure of thieno[2,3-b]pyridine scaffold

Despite their aforementioned promising biological activities, the thienopyridine core has only received scanty attention as scaffold for the design of AR ligands. 3,5-Dicyanopyridine derivatives which serve as intermediates in the synthesis of thieno[2,3-b]pyridine derivatives, were themselves found to exhibit interesting affinity for ARs. Due to chemical similarity between the 3,5-dicyanopyridin core and the thieno[2,3-b]pyridine core, we envisaged that a suitably substituted thieno[2,3-b]pyridine core could lead to derivatives which may exhibit AR affinity. Notably, bicyclic scaffolds such as benzofurans [43], tetralones and indanones were previously associated with affinity for ARs.

The main aim of this research study was to design, synthesise, characterise, and evaluate novel and known amino-3,5-dicyanopyridines (intermediates) and thieno[2,3-b]pyridines (target compounds) as potent and selective A₁ AR agonists for the potential treatment of neurological conditions, such as epilepsy. Modifications at R₂ and the aryl position on the thieno[2,3-b]pyridines scaffold were influenced by the lead compounds amino-3,5-dicyanopyridine derivatives which displayed good affinity at A₁ AR (Fig. 2). The proposed modifications included thiophene ring closure (from lead compound) resulting in a fused 5-membered (thiophene) heterocyclic ring structure. Different functional groups were substituted at the meta and para positions of the 4-phenyl ring (R₂) and different aryl groups were substituted at position 2 (Fig. 2). The structure-activity relationship (SAR) of the synthesised compounds were evaluated in relation to A₁ and A_2A AR affinity.

Fig. 2.

Synthesis of thieno[2,3-b]pyridine derivatives from lead compounds and modification on the thieno[2,3]pyridine scaffold

Results and discussion

Chemistry

The synthesis of the amino-3,5-dicyanopyridine derivatives (intermediates) 6a–6p was done by multicomponent condensation of malononitrile (MN) with hydrogen sulphide, a corresponding aldehyde, and a suitable halide in the presence of trimethylamine (Et₃N) as catalyst [44]. As depicted in Scheme 1, initial addition of hydrogen sulphide to MN gives cyanothioacetamide (1) which reacts with the aldehyde according to a Knoevenagel condensation reaction to yield 2. Further addition of MN results in 3 which undergoes chemoselective intramolecular cyclisation to 3,4-substituted phenyl-2,6-diamino-3,5-dicyano-4H-thiopyran (4). Recyclisation of the latter by the action of alkali (potassium hydroxide (KOH), dimethylformamide (DMF)) leads to pyridine-2-thiolate (5). The subsequent regioselective alkylation of 5 at the sulphur atom with a suitable halide results in a sulphide (6). According to the method adopted from [44], the sulphide was supposed to undergo intramolecular cyclisation in the presence of an alkali (KOH) to yield a thienopyridine (7)—a fused pyridine and thiophene ring heterocyclic compound—but all reactions except the one that yielded compound 7a, did not go to completion. Instead, the method produced the intermediate compounds, namely amino-3,5-dicyanopyridine derivatives. Modifications such as increasing the KOH concentration and contact time with the reaction mixture were made without success to try and bring the reactions to completion. Otherwise ring closure reactions were performed to convert the synthesised intermediate compounds to thieno[2,3-b]pyridine derivatives using Scheme 2, where either 2–3 drops of KOH were added to a solution of amino-3,5-dicyanopyridines in DMF and then the reaction was left to stand for several hours [45] or through heating a solution of amino-3,5-dicyanopyridines in ethanol (EtOH) containing KOH under reflux for 3 h [46]. Only 3 compounds, 7b–7d were obtained through these attempts. Details of unsuccessful attempts have been summarised (see supplementary material).

Scheme 1.

Reaction route for preparation of target thieno[2,3-b]pyridine derivatives [44]

Scheme 2.

Synthesis of thieno[2,3-b]pyridines from intermediates

From observation, only compounds with a carbonyl group at the aryl position managed to go to completion to thieno[2,3-b]pyridine derivatives (target compounds). This seems to be in line with the adopted method from [44, 45] since they used α-halo carbonyl compound as an alkylating agent to obtain thieno[2,3-b]pyridine derivatives in an one pot system. Most previously reported synthetic routes for thieno[2,3-b]pyridine derivatives involved the use of α-halo carbonyl compound as well [32, 47, 48]. It seems that the presence of a carbonyl compound at the aryl position has an influence on the intramolecular cyclisation of the intermediate compounds compared to aryl halides. This may be due to the fact that α-halo compounds are bifunctional since they can behave as both an electrophile and nucleophile in carbonyl condensation reactions. The target thieno[2,3-b] pyridines that were synthesised was based on intermediates with A₁ AR activity, hence the choice of halides used. Also, ring closure may have been accomplished with these compounds (7a–7d) due to presence of less bulky constituent (-CONH₂) at the aryl position as compared to other compounds with aromatic constituents at the same position. For Compounds 6q–6s and 7a, readily available cyanothioacetamide was used as starting material. One of the key starting materials for compounds 6q–6s, 4-(chloromethyl)-2-(4-chlorophenyl)thiazole was synthesised by refluxing a mixture of 4-chlorobenzothioamide and 1,3-dichloroacetone in absolute EtOH for 2 h (Scheme 3) [49].

Scheme 3.

Synthesis of 4-(chloromethyl)-2-(4-chlorophenyl)thiazole

The test compounds were obtained in relatively poor yields (6a, 6c–l, 6n–s and 7a–d: 11.8–66.4%; with the exception of 6b and 6m: >80%), purified by recrystallisation from a suitable solvent (either EtOH, methanol (MeOH) or hexane). The structure, molecular mass and purity of these compounds were verified by hydrogen-1 / protium (¹H) and carbon-13 (¹³C) nuclear magnetic resonance (NMR) spectra, mass spectroscopy and HPLC (see supplementary material). It should be noted that, protons on the NH₂-group (e.g., 6l) and the OH-group (e.g., 6k) are not always visible on a ¹H NMR spectrum as protons attached to a N-atom (or O-atom) are acidic, and thus, exchangeable [50]. Halogen-carbon bonds tend to cause splitting of ¹³C NMR chemical shifts (e.g., 6p and 7a) due to deshielding by the F-atom on the directly bonded carbon nucleus [51] which results in multiple carbon peaks. This has the potential of causing difficulty in interpreting ¹³C NMR spectra of fluorinated organic compounds.

Biology

In vitro evaluation

Radioligand binding assays

A total of 23 test compounds were synthesised (6a–s and 7a–d); 7 of these compounds were novel (6d and 6k–p), while 4 compounds (7a–d) have been synthesised before but have never been tested for AR affinity. The affinities of the test compounds 6a–s and 7a–d at rat (r) A₁ and A_2A ARs were determined by radioligand binding assays and are expressed as inhibition constant (K_i, nM) values (Table 1). All test compounds displayed specific binding values <20% at a maximum tested concentration of 100 μM (rA₁ screening), and therefore, all underwent full biological assay for determination of Ki values (nM). Compounds 6a–j and 7a–d displayed specific binding values <20% at a maximum tested concentration of 100 μM (rA_2A screening) and hence qualified for full rA_2A radioligand binding assay, unlike compounds 6k–s with specific binding values >20%. The radioligand binding assays were validated with N₆-cyclopentyladenosine (CPA) (A₁ agonist), 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) (A₁ antagonist), istradefylline (A_2A antagonist) and caffeine (A₁/A_2A antagonist) as reference compounds and results were compared to literature values as shown by Table 1.

Table 1.

K_i values of test compounds and reference compounds at rat A₁ and A_2A ARs

			Ki ± SEM (nM)a (Specific binding (%))b
#	R2	aryl	rA1c vs 1 nM [3H]DPCPX	rA2Ad vs 4 nM [3H]NECA	rA1c + 0.1 mM GTP vs 1 nM [3H]DPCPX	GTP shifte	SIf
Amino-3,5-dicyanopyridines
6a			139 ± 18.8a	1473 ± 256a	–	–	11
6b			0.213 ± 0.019a (2.9)g	48.0 ± 11.1a (35)g	–	–	255
6c			0.076 ± 0.002a (0.49)g (0.21)h	48.3 ± 10.1a (71)g (52)h	0.069 ± 0.006a	1	636
6d			10.3 ± 0.643a	1205 ± 367a	11.3 ± 0.663a	1	117
6e			60.4 ± 3.83a	338 ± 79.1a	–	–
6f			48.0 ± 4.36a	751 ± 12.0a	–	–	16
6g			26.6 ± 6.75a	429 ± 55.0a	–	–	16
6h			7.54 ± 0.768a (4.12)i	(581)i	–	–
6i			4.57 ± 0.284a	634 ± 94.3a	–	–	139
6j			Not determined (3.5)g	20.6 ± 6.56a (15)g	–	–
6k			8.82 ± 0.760a	(22)b	–	–	–
6l			21.0 ± 5.56a	(27)b	–	–	–
6m			0.179 ± 0.013a	(80)b	–	–	–
6n			0.831 ± 0.076a	(35)b	1.94 ± 0.509a	2	–
6o			1.64 ± 0.228a	(25)b	2.25 ± 0.159a	1	–
6p			0.430 ± 0.012a	(30)b	–	–	–
6q			0.383 ± 0.069a (1.4)j	(44)b	1.82 ± 0.582a	5	–
6r			1.36 ± 0.040a (1.5)j	(44)b	–	–	–
6s			4.06 ± 0.759a (5.0)j	(27)b	–	–	–
Thieno[2,3-b]pyridines
7a			1008 ± 58.3a	308 ± 93.6a
7b			556 ± 28.1a	561 ± 12.1a	–	–
7c			61.9 ± 2.11a	1062 ± 126a	145 ± 28.8a	2
7d			305 ± 15.3a	162 ± 24.4a	–	–
Reference compounds
CPA (A1 agonist)			6.5 ± 0.4a (15.3)k (7.9)l	–	36.5 ± 2.28a	6	–
DPCPX (A1 antagonist)			0.5 ± 0.1a (0.6)k (0.3)m	–	0.4 ± 0.032a	1	–
Istradefylline (A2A antagonist)			–	3 ± 0.9a (13; 2.2)n (11.1)o	–	–	–
Caffeine (A1/A2A antagonist)			52 800 ± 7 400a (44 000)p (41 000)q (26 000)r	27 800 ± 5 100a (43 000)q (22 000)r (33 000)k	–	–	0.5

