4-amino-5-aryl-6-arylethynylpyrimidines: structure-activity relationships of non-nucleoside adenosine kinase inhibitors.

A series of non-nucleoside adenosine kinase (AK) inhibitors is reported. These inhibitors originated from the modification of 5-(3-bromophenyl)-7-(6-morpholin-4-ylpyridin-3-yl)pyrido[2,3-d]pyrimidin-4-ylamine (ABT-702). The identification of a linker that would approximate the spatial arrangement found between the pyrimidine ring and the aryl group at C(7) in ABT-702 was a key element in this modification. A search of potential linkers led to the discovery of an acetylene moiety as a suitable scaffold. It was hypothesized that the aryl acetylenes, ABT-702, and adenosine bound to the active site of AK (closed form) in a similar manner with respect to the orientation of the heterocyclic base. Although potent acetylene analogs were discovered based on this assumption, an X-ray crystal structure of 5-(4-dimethylaminophenyl)-6-(6-morpholin-4-ylpyridin-3-ylethynyl)pyrimidin-4-ylamine (16a) revealed a binding orientation contrary to adenosine. In addition, this compound bound tightly to a unique open conformation of AK. The structure-activity relationships and unique ligand orientation and protein conformation are discussed.

Crystal structures of human adenosine kinase inhibitor complexes reveal two distinct binding modes.

Adenosine kinase (AK) is an enzyme responsible for converting endogenous adenosine (ADO) to adenosine monophosphate (AMP) in an adenosine triphosphate- (ATP-) dependent manner. The structure of AK consists of two domains, the first a large alpha/beta Rossmann-like nucleotide binding domain that forms the ATP binding site, and a smaller mixed alpha/beta domain, which, in combination with the larger domain, forms the ADO binding site and the site of phosphoryl transfer. AK inhibitors have been under investigation as antinociceptive, antiinflammatory, and anticonvulsant as well as antiinfective agents. In this work, we report the structures of AK in complex with two classes of inhibitors: the first, ADO-like, and the second, a novel alkynylpyrimidine series. The two classes of structures, which contain structurally similar substituents, reveal distinct binding modes in which the AK structure accommodates the inhibitor classes by a 30 degrees rotation of the small domain relative to the large domain. This change in binding mode stabilizes an open and a closed intermediate structural state and provide structural insight into the transition required for catalysis. This results in a significant rearrangement of both the protein active site and the orientation of the alkynylpyrimidine ligand when compared to the observed orientation of nucleosidic inhibitors or substrates.

5-(3-Bromophenyl)-7-(6-morpholin-4-ylpyridin-3-yl)pyrido[2,3-d]pyrimidin-4-ylamine: structure-activity relationships of 7-substituted heteroaryl analogs as non-nucleoside adenosine kinase inhibitors.

4-Amino-5,7-disubstituted pyridopyrimidines are potent, non-nucleoside inhibitors of adenosine kinase (AK). We recently identified a potent, orally efficacious analog, 4 containing a 7-pyridylmorpholine substituted ring system as the key structural element of this template. In this report, we disclose the pharmacologic effects of five- and six-membered heterocyclic ring replacements for the pyridine ring in 4. These replacements were found to have interesting effects on in vivo efficacy and genotoxicity as well as in vitro potency. We discovered that the nitrogen in the heterocyclic ring at C(7) is important for the modulation of mutagenic side effects (Ames assay).

Synthesis and biological evaluation of pteridine and pyrazolopyrimidine based adenosine kinase inhibitors.

Three new approaches have been tested to modify existing pyridopyrimidine and alkynylpyrimidine classes of nonnucleoside adenosine kinase inhibitors 2 and 3. 4-Amino-substituted pteridines 8a-e were generally less active than corresponding 5- and 6-substituted pyridopyrimidines 2. Pyrazolopyrimidine 13c with IC(50)=7.5 nM was superior to its open chain alkynylpyrimidine analog 13g (IC(50)=22 nM) while pyrrolopyrimidines such as 17a were inactive.

5,6,7-trisubstituted 4-aminopyrido[2,3-d]pyrimidines as novel inhibitors of adenosine kinase.

The synthesis and structure-activity relationship of a series of 5,6,7-trisubstituted 4-aminopyrido[2,3-d]pyrimidines as novel nonnucleoside adenosine kinase inhibitors is described. A variety of alkyl, aryl, and heteroaryl substituents were found to be tolerated at the C5, C6, and C7 positions of the pyridopyrimidine core. These studies have led to the identification of analogues that are potent inhibitors of adenosine kinase with in vivo analgesic activity.

Adenosine kinase inhibitors: polar 7-substitutent of pyridopyrimidine derivatives improving their locomotor selectivity.

We have discovered that polar 7-substituents of pyridopyrimidine derivatives affect not only whole cell AK inhibitory potency, but also selectivity in causing locomotor side effects in vivo animal models. We have identified compound, 1o, which has potent whole cell AK inhibitory potency, analgesic activity and minimal reduction of locomotor activity.