^aInhibition constant (K_i, nM) represented as the mean ± standard error of the mean (SEM), n = 3 samples

^bSpecific binding (%) of the radioligand at a maximum tested concentration of 100 µM is represented as the mean, n = 2 samples

^crA₁: rat whole brain membranes expressing adenosine A₁ receptor

^drA_2A: rat striatal membranes expressing adenosine A_2A receptor

^eGTP shift calculated by dividing the K_i (nM) in the presence of 0. 100 µM GTP by the K_i (nM) in the absence of 100 µM GTP

^fSelectivity index (SI) for the adenosine A₁ receptor subtype calculated by dividing the rA_2AK_i (nM) by the rA₁K_i (nM)

^gLiterature value: human adenosine A₁ receptor and [³H]DPCPX; human adenosine A_2A receptor and [³H]ZM241385 [56]

^hLiterature value: rat adenosine A₁ receptor and [³H]DPCPX; rat adenosine A_2A receptor and [³H]ZM241385 [56]

ⁱLiterature value: human adenosine A₁ receptor and [³H]DPCPX; human adenosine A_2A receptor and [³H]ZM241385 [79]

^jLiterature value: human adenosine A₁ receptor and [³H]DPCPX [57]

^kLiterature value: rat adenosine A₁ receptor and [³H]DPCPX [26]

^lLiterature value: rat adenosine A₁ receptor and [³H]DPCPX [75]

^mLiterature value: rat adenosine A₁ receptor and [³H]DPCPX [52]

ⁿLiterature value: rat adenosine A₁ receptor and [³H]DPCPX [80]

^oLiterature value: rat adenosine A₁ receptor and [³H]DPCPX [60]

^pLiterature value: rat adenosine A₁ receptor and [³H]DPCPX [62]

^qLiterature value: rat adenosine A₁ receptor and [³H]DPCPX [63]

^rLiterature value: rat adenosine A₁ receptor and [³H]DPCPX [81]

Structure-activity relationships (SAR)

Modifications were made at the R² and aryl positions of the test compounds to assess how different substituents can influence both rA₁ and rA_2A ARs binding affinity as well as selectivity. As shown in Table 1, all test compounds displayed greater affinity toward the rA₁ than rA_2A AR. Compound 6c had the best rA₁ AR affinity (K_i = 0.076 nM) of the present series. The latter compound together with 6b displayed better rA_2A AR affinity than the other test compounds with K_i values of 48.3 nM (6c) and 48.0 nM (6b), respectively, but remain selective for the rA₁ AR. Comparing amino-3,5-dicyanopyridines (6a–s) and thieno[2,3-b]pyridines (7a–d), it is evident that there was a significant decrease in both rA₁ and rA_2A AR affinity from the open ring structures to the closed ring structures. The only thieno[2,3]pyridine derivative that showed moderately good rA₁ AR affinity is compound 7c (rA₁K_i = 61.9 nM). The general poor activity of thieno[2,3-b]pyridines relative to amino-3,5-dicyanopyridins suggest that the ring closure affects binding to the receptors, perhaps, due to steric hindrance. (This may be confirmed by molecular docking studies in the future.)

SAR for amino-3,5-dicyanopyrines (intermediates)

For compounds 6a, 6b, 6l and 6s, the 4-methoxyphenyl group was maintained at position R² and different functional groups were substituted at the aryl position. Compound 6b with a methylpyridine substituent at the aryl position exhibited low nanomolar activity toward the rA₁ AR (K_i = 0.213 nM) as well as selectivity for the rA₁ AR over the rA_2A AR (SI = 636). Affinity for the rA₁ and/or rA_2A ARs decreased when introducing a 4-chlorophenylthiazole group (6s: rA₁K_i = 4.06 nM), benzoic acid substituent (6l: rA₁K_i = 21.0 nM) and a carbonyl containing substituent (6a: rA₁K_i = 139 nM) which displayed the lowest affinity for rA1 AR. In terms of selectivity, 6b also showed affinity toward the rA_2A AR (K_i = 48.0 nM) while compounds 6l and 6s were more selective towards rA₁ ARs, as seen from the calculated SIs.

Replacing 4-methoxyphenyl with 3-methoxyphenyl at position R² while maintaining the same aryl functional groups as 6a, 6b and 6l above was also explored. Comparison of compound 6d to 6a (aryl = -CONH₂) showed a significant increase in binding affinity towards rA₁ ARs (6d: rA₁K_i = 10.3 nM vs 6a: rA₁K_i = 139 nM) but had no effect on rA_2A AR affinity. In general, the presence of the 3-methoxyphenyl substituent resulted in increased affinity for rA₁ ARs but had no influence on rA_2A AR affinity as shown by 6c vs 6b and 6o vs 6l. Again, the substituents at the aryl position had a similar effect on affinity as observed with the 4-methoxyphenyl containing compounds 6a, 6l and 6s (in decreasing order of affinity: 6c (methylpyridine) > 6o (benzoic acid substituent) > 6d (carbonyl containing substituent)). From these results it is evident that 3-methoxyphenyl is favoured over 4-methoxyphenyl in terms of rA₁ AR binding affinity.

For compounds 6e, 6m, 6n and 6q, 4-hydroxyphenyl was introduced at the R² position while maintaining almost all the same aryl groups mentioned earlier. Generally, these compounds displayed rA₁ AR affinity of 1 nM or smaller (except 6e, aryl = CONH₂), with 6m (aryl = -methylpyridine) being the best with rA₁K_i = 0.179 nM of these compounds. Comparing 4-methoxyphenyl and 4-hydroxyphenyl substitutions (6a vs 6e and 6b vs 6m) showed that with the latter, rA₁ AR activity increased slightly. Looking at 6m (aryl = methylpyridine) and 6n (aryl = -methylbenzene), it appears that the introduction of a N-atom in compound 6m had a positive influence in rA₁ AR affinity.

Replacing 4-hydroxyphenyl with 3-hydroxyphenyl at position R² was also studied (6e vs 6f and 6m vs 6j), although a definite trend could not be observed with the limited data at hand. Comparison of 6f (3-hydroxyphenyl) to its 4-methoxyphenyl substituted counterpart 6d showed a four-fold decrease in rA₁ AR affinity, although rA₁ selectivity was maintained.

Comparison of 6a, 6d, 6e and 6f showed that the meta position is preferred to the para position whether OCH₃- or OH-group substitution is incorporated, and furthermore, it seems that a OCH₃-group is preferred to an OH-group.

Comparing 6l and 6k with 4-OCH₃ and 4-SCH₃ revealed that introducing a sulphur component increased binding affinity for compound 6k (K_i = 8.82 nM) as compared to 6l (21.2 nM).

Compounds, 6q, 6r and 6s with the same (4-chlorophenyl)thiazole aryl substituent were also explored. All these compounds displayed rA₁ AR affinity but had no mentionable affinity for rA_2A ARs. Compound 6q (R = 4-OCH₂CH₂OH) had the best affinity of these compouns with K_i = 0.383 nM. SARs of amino-3,5-dicyanopyridines against rA₁ AR are summarised in Fig. 3.

Fig. 3.

Structure-activity relationship of amino-3,5-dicyanopyridines against rA₁ AR

SAR for thieno[2,3-b]pyridines (target compounds)

Thieno[2,3-b]pyridine derivatives 7a–d displayed poor affinity towards rA₁ ARs compared to their corresponding intermediate amino-3,5-dicyanopyridines (Fig. 4). These compounds all had a -CONH₂-group at the aryl position. The results indicate that ring closure from the intermediate open ring to fused ring structures decreased activity towards both rA₁ and rA_2A ARs. This corresponds with a study by [7] in which intramolecular cyclisation of the 6-amino-3,5-dicyanopyridines, specifically BAY606583 (a potent A_2B receptor agonist) was evaluated. The study revealed that the bicyclic compound (thieno[2,3]pyridine derivative) that resulted after intramolecular cyclisation of BAY60 6586 bind none of the ARs suggesting that molecular stiffening decreases AR binding affinity.

Fig. 4.

Structure-activity relationships of amino-3,5-dicyanopyridines (6a, 6f, 6g) vs thieno[2,3]pyridines (7a, 7c, 7d)

GTP shift assays

The type of binding affinities that test compounds 6c, 6d, 6n, 6o, 6q and 7c displayed at the rA₁ AR were determined through guanosine 5'-triphosphate (GTP) shift assays, as described in literature [52–54]. These test compounds were selected as they possessed the best rA₁ AR affinity among the investigated test compounds (Table 1). The theory of a GTP assay is that competition curve of an antagonist will be unaffected by GTP, thus resulting in a calculated GTP shift of approximately 1 [54]. Agonists’ curves, on the other hand, will be shifted towards the right in the presence of GTP [55]. GTP shifts were calculated by dividing the rAKi values of compounds reported in the presence of GTP by the rARKi values obtained in the absence of GTP and the results are summarised in Table 1. Compounds 6c, 6d and 6o behaved as antagonists (interestingly, all these compounds contained a 3-OCH₃ group at position R²), while 6n, 6q and 7c behaved as agonists (Fig. 5). Contradictory to the present results, Guo et al. [56] found 6c to be a partial agonist and not an antagonist. Notably, Louvel and co-workers [57] also found 6q to be a full agonist in accordance with the present results.

Fig. 5.

The binding curves of test compounds 6d and 6q in the presence and absence of 100 µM GTP using [³H]DPCPX as radioligand in rat whole brain membranes expressing adenosine A₁ receptors as representative cases for adenosine A₁ receptor antagonistic and agonistic activity. A Calculated GTP shift: 1.09 (antagonist); B Calculated GTP shift: 4.74 (agonist)

In silico evaluation

The physiochemical properties, pharmacokinetic profiles, drug-likeness and medicinal chemistry friendliness of compounds 6c, 6d, 6n, 6o, 6q and 7c were predicted through the free online web tool SwissADME (https://swissadme.ch). The prediction is based on the chemical structures of the compounds. The results are in the supplementary material.

The bioavailability radar (which takes in to account the physicochemical properties lipophilicity, size, polarity, solubility, flexibility and saturation) for compounds 6b, 6d, 6m, 6o, 6q and 7c may be seen in the supplementary material (Fig. S2). Almost all compounds fall within the optimal ranges of lipophilicity, size, solubility, and flexibility parameters except compound 6q which exceeded the optimal size of the molecule (150–500 g/mol) since it has a molecular weight (MW) of 520.03 g/mol. All these compounds failed the saturation parameter since all have a lower fraction of carbon atoms in the sp3 hybridization (Csp3 > 0.25) and high polarity values (TPSA > 130 Ų). These compounds are considered to be too polar with a low degree of saturation and consequently predicted not to be orally bioavailable. The LogP value of a compound using a logP of a reference compound (XLOGP3) (<5.0) of compound 6q slightly exceeded the limit (5.1) proving to be the most lipophilic.

In terms of water solubility (Log S), compound 6c, 6m, 6o and 7c are predicted to be moderately soluble to poorly soluble in water. Compound 6d was predicted to be soluble to moderately soluble and compound 6q was classified as poorly soluble. Poor water solubility of compound 6q may be attributed to its high MW and the presence of lipophilic halogen (Cl) as part of the aryl substituent. Water solubility is the most important in terms of achieving desired drug concentration in systemic circulation for pharmacological response [58]. It must be understood that poorly water-soluble drugs have slow drug absorption leading to inadequate and variable bioavailability and gastrointestinal mucosal toxicity [58]. Solubility improvement techniques need to be employed for future formulation development especially for compound 6q (capadenoson), since any drug to be absorbed must be present in an aqueous solution at the site of absorption.

The BOILED-Egg predictive model allows evaluation of passive gastrointestinal (GI) absorption and brain penetration (BBB). Compounds 6c, 6d, 6m, 6o, 6q and 7c are all predicted to have low GI absorption and no blood brain barrier (BBB) permeability, probably because of a high topological polar surface area (TPSA) (>130), although some sources recommend TSPA < 140Ų (e.g. 6c) to be adequate for high probability of good intestinal permeability [59]. This may also be attributed to the high polarity of these compounds. Interestingly, a study by [60, 61] indicated that compound 6q (capadenoson), showed hints of CNS effects in humans. Compound 6c as well has been considered to possess high BBB permeability by [56, 62, 63] despite this prediction. The prediction of permeability glycoprotein (P-gp) substrate indicates that only compound 6q can be actively effluxed by P-gp while compounds 6c, 6m, 6o and 7c are not substrates of this efflux mechanism. The potential interaction of compounds 6c, 6d, 6m, 6o, 6q and 7c with cytochromes P450 (CYP) isoenzymes was also evaluated. This is important for determination of drug-drug interactions and adverse effects due to low drug clearance leading to accumulation of the drug [50, 51]. Generally, all the compounds are inhibitors of CYP isoforms (CYP1A2, CYP2C19, CYP2C9, CYP3A4) with a few exceptions, but they did not affect CYP2D6 except compound 6q.

SwissADME also provides qualitative assessment of drug-likeness which predicts a molecule’s chance to be classified as an oral drug candidate [64] by implementing different rule-based filters [65–69]. Additionally, compounds 6c, 6d, 6m, 6o, 6q and 7c all had a bioavailability score of 0.55 (the probability that a compound will have >0% bioavailability in rat or measurable Caco-2 permeability) [64].

The medical chemistry friendliness of compounds was assessed by identifying pan assay interference compounds (PAINS) [70] and structural alerts [71]. All compounds passed both PAINS and Brenk tests as no alerts were raised. This means that these compounds may not affect any bioassays [72] and generally have good pharmacokinetics properties with an acceptable toxic level [73]. Interestingly, only one compound (6d) passed the lead-likeness test, and hence, can be used as a lead compound in drug discovery processes. Compounds 6c, 6m, 6o, 6q and 7c all had higher MW (>350) as well as high partition coefficient (logP) values (XLOGP: >3.5). Structural optimisation for these chemical scaffolds is needed, most probably by decreasing size, polarity and/or lipophilicity.

Conclusion

The aim of this study was to investigate use of amino-3,5-dicyanopyridine and thieno[2,3-b]pyridine derivatives as potential AR agonists. A total of 23 test compounds were synthesised (6a–s and 7a–d) and 7 of these were novel (6d and 6k–p), while 4 compounds (7a–d) have been synthesised before but have never been tested for AR affinity.

Overall, amino-3,5-dicyanopyridine displayed superior activity towards rA₁ ARs compared to thieno[2,3]pyridines. The general poor activity of thieno[2,3-b]pyridines suggest that the intramolecular cyclisation results in molecular stiffening or rigidity which negatively affects binding to the receptors, perhaps, due to steric hindrance. On the R² substitution, it was observed that 3- and 4-methoxyphenyl groups favoured rA₁ AR binding compared to their 3- and 4-hydroxyphenyl counterparts. Looking at the aryl substitution, the methylpyridine substituent displayed the overall best rA₁ AR affinity. Novel compounds (6d, 6k, 6l, 6m, 6n, 6o and 6p) proved to be highly selective with low nanomolar rA₁ AR affinity (K_i values between 0.179 nM and 21.0 nM). The only thieno[2,3-b]pyridine derivative that displayed moderately good rA₁ AR activity (K_i = 61.9 nM) has been investigated as a TGF-β receptor kinase inhibitor for the treatment of tumours and now AR affinity may be included.

Compounds 6n, 6q and 7c acted as potent, highly selective agonists at A₁ ARs; however, compounds 6c, 6d and 6o (notably all containing a 3-OCH₃ group at position R²) behaved as rA₁ antagonists.

Upon in silico evaluation, the SwissADME profiles of the test compounds raised concern about their bioavailability; therefore, it may be advisable to confirm BBB permeation via in vitro evaluation of promising test compounds before further structure optimisation.

The high affinity and selectivity for the rA₁ AR displayed by the amino-3,5-dicyanopyridine scaffold showed that, if correctly modified, it may produce highly potent AR ligands which can be used in development of treatment for epilepsy.

Experimental

Chemistry

Materials and methods

Unless otherwise noted, all starting materials and solvents were purchased from commercial manufacturers (Sigma-Aldrich and AmBeed) and used without further purification. Thin layer chromatography (TLC) silica gel 60 F254 aluminium sheets from Merck was used to monitor reaction progress. Melting points (mp) were determined on a Buchi M-545 melting point apparatus. Mp for compounds 6h, 6i, 6j, 7c and 7b were obtained through differential scanning calorimetry (DSC) analysis using Mettler Toledo analyser. ¹H and ¹³C NMR spectra were recorded on a Bruker Avance III 600 spectrometer at frequencies of 600 and 151 megahertz (MHz) respectively, using DMSO-d6 (deuterated dimethyl sulfoxide) as solvent and tetramethylsilane (TMS - Si(CH₃)₄) as reference. Chemical shifts were reported in parts per million (ppm) in relation to the solvent peak (DMSO-d6: residual CH₃ at 2.50 ppm for ¹H NMR and 39.52 ppm for ¹³C NMR). Spin multiplicities were indicated as follows: singlet (s), doublet (d), triplet (t), quartet (q), doublet of doublets (dd), triplet of doublets (td), double double doublet (ddd) and multiplet (m). Coupling constant (J) values were reported in Hertz (Hz). High-resolution mass spectra (HRMS) were recorded on a Bruker micrOTOF-Q II mass spectrometer in atmospheric chemical ionisation (APCI) mode. High-performance liquid chromatography (HPLC) analyses were done on Shimadzu Nexera-i LC-2040C 3D Plus HPLC system to determine the purity of test compounds.

Synthesis of test compounds

General procedure for the synthesis of 6a–6p

2-((6-amino-3,5-dicyano-4-(4-methoxyphenyl)pyridin-2-yl)thio)acetamide (6a)

Three drops of trimethylamine were added to a solution of MN (0.629 ml,10 mmol) in 20 mL of EtOH, while stirring with magnetic stirrer. The reaction mixture was cooled to 10 °C and H₂S generated by reaction between iron sulphide (FeS) (12.081 g) and hydrochloric acid (HCl) (90 ml) was passed through the mixture for 24 h to produce cyanothioacetamide (1). The reaction mixture was then stirred for 15–20 min before adding 3-methoxybenzaldehyde (1.217 ml, 10 mmol) while stirring at room temperature to produce 2a. After obtaining a homogeneous mixture, more MN (0.629 ml, 10 mmol) was added and the mixture stirred until it became homogeneous again. It was then left to stand at room temperature for 12–14 h to obtain 2,6-diamino-4-methoxy-4H-thiopyran-3,5-dicarbonitrile (4a). The mixture was then diluted with an equal volume of DMF and 10% aqueous KOH (5.6 mL, 10 mmol) and left to stand for 24 h to produce potassium 6-amino-3,5-dicyano-4-methoxy-1,4-dihydropyridine-2-thiolate (5a). 2-bromoacetamide (10 mmol, 1.382 g) was added to the mixture and continuously stirred for 3 h, after which 10% aqueous KOH (5.6 mL, 10 mmol) was added again. After 2–4 h, ice was added and the resulting precipitate was filtered off, washed with distilled H₂O, EtOH and hexane, dried (30 °C) and recrystallised from MeOH to yield the title compound 6a as whitish powder (0.988 g, 29.1%): Rf: 0.77 (DCM/PE/EtOAc 10:1:1); mp: 237.2–238.0 °C; ¹H NMR (600 MHz, DMSO) δ 7.93 (s, 2H), 7.49 (d, J = 8.7 Hz, 2H), 7.46 (s, 1H), 7.19 (s, 1H), 7.12 (d, J = 8.7 Hz, 2H), 3.89 (s, 2H), 3.85 (s, 3H); ¹³C NMR (151 MHz, DMSO) δ 168.8 (C,-CO), 166.1 (C, C-6), 160.8 (C, C-2), 159.6 (C, C-4'), 158.0 (C, C-4), 130.1 (C, C1'), 125.7 (CH, C-2',C-6'), 115.3 (CH, C-3', C-5'), 115.3 (C, CN), 114.1 (C, CN), 93.3 (C, C-5), 85.9 (C, C-3), 55.3 (CH₃, OCH₃), 33.3 (CH₂, S-CH₂-); APCI-HRMS m/z: calculated for C₁₆H₁₄N₅O₂S [M + H] + 340.0863, found 340.0871; Purity (HPLC, λ = 280): 100%

2-amino-4-(4-methoxyphenyl)-6-(((6-methylpyridin-2-yl)methyl)thio)pyridine-3,5-dicarbonitrile (6b)

Prepared as for 6a from 4-methoxybenzaldehyde (1.217 ml, 10 mmol) and 2-(bromomethyl)-6-methylpyridine (1.862 g, 10 mmol) to yield 6b which was recrystallised from MeOH as white flakes (3.418 g, 88.2%): Rf: 0.77 (PE:EtOAc 1:1); mp: 168.5–171.9 °C; ¹H NMR (600 MHz, DMSO) δ 8.00 (s, 2H), 7.63 (t, J = 7.6 Hz, 1H), 7.48 (d, J = 8.7 Hz, 2H), 7.42 (d, J = 7.6 Hz, 1H), 7.12 (dd, J = 24.1, 8.1 Hz, 3H), 4.56 (s, 2H), 3.84 (s, 3H), 2.46 (s, 3H); ¹³C NMR (151 MHz, DMSO) δ 166.1 (C, C-6), 160.8 (C, C-2), 159.6 (C, C-4'), 158.1 (C, C-4), 157.7 (C, C-2″), 155.73 (C, C-6″), 137.0 (CH, C-4″), 130.1 (C, C-1'), 125.8 (CH, C-2', C-6'), 121.8 (CH, C-5″), 120.6 (CH, C-3″), 115.4 (CH, C-3', C-5'), 115.4 (C, CN), 114.0 (C, CN), 93.2 (C, C-5), 85.9(C. C-3), 55.3 (CH₃, OCH₃), 35.3 (CH₂, -SCH₂-), 23.9 (CH₃, -methylpyridine); APCI- HRMS m/z: calculated for C₂₁H₁₈N₅OS [M + H] + 388.1227, found 388.1208; Purity (HPLC, λ = 254): 100%

2-amino-4-(3-methoxyphenyl)-6-(((6-methylpyridin-2-yl)methyl)thio)pyridine-3,5-dicarbonitrile (6c)

Prepared as for 6a from MN (0.315 ml, 5 mmol), 3-methoxybenzaldehyde (5 mmol, 0.608 ml) and 2-(bromomethyl)-6-methylpyridine (0.933 g, 5 mmol) to yield 6c which was recrystallised from MeOH as white solid (0.505 g, 26.1%): Rf: 0.64 (DCM/PE/EtOAc 10:1:1); mp: 175.5–176.7 °C; ¹H NMR (600 MHz, DMSO) δ 8.05 (s, 2H), 7.63 (t, J = 7.7 Hz, 1H), 7.50–7.40 (m, 2H), 7.17–7.08 (m, 3H), 7.07–7.05 (m, 1H), 4.57 (s, 2H), 3.80 (s, 3H), 2.46 (s, 3H); ¹³C NMR (151 MHz, DMSO) δ 166.1 (C, C-6), 159.5 (C, C-2), 159.0 (C, C-3'), 158.1 (C, C-4), 157.7 (C, C-2″), 155.7 (C, C-6″), 137.0 (C, C-1'), 135.1 (CH, C-4″), 129.9 (CH, C-5'), 121.8 (CH, C-3″), 120.6 (CH, C-5″), 120.4 (CH, C-6'), 115.8 (CH, C-4'), 115.1 (CH, C-2'), 115.0 (C, CN), 114.0 (C, CN), 93.2 (C, C-5), 86.0 (C, C-3), 55.3 (CH₃, OCH₃), 35.3 (CH₂, -SCH₂-), 23.9 (CH₃, methylpyridine). APCI-HRMS m/z: calculated for C₂₁H₁₈N₅OS [M + H] + 388.1227, found 388.1208; Purity (HPLC, λ = 254): 100%

2-((6-amino-3,5-dicyano-4-(3-methoxyphenyl)pyridin-2-yl)thio)acetamide (6d)

Prepared as for 6c from 3-methoxybenzaldehyde (5 mmol, 0.608 ml) and 2-bromoacetamide (0.691 g, 5 mmol) to yield 6d which was recrystallised from MeOH as yellowish powder (0.537 g, 31.6%): Rf: 0.78 (DCM/PE/EtOAc 10:1:1); mp: 232.3–237.0 °C; ¹H NMR (600 MHz, DMSO) δ 7.98 (s, 2H), 7.53–7.42 (m, 2H), 7.20 (s, 1H), 7.14–7.09 (m, 2H), 7.08–7.04 (m, 1H), 3.89 (s, 2H), 3.81 (s, 3H); ¹³C NMR (151 MHz, DMSO) δ 168.8 (C, CO), 166.0 (C, C-6), 159.4 (C-C2), 159.0 (C, C-3'), 158.1 (C, C-4), 135.1 (C, C-1'), 129.9 (CH, C-5'), 120.4 (CH, C-6'), 115.8 (C, C-4'), 115.1 (C, CN), 115.0 (CH, C-2'), 114.0, (C, CN) 93.3 (C, C-5), 86.0 (C, C-5), 55.3 (CH₃, OCH₃), 33.3 (CH₂, -S-CH₂-); APCI-HRMS m/z: calculated for C₁₆H₁₄N₅O₂S [M + H] + 340.0863, found 340.0844; Purity (HPLC, λ = 254): 100%

2-((6-amino-3,5-dicyano-4-(4-hydroxyphenyl)pyridin-2-yl)thio)acetamide (6e)

Prepared as for 6c from 4-hydroxybenzaldehyde (0.614 g, 5 mmol) and 2-bromoacetamide (0.692 g, 5 mmol) to yield compound 6e which was recrystallised from MeOH as cream white powder (0.650 g, 40.0%): Rf: 0.15 (EtAOc only); mp: 267.6–268.0 °C; ¹H NMR (600 MHz, DMSO) δ 10.03 (s, 1H), 7.89 (s, 2H), 7.46 (s, 1H), 7.40–7.34 (m, 2H), 7.19 (s, 1H), 6.95–6.89 (m, 2H), 3.88 (s, 2H); ¹³C NMR (151 MHz, DMSO) δ 168.9 (C, CO), 166.1 (C, C-6), 159.7 (C, C-2), 159.4 (C, C-4), 158.3 (C, C-4'), 130.1 (C, C-1'), 124.1 (CH, C-2', C-6'), 115.4 (CH, C-3', C-5'), 115.4 (C, CN, 115.4 (C, CN), 93.2 (C, C-5), 85.8 (C, C-3), 33.3 (CH₂, -SCH₂-); APCI-HRMS m/z: calculated for C₁₅H₁₂N₅O₂S [M + H] + 326.0706, found 326.0688; Purity (HPLC, λ = 254): 100%

2-((6-amino-3,5-dicyano-4-(3-hydroxyphenyl)pyridin-2-yl)thio)acetamide (6f)

Prepared as for 6c from 3-hydroxybenzaldehyde (0.612 g, 5 mmol) and 2-bromoacetamide (0.690 g, 5 mmol) to yield 6f which was recrystallised from ethanol as light brown solid (0.735 g, 45.2%): Rf: 0.28 (DCM/MeOH: 10:1); mp: 248.1–248.4 °C; ¹H NMR (600 MHz, DMSO) δ 9.83 (s, 1H), 7.95 (d, J = 6.9 Hz, 2H), 7.47 (s, 1H), 7.35 (t, J = 7.9 Hz, 1H), 7.20 (s, 1H), 6.95 (ddd, J = 8.2, 2.4, 0.8 Hz, 1H), 6.92–6.88 (m, 1H), 6.88–6.85 (m, 1H), 3.89 (s, 2H); ¹³C NMR (151 MHz, DMSO) δ 168.8 (C, CO), 166.1 (C, C-6), 159.5 (C, C-2), 158.3 (C, C-4), 157.3 (C, C-3'), 135.0 (C, C-1'), 129.9 (CH, C-5'), 118.8 (CH, C-6'), 117.2 (CH, C-4'), 115.1 (CH, C-2'), 115.0 (C, CN), 115.0 (C, CN), 93.2 (C, C-5), 85.9 (C, C-3), 33.3 (CH₂, -SCH₂-); APCI-HRMS m/z: calculated for C₁₅H₁₂N₅O₂S [M + H] + 326.0706, found 326.0689; Purity (HPLC, λ = 254): 100%

2-((6-amino-4-(benzo[d][1,3]dioxol-5-yl)-3,5-dicyanopyridin-2-yl)thio)acetamide (6g)

Prepared as for 6c from piperonaldehyde (0.752 g, 5 mmol) and 2-bromoacetamide (0.690 g, 5 mmol) to yield 6g which was recrystallised from MeOH as light orange solid (0.682 g, 38.6%): Rf: 0.40 (DCM/MeOH: 10:1); mp: 244.5–245.1 °C; ¹H NMR (600 MHz, DMSO) δ 7.93 (s, 2H), 7.46 (s, 1H), 7.19 (s, 1H), 7.14 (d, J = 1.8 Hz, 1H), 7.10 (d, J = 8.0 Hz, 1H), 7.02 (dd, J = 8.0, 1.8 Hz, 1H), 6.15 (s, 2H), 3.88 (s, 2H).; ¹³C NMR (151 MHz, DMSO) δ 168.8 (C, CO), 166.0 (C, C-6), 159.5 (C, C-2), 157.9 (C, C-4), 148.9 (C, C-3',C-4'), 147.3 (C, C-1'), 127.2 (C, C-6'), 122.9 (CH, C-2'), 115.2 (CH, C-5'), 108.8, (C, CN) 108.5 (C, CN), 101.7 (CH₂, C at dioxol), 93.4 (C, C-5), 86.1 (C, C-3), 33.3 (CH₂, -SCH₂-); APCI-HRMS m/z: calculated for C₁₆H₁₂N₅O₃S [M + H] + 354.0655, found 354.0635; Purity (HPLC, λ = 254): 100%

3-(((6-amino-3,5-dicyano-4-(furan-2-yl)pyridin-2-yl)thio)methyl)benzoic acid (6h)

Prepared as for 6c from furan-2-carbaldehyde (0.414 g, 5 mmol) and 3-(bromomethyl)benzoic acid (1.081 g, 5 mmol) to yield 6h which was recrystallised from acetone as cream white powder (0.341 g, 18.1%):Rf: 0.38 (DCM:MeOH 10:1); mp: 279.59 °C; ¹H NMR (600 MHz, DMSO) δ 8.10–8.03 (m, 2H), 7.86 (d, J = 6.3 Hz, 2H), 7.57 (d, J = 7.0 Hz, 1H), 7.37 (d, J = 3.6 Hz, 1H), 7.30 (t, J = 7.6 Hz, 1H), 6.81 (dd, J = 3.6, 1.7 Hz, 1H), 4.50 (s, 2H).; ¹³C NMR (151 MHz, DMSO) δ 167.4 (C, COOH), 160.1 (C, C-6), 146.5 (C, C-2), 145.1 (C, C-4, C-2'), 143.7 9CH, C-5', 136.5 (C-3'), 127.7 (C, C-4″, C-1″), 116.3 (CH, C-5″,C-6″, C-2″), 115.7 (C, CN), 115.7 C, CN), 112.8 (CH, C-3', C-4'), 89.2 (C, C-5), 81.6 (C, C-3), 33.4 (CH₂, -SCH₂); APCI-HRMS m/z: calculated for C₁₉H₁₃N₄O₃S [M + H] + 377.0703, found 377.0687; Purity (HPLC, λ = 254): 100%

3-(((6-amino-3,5-dicyano-4-phenylpyridin-2-yl)thio)methyl)benzoic acid (6i)

Prepared as for 6c from benzaldehyde (0.460 ml, 5 mmol) and 3-(bromomethyl)benzoic acid (1.083 g, 5 mmol) to yield 6i which was recrystallised from hexane as white fluffy solid (0.601 g, 31.1%): Rf: 0.89 (DCM:MeOH 10:1); mp: 200 °C; ¹H NMR (600 MHz, DMSO) δ 8.01 (s, 2H), 7.78 (d, J = 7.6 Hz, 1H), 7.58–7.49 (m, 5H), 7.44 (d, J = 7.5 Hz, 1H), 7.22 (t, J = 7.6 Hz, 1H), 4.51 (s, 2H); ¹³C NMR (151 MHz, DMSO) δ 166.4 (C-COOH), 159.5 (C, C-6), 158.3 (C, C-2), 135.7 (C, C-4), 133.9 (C, C-3″), 130.3 (C, C-1'), 130.2 (CH, C-4″), 128.6 (C, C-1″), 128.3 (CH, C-3', C-5', C-4', C2″) 128.0 (CH, C-5″, C-6″), 127.1 (CH, C-2', C-4'), 115.1 (C, CN), 115.1 (C-CN), 93.2 (C, C-5), 85.9 (C, C-3), 33.6 (C, -SCH₂); APCI-HRMS m/z: calculated for C₂₁H₁₅N₄O₂S [M + H] + 354.0655, found 354.0635; Purity (HPLC, λ = 254): 100%

2-amino-4-(3-hydroxyphenyl)-6-(((6-methylpyridin-2-yl)methyl)thio)pyridine-3,5-dicarbonitrile (6j)

Prepared as for 6c from 3-hydroxybenzaldehyde (0.610 g, 5 mmol) and 2-(bromomethyl)-6-methylpyridine (0.933 g, 5 mmol) to yield 6j which was recrystallised from MeOH as light yellow powder (0.580 g, 31.1%): Rf: 0.73 (DCM/MeOH 10:1); mp: 231.22 °C; ¹H NMR (600 MHz, DMSO) δ 9.82 (s, 1H), 8.04 (s, 2H), 7.63 (t, J = 7.7 Hz, 1H), 7.43 (d, J = 7.7 Hz, 1H), 7.34 (t, J = 7.9 Hz, 1H), 7.15 (d, J = 7.7 Hz, 1H), 6.94 (ddd, J = 8.2, 2.4, 0.7 Hz, 1H), 6.91–6.83 (m, 2H), 4.56 (s, 2H), 2.46 (s, 3H); ¹³C NMR (151 MHz, DMSO) δ 166.1 (C, C-6), 159.5 (C, C-2), 158.4 (C, C-4), 157.7 (C, C-2″), 157.3 (C, C-6″), 155.7 (C, C-3'), 137.0 (C, C-1'), 135.0 (CH, C-4″), 129.8 (CH, C-5'), 121.8 (CH, C-3″), 120.6 (CH, C-5″), 118.8 (C, C-6'), 117.2 (CH,C-4'), 115.1 (CH, C-2'), 115.0 (C, CN), 115.0 (C, CN), 93.1, (C, C-5) 85.8 (C, C-3), 35.4 (CH₂, -SCH₂-), 23.9 (CH₃, Methylpyridine); APCI-HRMS m/z: calculated for C₂₀H₁₆N₅OS [M + H] + 374.1070, found 354.1063; Purity (HPLC, λ = 254): 100%

3-(((6-amino-3,5-dicyano-4-(4-(methylthio)phenyl)pyridin-2-yl)thio)methyl)benzoic acid (6k)

Prepared as for 6c from 4-(methylthio)benzaldehyde (0.664 ml, 5 mmol) and 3-(bromomethyl)benzoic acid (1.080 g, 5 mmol) to yield 6k which was recrystallised from MeOH as yellowish powder (0.580 g, 31.1%): Rf: 0.69 (DCM/PE/EtOAc 10:1:1); mp: 193.2–193.3 °C; ¹H NMR (600 MHz, DMSO) δ 8.05 (s, 1H), 7.83 (d, J = 7.6 Hz, 1H), 7.73 (d, J = 7.5 Hz, 1H), 7.47 (dd, J = 6.1, 4.2 Hz, 2H), 7.44–7.35 (m, 3H), 4.57 (s, 2H), 2.53 (s, 3H); ¹³C NMR (151 MHz, DMSO) δ 166.01 (C, COOH), 163.8 (C, C-6), 159.6 (C, C-2), 157.9 (C, C-4), 141.7 (C, C-3″, C-4'), 130.1 (C, C-4'), 129.8 (C, C-4″), 129.1 (C, C-1″), 128.3 (CH, C-2″), 128.2 (CH, C-5″, C-6″), 125.2 (CH, C-2', C-3', C-5', C-6'), 115.4 (C, CN, CN), 93.1 (C, C-5), 85.9 (C, C-3), 32.9 (CH₂, -SCH₂-), 14.0 (CH₃, SCH₃). APCI-HRMS m/z: calculated for C₂₂H₁₇N₄O₂S₂ [M + H] + 3433.0787, found 433.0784; Purity (HPLC, λ = 254): 100%

3-(((6-amino-3,5-dicyano-4-(4-methoxyphenyl)pyridin-2-yl)thio)methyl)benzoic acid (6l)

Prepared as for 6c from 4-methoxybenzaldehyde (0.608 ml, 5 mmol) and 3-(bromomethyl)benzoic acid (1.080 g, 5 mmol) to yield 6l which was recrystallised from MeOH as white solid (0.311 g, 14.9%): Rf: 0.97 (DCM/PE/EtOAc 10:1:1); mp: 226.5–226.6 °C; ¹H NMR (600 MHz, DMSO) δ 8.07 (s, 1H), 7.87 (d, J = 7.2 Hz, 1H), 7.57 (d, J = 6.7 Hz, 1H), 7.47 (d, J = 8.7 Hz, 2H), 7.28 (t, J = 7.6 Hz, 1H), 7.08 (d, J = 8.8 Hz, 2H), 4.50 (s, 2H), 3.82 (s, 3H); ¹³C NMR (151 MHz, DMSO) δ 166.3 (C, COOH), 160.8 (C, C-6), 159.7 (C, C-2), 158.1 (C, C-4'), 136.6 (C, C-4), 130.4, (C, C-3″) 130.3 (CH, C-4″), 128.4 (C, C-1″, C-1'), (128.4 (CH, C-2', C-6'), 127.7 (CH, C-2″), 125.8 (CH, C-5″, C-6″), 115.6 (CH, C-3', C-5'), 115.5 (C, CN), 114.1, (C, CN), 93.2 (C, C-5), 85.9 (C, C-3), 55.4 (CH₃, OCH₃), 33.3 (CH₂, -SCH₂-); APCI-HRMS m/z: calculated for C₂₂H₁₇N₄O₃S [M + H] + 417.1016, found 417.1009; Purity (HPLC, λ = 254): 100%

2-amino-4-(4-hydroxyphenyl)-6-(((6-methylpyridin-2-yl)methyl)thio)pyridine-3,5-dicarbonitrile (6 m)

Prepared as for 6c from 4-hydroxybenzaldehyde (0.611 g, 5 mmol) and 2-(bromomethyl)-6-methylpyridine (0.932 g, 5 mmol) to yield 6m which was recrystallised from MeOH as creamy white powder (1.611 g, 86.3%): Rf: 0.68 (PE:EtOAC 1:1); mp: 190.8–191.7 °C; ¹H NMR (600 MHz, DMSO) δ 10.09 (s, 1H), 7.63 (t, J = 7.7 Hz, 1H), 7.43 (d, J = 7.6 Hz, 1H), 7.41–7.33 (m, 2H), 7.14 (d, J = 7.7 Hz, 1H), 6.94–6.87 (m, 2H), 4.54 (s, 2H), 2.45 (s, 3H); ¹³C NMR (151 MHz, DMSO) δ 166.2 (C, C-6), 159.8 (C, C-2), 159.5 (C, C-4), 158.4 (C, C-4'), 157.8 (C, C-2″), 155.9 (C, C-6″), 137.1 (CH, C-4″), 130.3 (C, C-1'), 124.1 (CH, C-2', C-6'), 121.9 (CH, C-3″), 120.8 (CH, C-5″), 115.7 (CH, C-3', C-5'), 115.6 (C, CN), 115.4 (C, CN), 93.1 (C, C-5), 85.8 (C, C-3), 35.4 (CH₂, -SCH₂-), 24.0 (CH₃, Methylpyridine); APCI-HRMS m/z: calculated for C₂₀H₁₆N₅OS [M + H] + 374.1070, found 374.1061; Purity (HPLC, λ = 254): 100%

2-amino-4-(4-hydroxyphenyl)-6-((3-methylbenzyl)thio)pyridine-3,5-dicarbonitrile (6n)

Prepared as for 6c from 4-hydroxybenzaldehyde (0.612 g, 5 mmol) and 1-(bromomethyl)-3-methylbenzene (0.678 ml, 5 mmol) to yield 6n which was recrystallised from MeOH as light yellowish powder (0.532 g, 28.6%): Rf: 0.75 (PE/EtOAC 1:1); mp: 224.9–226.1 °C;¹H NMR (600 MHz, DMSO) δ 10.06 (s, 1H), 7.39–7.33 (m, 2H), 7.33–7.26 (m, 2H), 7.20 (t, J = 7.6 Hz, 1H), 7.06 (d, J = 7.5 Hz, 1H), 6.93–6.85 (m, 2H), 4.45 (s, 2H), 2.28 (s, 3H); ¹³C NMR (151 MHz, DMSO) δ 166.3 (C, C-6), 159.7 (C, C-2), 159.4 (C, C-4), 158.4 (C, C-4'), 137.6 (C, C-1″), 137.3 (C, C-3″), 130.3 (C, C-1'), 129.9 (CH, C-2', C-6'), 128.3 (CH, C-5″), 127.9 (CH, C-4″), 126.4 (CH, C-2″), 124.2 (CH, C-6″), 115.6 (CH,C-3', C-5'), 115.6 (C, CN), 115.4 (C, CN), 93.1 (C, C-5), 85.7 (C, C-3), 33.2 (CH₂, -S-CH₂-), 20.9 (CH₃, Methylbenzyl); APCI-HRMS m/z: calculated for C₂₁H₁₇N₄OS [M + H] + 373.1118, found 373.1111; Purity (HPLC, λ = 254): 100%

3-(((6-amino-3,5-dicyano-4-(3-methoxyphenyl)pyridin-2-yl)thio)methyl)benzoic acid (6o)

Prepared as for 6c from 3-methoxybenzaldehyde (0.608 ml, 5 mmol) and 3-(bromomethyl)benzoic acid (1.083 g, 5 mmol) to yield 6o which was recrystallised from MeOH as cream white powder (0.490 g, 23.5%):Rf: 0.70 (DCM/PE/EtOAc 10:1:1); mp: 240.0–241.1 °C; ¹H NMR (600 MHz, DMSO) δ 8.04 (s, 1H), 7.83 (d, J = 7.7 Hz, 1H), 7.69 (d, J = 7.6 Hz, 1H), 7.48–7.42 (m, 1H), 7.37 (t, J = 7.7 Hz, 1H), 7.14–7.04 (m, 3H), 4.57 (s, 2H), 3.79 (s, 3H); ¹³C NMR (151 MHz, DMSO) δ 166.0 (C, COOH), 159.5 (C, C-6), 159.0 (C, C-2), 158.2 (C, C-3'), 137.5 (C, C-4), 135.2 (C, C-1', C-3″), 132.5 (C,C-4″), 130.1 (C, C-5', C-1″), 130.0 (CH, C-2″), 128.2 (CH, C-5″, C6″), 120.5 (CH, C-6'), 115.9 (CH, C-4'), 115.2 (C, CN), 115.2 (C, CN), 114.0 (CH, C-2'), 93.3 (C, C-5), 86.1 (C, C-3), 55.3 (CH₃, OCH₃), 32.9 (CH₂, -SCH₂-); APCI-HRMS m/z: calculated for C₂₂H₁₆N₄O₃S [M + H] + 417.1016, found 417.1012; Purity (HPLC, λ = 254): 100%

2-amino-4-(4-fluorophenyl)-6-(((6-methylpyridin-2-yl)methyl)thio)pyridine-3,5-dicarbonitrile (6p)

Prepared as for 6c from 4-fluorobenzaldehyde (0.536 ml, 5 mmol) and 2-(bromomethyl)-6-methylpyridine (0.932 g, 5 mmol) to yield 6o which was recrystallised from MeOH as white powder (0.222 g, 11.8%): Rf: 0.84 (PE:EtOAC 1:1); mp: 216.8–216.9 °C; ¹H NMR (600 MHz, DMSO) δ 8.33 (s, 2H), 7.62 (dt, J = 8.9, 6.5 Hz, 3H), 7.48–7.36 (m, 3H), 7.14 (d, J = 7.7 Hz, 1H), 4.55 (s, 2H), 2.45 (s, 3H); ¹³C NMR (151 MHz, DMSO) δ 166.2 C, C-6), 164.0 (C, C-2), 162.3 (C, C-4), 159.5 (C, C-2″), 157.8 (C, C-6″), 157.5 (CH, C-5″), 155.8 (CH, C-3″), 137.1 (CH, C-4″), 131.1 (d, J = 8.8 Hz,C, C-1'), 131.1 (C, C-6'), 130.3 (d, J = 3.0 Hz, CH, C-2', C-6'), 121.9 (C, CN), 120.8 (C, CN), 115.8 (d, J = 22.0 Hz, C, C-4'), 115.2 (d, J = 11.2 Hz, CH, C-3', C-5'), 93.3 (C, C-5), 86.1 (C, C-5), 35.4 (CH₂, -SCH₂-), 24.0 (CH₃, Methylpyridine); APCI-HRMS m/z: calculated for C₂₀H₁₅FN₅S [M + H] + 376.1027, found 376.1039; Purity (HPLC, λ = 254): 100% General procedure for the synthesis of 6q–6s and 7a:

2-amino-6-(((2-(4-chlorophenyl)thiazol-4-yl)methyl)thio)-4-(4-(2-hydroxyethoxy)phenyl)pyridine-3,5-dicarbonitrile (6q)

Cyanothioacetamide (0.503 g, 5 mmol) was dissolved in 10 ml EtOH. The reaction mixture was then stirred for 15–20 min before adding 4-(2-hydroxyethoxy)benzaldehyde (0.696 ml, 5 mmol) while stirring at room temperature. After obtaining a homogeneous mixture, MN (0.315 ml, 5 mmol) was added and the mixture stirred until it became homogeneous again, then left to stand at room temperature for 12–14 h. The mixture was then diluted with an equal volume of DMF and 10% aqueous KOH (2.8 ml, 5 mmol) and left to stand for 24 h. 4-(Chloromethyl)-2-(4-chlorophenyl)thiazole (1.221 g, 5 mmol) was added to the mixture and continuously stirred for 3 h, after which 10% aqueous KOH (5.6 mL, 10 mmol) was added again. After 2–4 h, ice was added and the resulting precipitate was filtered off, washed with distilled H₂O, EtOH and hexane, dried (30 °C) and recrystallised from MeOH to yield the title compound 6q as light brown powder (1.355 g, 52.1%): Rf: 0.82 (DCM/PE/EtOAc 10:1:1); mp: 162.1–163.5 °C; ¹H NMR (600 MHz, DMSO) δ 7.97–7.92 (m, 2H), 7.89 (s, 1H), 7.59–7.53 (m, 2H), 7.50–7.42 (m, 2H), 7.15–7.02 (m, 2H), 4.86 (t, J = 5.5 Hz, 1H), 4.64 (s, 2H), 4.08 (t, J = 5.0 Hz, 2H), 3.74 (dd, J = 10.1, 5.2 Hz, 2H); ¹³C NMR (151 MHz, DMSO) δ 165.8 (C, C-2″), 165.6 (C, C-6), 160.3 (C, C-2), 159.7 (C, C-4'), 158.1 (C, C-4), 152.4 (C, C-4″), 134.8 (C, C-1″'), 131.6 (C, C-4″'), 130.1 (C, C-1'), 129.2 (CH, C-3'", C5"'), 127.7 (CH, C-2', C-6'), 125.7 (CH, C-2"', C-6"'), 118.7 (CH, C-5"), 115.3 (CH, C-3', C-5'), 115.3 (C, CN), 114.5 (C, CN), 93.4 (C, C-5), 85.9 (C, C-3), 69.7(CH₂, OCH₂CH₂OH), 59.4 (CH₂, OCH₂CH₂OH), 29.2 (CH₂, -SCH₂-); APCI-HRMS m/z: calculated for C₂₅H₁₉ClN₅O₂S₂ [M + H] + 520.0663, found 520.0651; Purity (HPLC, λ = 254): 100%

2-amino-6-(((2-(4-chlorophenyl)thiazol-4-yl)methyl)thio)-4-(4-hydroxyphenyl)pyridine-3,5-dicarbonitrile (6r)

Prepared as for 6q from 4-hydroxybenzaldehyde (0.612 g, 5 mmol) and 4-(chloromethyl)-2-(4-chlorophenyl)thiazole (1.220 g, 5 mmol) to yield 6r which was recrystallised from MeOH as light pink powder (1.203 g, 50.6%): Rf: 0.81(DCM/PE/EtOAc 10:1:1); mp: 233.8–234.6 °C; ¹H NMR (600 MHz, DMSO) δ 10.06 (s, 1H), 7.97–7.92 (m, 2H), 7.90 (s, 1H), 7.58–7.52 (m, 2H), 7.39–7.33 (m, 2H), 6.92–6.87 (m, 2H), 4.63 (s, 2H); ¹³C NMR (151 MHz, DMSO) δ 165.9 (C, C-2″), 165.6 (C, C-6), 159.8 (C, C-2), 159.4 (C, C-4), 158.4 (C, C-4'), 152.5 (C, C-4″), 134.8 (C, C-1″'), 131.6 (C, C-4″'), 130.2 (C, C-1'), 129.3 (CH, C-2', C-6'), 127.8 (CH, C-2″', C-6″'), 124.1 (CH, C-3″', C-5″'), 118.8 (CH, C-3', C-5'), 115.6 (CH, C-5″), 115.5 (C, CN), 115.4 (C, CN), 93.3 (C, C-5), 85.8 (C, C-3), 29.3 (CH₂, -SCH₂-); APCI-HRMS m/z: calculated for C₂₃H₁₅ClN₅OS₂ [M + H] + 476.0401, found 476.0386; Purity (HPLC, λ = 254): 100%

2-amino-6-(((2-(4-chlorophenyl)thiazol-4-yl)methyl)thio)-4-(4-methoxyphenyl)pyridine-3,5-dicarbonitrile (6 s)

Prepared as for 6q from 4-methoxybenzaldehyde (0.608 ml, 5 mmol) and 4-(chloromethyl)-2-(4-chlorophenyl)thiazole (1.222 g, 5 mmol) to yield 6s which was recrystallised from MeOH as cream white solid (0.850 g, 34.9%): Rf: 0.72 (DCM/PE/EtOAc 10:1:1); mp: 238.2–240.4 °C; ¹H NMR (600 MHz, DMSO) δ 8.06 (s, 2H), 7.96–7.92 (m, 2H), 7.89 (s, 1H), 7.58–7.53 (m, 2H), 7.50–7.46 (m, 2H), 7.13–7.08 (m, 2H), 4.64 (s, 2H), 3.83 (s, 3H); ¹³C NMR (151 MHz, DMSO) δ 165.8 (C, C-2″), 165.6 (C, C-6), 160.8 (C, C-2), 159.7 (C, C-4'), 158.0 (C, C-4), 152.4 (C, C-4″), 134.8 (C, C-1″'), 131.6 (C, C-4″'), 130.1 (C, C-1'), 129.2 (CH, C-2', C-6'), 127.7 (CH, C-″'3, C-5″'), 125.7 (CH, C-2″', C-6″'), 118.7 (CH, C-5″), 115.4 (CH, C-3', C-5'), 115.3 (C, CN), 114.0 (C, CN), 93.4 (C, C-5), 85.9 (C, C-3), 55.3 (CH₃, OCH₃), 29.3 (CH₂, -SCH₂-); APCI-HRMS m/z: calculated for C₂₄H₁₇ClN₅OS₂ [M + H] + 490.0558, found 490.0538; Purity (HPLC, λ = 254): 100%

3,6-diamino-5-cyano-4-(4-fluorophenyl)thieno[2,3-b]pyridine-2-carboxamide (7a)

Prepared as for 6q from 4-fluorobenzaldehyde (0.536 ml, 5 mmol) and 2-bromoacetamide (0.693 g, 5 mmol) to yield compound 7a which was recrystallised from MeOH as yellow to greenish solid (0.687 g, 42.0%): Rf: 0.77 (DCM/PE/EtOAc 10:1:1); mp: 257.5–260.7 °C; ¹H NMR (600 MHz, DMSO) δ 7.72–7.53 (m, 2H), 7.52–7.39 (m, 2H), 7.33 (s, 2H), 6.99 (s, 2H), 5.63 (s, 2H); ¹³C NMR (151 MHz, DMSO) δ 166.7 (C, CO), 163.6 (C, C-6), 163.4 (C, between position 1 & 7 of thienopyridine), 162.0 (C, C-4), 158.3 (C, C-3), 151.3 (C, C-2), 146.1 (C, C-1'), 130.60 (d, J = 8.5 Hz, C-2',C-6'), 129.91 (d, J = 3.1 Hz), C-3', C-5'), 116.22–115.68 ((m), C-4'), 114.2 (C, between position 3 & 4 of thienopyridine), 93.3 (C, CN), 90.4 (C-C5); APCI-HRMS m/z: calculated for C₁₅H₁₁FN₅OS [M + H] + 328.0663, found 328.0652; Purity (HPLC, λ = 254): 100% General procedure for synthesis of 7b and 7c

3,6-diamino-5-cyano-4-(4-methoxyphenyl)thieno[2,3-b]pyridine-2-carboxamide (7b)

Prepared by dissolving 2-((6-amino-3,5-dicyano-4-(4-methoxyphenyl)pyridin-2-yl)thio)acetamid (6a) (0.203 g, 0.598 mmol) in 10 ml DMF. Two or three drops of 10% aqueous KOH was then added to the solution. The reaction mixture was maintained at room temperature for 24 h. Product was precipitated by adding ice. The resulting precipitate was filtered off, washed with distilled H₂O, EtOH and hexane, dried (30 °C) and recrystallised from MeOH to yield 7b as yellow powder (0.054 g, 31.8%): Rf: 0.75 (DCM/PE/EtOAc 10:1:1); mp: 263.9–264.9 °C; ¹H NMR (600 MHz, DMSO) δ 7.42 (d, J = 8.4 Hz, 2H), 7.30 (s, 2H), 7.14 (d, J = 8.4 Hz, 2H), 6.99 (s, 2H), 5.70 (s, 2H), 3.85 (s, 3H); ¹³C NMR (151 MHz, DMSO) δ 166.8 (C, CO), 163.4 (C, C-6), 160.2 (C, C-4'), 158.5 (C, between position 1 & 7 of thienopyridine), 152.3 (C, C-4), 146.3 (C, C-3), 129.7 (CH, C-2', C-6'), 125.5 (C, C-2), 116.0 (C, C-1'), 114.4 (C, between position 3 & 4 of thienopyridine), 114.3 (CH, C-3',C-5'), 92.9 (C, CN), 90.5 (C, C-5), 55.3 (CH₃, OCH₃); APCI-HRMS m/z: calculated for C₁₆H₁₄N₅O₂S [M + H] + 340.0863, found 340.0853; Purity (HPLC, λ = 254): 93.8%

3,6-diamino-4-(benzo[d][1,3]dioxol-5-yl)-5-cyanothieno[2,3-b]pyridine-2-carboxamide (7c)

Prepared as for 7b from 2-((6-amino-4-(benzo[d][1,3]dioxol-5-yl)-3,5-dicyanopyridin-2-yl)thio)acetamide (6g) (0.134 g, 0.379 mmol) to yield 7c which was washed with distilled H₂O and hexane and dried without further purification to yield an orange solid (0.034 g, 19.3%): Rf: 0.56 (DCM/PE/EtOAc 10:1:1); mp: 296.82 °C; ¹H NMR (600 MHz, DMSO) δ 7.28 (s, 1H), 7.15–7.03 (m, 1H), 7.03–6.91 (m, 1H), 6.06 (dd, J = 118.5, 114.5 Hz, 2H); ¹³C NMR (151 MHz, DMSO) δ 166.8 (C, CO), 158.4 (C, C-6), 152.0 (C, between position 1 & 7 of thienopyridine), 148.5 (C, C-4), 147.5 (C, C3', C4'), 146.3 (C, C-3), 126.8 (C, C-1'), 122.0 (C, C-2), 115.9 (C, between position 3 & 4 of thienopyridine), 114.4 (CH, C-6'), 108.8 (CH, C-2'), 108.7 (C, CN), 101.7 (CH, C-5'), 92.8 (CH₂, at dioxol), 90.5 (C, C-5); APCI-HRMS m/z: calculated for C₁₆H₁₂N₅O₃S [M + H] + 354.0655, found 354.0647; Purity (HPLC, λ = 254): 62.6%

3,6-diamino-5-cyano-4-(4-methoxyphenyl)thieno[2,3-b]pyridine-2-carboxamide (7d)

Prepared by refluxing a solution of 2-((6-amino-3,5-dicyano-4-(3-hydroxyphenyl)pyridin-2-yl)thio)acetamide (6f) (0.201 g, 0.518 mmol) and KOH (0.080 g, 1.426 mmol) in EtOH (10 ml) for 6 h. After cooling, ice-cold water was added to the reaction mixture to precipitate the product. The resulting precipitate was filtered off, washed with distilled H₂O, EtOH and hexane, dried (30 °C) and recrystallised from MeOH to yield 7d as greenish solid (0.163 g, 66.4%): Rf: 0.15 (DCM/PE/EtOAc 10:1:1); mp: 285.12 °C; ¹H NMR (600 MHz, DMSO) δ 9.97 (s, 1H), 7.39 (t, J = 7.7 Hz, 1H), 7.31 (s, 2H), 6.98 (s, 3H), 6.90–6.76 (m, 2H), 5.70 (s, 2H); ¹³C NMR (151 MHz, DMSO) δ 166.7 (C,CO), 163.3 (C, C-6), 158.4 (C, C, between position 1 & 7 of thienopyridine), 157.6 (C, C-3'), 152.2 (C, C-4), 146.0 (C, C-1'), 134.8 (C, C-3), 130.4 (CH, C-5'), 118.3 (C, C-2), 116.9 (C, C between position 3 & 4 of thienopyridine), 115.8 (CH, C-6'), 114.7 (CH, C-4'), 113.9 (CH, C-2'), 93.0 (C, CN), 89.9 (C, C-5); APCI-HRMS m/z: calculated for C₁₅H₁₂N₅O₂S [M + H] + 326.0706, found 326.0690; Purity (HPLC, λ = 254): 100%

Biology

In vitro evaluation

Materials and methods

All reagents were commercially available and purchased from various manufacturers. Radioligands [³H]DPCPX (120 Ci/mmol) and [³H]NECA (27.1 Ci/mmol) were obtained from PerkinElmer. Adenosine deaminase from bovine spleen (157 units/mg, 5.9 mg/ml, or 130 units/mg, 6.8 mg/ml), CPA, DPCPX, istradefylline, caffeine and anhydrous magnesium chloride (MgCl₂) were all obtained from Sigma-Aldrich. Radioactivity was counted by a PerkinElmer Tri-CARB 2810 TR liquid scintillation analyser.

Ethics

The collection of tissue samples for the A₁ and A_2A AR radioligand binding assays were approved by the Health Sciences Ethics Office for Research, Training and Support, North-West University (NWU-00418-21-A5) and were performed in accordance with the guidelines of the South African National Standard (SANS) document (The care and use of animals for scientific purposes).

Tissue samples

Male Sprague–Dawley rats whole brain membranes (including striata and excluding cerebellum and brain stem) and rat striatal membranes were used for the A₁ and A_2A AR radioligand binding assays, respectively, and prepared as described in literature [53]. Upon dissection, the tissue samples were snap frozen with liquid nitrogen and stored at −70 °C. The samples were later thawed on ice, weighed and disrupted for 90 s (whole brain) or 30 s (striata) with the aid of a Polytron homogeniser (model: Polytron PT 10-35 GT) in 10 volumes of ice-cold 50 mM Tris buffer (pH 7.7 at 25 °C). The resulting homogenate was centrifuged at 20,000 g for 10 min at 4 °C and the pellet was resuspended in 10 volumes of ice-cold Tris buffer, again with the aid of a Polytron homogeniser as above. The resulting suspension was recentrifuged and the pellet obtained was suspended in Tris buffer (pH 7.7 at 25 °C) to a volume of 5 mL/g original tissue weight. The whole brain and striatal membranes were aliquoted into microcentrifuge tubes and stored at −70 °C until needed. Protein concentration of the rat brain tissues was determined according to the Bradford protein assay, using bovine serum albumin as reference standard [74].

Adenosine A₁ and A_2A receptor radioligand binding assays

The degree of binding affinity the test compounds showed toward A₁ and A_2A ARs were determined through radioligand binding assays, as previously described in literature [53, 74–76]. The A₁ AR radioligand binding assay used rat whole brain membranes (expressing A₁ ARs) and 0.1 nM 1,3-[³H]-dipropyl-8-cyclopentylxanthine ([³H]DPCPX) as radioligand while the A_2A AR radioligand binding assay used rat striatal membranes (expressing A_2A ARs) and 4 nM [³H]-5′-N-ethylcarboxamidoadenosine ([³H]NECA) as radioligand. In A_2A AR radioligand binding assays, 50 nM CPA was also added to reduce the binding of [³H]NECA to adenosine A₁ receptors and 10 mM MgCl₂ was also included to increase radioligand binding and decrease non-specific binding. Adenosine deaminase was included in both A₁ and A_2A binding assay to inactivate any remaining endogenous adenosine. The incubations were carried out in 4 mL polypropylene tubes that were precoated with Sigmacote (Sigma–Aldrich). All incubations were prepared with 50 mM Tris buffer (pH 7.7 at 25 °C) to a volume of 1 mL. Each incubation of the A₁ assay consisted of: (i) test compound (10 µL), (ii) 0.1 nM [³H]DPCPX (radioligand solution, 100 µL) and (iii) 120 µg rat whole brain membranes and 0.1 units/mL adenosine deaminase (membrane suspension, 890 µL). Whereas every incubation of the A_2A assay consisted of: (i) 120 µg rat striatal membranes, 0.2 units/mL adenosine deaminase. 10 mM MgCl₂ (membrane suspension, 790 µL), (ii) test compound (10 µL), (iii) 50 nM CPA (100 µL) and (iv) 4 nM [³H]NECA (radioligand solution, 100 µL). The final volume of all incubations contained 1 mL of 50 mM Tris.HCl buffer (pH 7.7, 25 °C) and 1% DMSO. (DMSO was used to prepare all stock solutions of the test compounds.) The incubations were vortexed and incubated for 60 min at 25 °C in a shaking waterbath. Half an hour after incubation was started, the incubations were vortexed again. The incubations were terminated via filtration through a prewetted 2.5 cm Whatman glass microfiber filter (grade GF/B) under reduced pressure using a Hoffeler vacuum system. The tubes were washed twice with 4 mL ice-cold Tris buffer and the filters were washed once more with 4 mL ice-cold Tris buffer. The damp filters were place in scintillation vials and 4 mL of scintillation fluid (Filter-Count) was added. The vials were shaken and incubated for 2 h before being counted (Packard Tri-CARB 2100 TR). Non-specific binding of [³H]DPCPX and [³H]NECA for the radioligand binding assays were defined as binding in the presence of 100 μM CPA or 10 µM DPCPX. Specific binding was defined as the total binding minus the non-specific binding.

GTP shift assays

The type of binding affinity at the rat A₁ AR displayed by test compounds was determined via a GTP shift assay, as described in literature [52–54, 77]. The membrane preparation was performed under the same conditions as described above for the adenosine A1 receptor radioligand binding assay (see Tissue samples). A GTP shift assay follows similar method as A₁ AR radioligand binding assay, but additionally 100 μM GTP was added. GTP is thought to act by uncoupling the receptors from their G-proteins which causes agonists of the receptor to lose binding affinity [78]. The incubations were carried out in 4 mL polypropylene tubes that were precoated with Sigmacote (Sigma–Aldrich). All incubations were prepared with 50 mM Tris buffer (pH 7.7 at 25 °C) to a volume of 1 mL. Each incubation (i) test compound (10 µL), (ii) 0.1 nM [³H]DPCPX (radioligand solution, 100 µL), (iii) 0.1 mM GTP and (iv) 120 µg rat whole brain membranes and 0.1 units/mL adenosine deaminase (membrane suspension, 790 µL). Non-specific binding was defined as binding in the presence of 10 μM DPCPX.

Statistical data analyses

All statistical data analyses were carried out with Microsoft Excel and GraphPad Prism Software. Sigmoidal dose response curves, from which IC₅₀ (half maximal inhibitory concentration) values were calculated, were obtained by plotting the specific binding against the logarithm of the test compounds’ concentrations. Subsequently, the IC₅₀ values were used to calculate the Ki values for the competitive inhibition of [³H]DPCPX (K_d = 0.36 nM) against rat whole brain membranes and [³H]NECA (K_d = 15.3 nM) against rat striatal membranes by the test compounds by means of the Cheng–Prusoff equation. All incubations were carried out in triplicate and the K_i values are expressed as the mean ± standard error of mean (SEM). GTP shifts were calculated by dividing the Ki values of compounds reported in the presence of GTP by the Ki values obtained in the absence of GTP.

Abbreviations

[³H]DPCPX

1,3-[³H]-dipropyl-8-cyclopentylxanthine

[³H]NECA

[³H]-5′-N-ethylcarboxamidoadenosine

¹³C

carbon-13

¹H

hydrogen-1 / protium

A₁ AR

adenosine A₁ receptor substype

A_2A AR

adenosine A_2A receptor subtype

A_2B AR

adenosine A_2B receptor subtype

A₃ AR

adenosine A₃ receptor subtype

AEDs

antiepileptic drugs

ADME

absorption, distribution, metabolism, and excretion

AMPA

α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid

APCI

atmospheric pressure chemical ionisation

AR

adenosine receptor

BBB

blood-brain barrier

BOILED-Egg

Brain Or IntestinaL EstimateD permeation

C = O

carbonyl

Ca²⁺

calcium ion

CADO

2-chloroadenosine

CCPA

2-chloro-N⁶-cyclopentyladenosine

Ci

curie

Cl

chlorine

CNS

central nervous system

CPA

N⁶-cyclopentyladenosine

CCPA

2-chloro-N6-cyclopentyladenosine

Csp³

Fraction of carbon atoms in the sp3 hybridization

CYP

cytochrome P450 enzyme system

d

doublet

DCM

dichloromethane

dd

doublet of doublets

ddd

double double doublet

DMF

dimethylformamide

DMSO-d₆

deuterated dimethyl sulfoxide

DPCPX

8-cyclopentyl-1,3-dipropylxanthine

DSC

differential scanning calorimetry

EtOAc

ethyl acetate

EtOH

ethanol

Et₃N

triethylamine

F

fluorine

FeS

iron sulphide

GI

gastrointestinal

GPCR

G protein-coupled receptor

GTP

guanosine 5'-triphosphate

H

hydrogen

HCl

hydrochloric acid

HPLC

high-performance liquid chromatography

HRMS

high-resolution mass spectrometry

Hz

hertz

IC₅₀

half maximal inhibitory concentration

J

coupling constant

K_i

inhibition constant

KOH

potassium hydroxide

LogP

partition coefficient

m

multiplet

MeOH

methanol

MgCl₂

magnesium chloride

MHz

megahertz

MN

malononitrile

mp

melting point

MW

molecular weight

m/z

mass-to-charge ratio

N

nitrogen

NH₂

amino

nM

nano molar

NMDA

N-methyl-D-aspartate

NMR

nuclear magnetic resonance

NWU

North-West University

OCH₃

methoxy

O

oxygen

OH

hydroxy

PAINS

pan assay interference compounds

PE

petroleum ether

P-gp

permeability glycoprotein

ppm (δ)

parts per million

PRE

pharmaco-resistant epilepsy

q

quartet

r

rat

Rf

retention factor

s

singlet

S

sulphur

SANS

South African National Standard

SAR

structure-activity relationships

SEM

standard error of the mean

SI

selectivity index

t

triplet

td

triple of doublets

TLC

thin layer chromatography

TMS

tetramethylsilane

TPSA

topological polar surface area

XLOGP3

LogP value of a compound using a logP of a reference compound

Compliance with ethical standards

Conflict of interest

The authors declare no competing interests.

Ethical approval

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. All procedures performed in studies involving animals were in accordance with the ethical standards of the institution or practice at which the studies were conducted.

Supplementary information

The online version contains supplementary material available at 10.1007/s00044-022-02908-9.