Synthesis and structure-activity relationships of 5-heteroatom-substituted pyridopyrimidines as adenosine kinase inhibitors.

Under stressful conditions, many cells release adenosine to minimize tissue damage. Inhibition of intracellular adenosine kinase (AK) increases the local extracellular concentration of adenosine and its effect on traumatized tissue. The synthesis and SAR of a new series of pyridopyrimidines for the inhibition of AK are described. It was found that a range of analogs with position five substituted by an amine or ether functionality increased aqueous solubility while retaining the in vitro potency of initial leads. A narrower range of analogs was active in vivo in a rat inflammatory hyperalgesia model.

Design, synthesis, and structure-activity relationship of 6-alkynylpyrimidines as potent adenosine kinase inhibitors.

Adenosine (ADO) is an extracellular signaling molecule within the central and peripheral nervous system. Its concentration is increased at sites of tissue injury and inflammation. One of the mechanisms by which antinociceptive and antiinflammatory effects of ADO can be enhanced consists of inhibition of adenosine kinase (AK), the primary metabolic enzyme for ADO. Novel nonnucleoside AK inhibitors based on 4-amino-6-alkynylpyrimidines were prepared, and the importance of the length of the linker at the 5-position for high affinity AK inhibition was demonstrated. Compounds with 2- and 3-atom linkers were the most potent AK inhibitors. Optimization of their physicochemical properties led to 31a and 37a that effectively reduced pain and inflammation in animal models.

Comparison of the ability of adenosine kinase inhibitors and adenosine receptor agonists to attenuate thermal hyperalgesia and reduce motor performance in rats.

Inhibitors of adenosine kinase (AK) enhance extracellular concentrations of the inhibitory neuromodulator adenosine (ADO) at sites of tissue hyperexcitability and produce antinociceptive effects in animal models of pain and inflammation. The present study compared the ability of several novel and selective AK inhibitors and ADO receptor-selective agonists to attenuate carrageenan-induced thermal hyperalgesia and to impair motor performance as measured by effects on exploratory motor activity and rotorod performance. The prototypical nucleoside AK inhibitor, 5'deoxy-5-iodotubercidin (5'd-5IT), dose-dependently blocked thermal hyperalgesia (ED(50)=0.2 micromol/kg ip) and was 4- and 75-fold less potent in reducing exploratory motor activity and rotorod performance, respectively. The antihyperalgesic effects of 5'd-5IT were fully blocked by the A(1) antagonist, cyclopentyltheophylline (CPT) and the A(2A) antagonist, 3,7-dimethyl-1-propargylxanthine (DMPX). Novel nucleoside and non-nucleoside AK inhibitors (A-134974, A-286501 and ABT-702) also potently (ED(50)=0.7-2 micromol/kg ip) blocked carrageenan-induced thermal hyperalgesia and were significantly less potent than 5'd-5IT in impairing motor performance. The systemic administration of N(6)-cyclopentyladenosine (CPA), an A(1) receptor-selective agonist, CGS 21680, an A(2A) receptor-selective agonist, and N(6)-ethylcarboxamidoadenosine (NECA), a nonselective ADO receptor agonist potently reduced (ED(50)=0.3-1.0 micromol/kg ip) thermal hyperalgesia. Unlike the AK inhibitors, however, these ADO receptor agonists produced significant antinociception only at doses that also decreased motor performance. These data demonstrate that AK inhibitors produce specific antihyperalgesic effects via an interaction with ADO A(1) and A(2A) receptors at doses that lack detectable effects on exploratory motor activity and rotorod performance and offer an improved separation between antinociceptive and motor impairing effects as compared to ADO receptor agonists.

Analgesic and anti-inflammatory effects of A-286501, a novel orally active adenosine kinase inhibitor.

Adenosine (ADO) is an inhibitory neuromodulator that can increase nociceptive thresholds in response to noxious stimulation. Inhibition of the ADO-metabolizing enzyme, adenosine kinase (AK) increases extracellular ADO concentrations at sites of tissue trauma and AK inhibitors may have therapeutic potential as analgesic and anti-inflammatory agents. N7-((1'R,2'S,3'R,4'S)-2',3'-dihydroxy-4'-amino-cyclopentyl)-4-amino-5-bromo-pyrrolo[2,3-a]pyrimidine (A-286501) is a novel and potent (IC50=0.47 nM) carbocyclic nucleoside AK inhibitor that has no significant activity (IC50 >100 microM) at other sites of ADO interaction (A1, A2A, A3 receptors, ADO transporter, and ADO deaminase) or other (IC50 value >10 microM) neurotransmitter and peptide receptors, ion channel proteins, neurotransmitter reuptake sites and enzymes, including cyclooxygenases-1 and -2. A-286501 showed equivalent potency to inhibit AK from several mammalian species and kinetic studies revealed that A-286501 was a reversible and competitive inhibitor with respect to ADO and non-competitive with respect to MgATP2-. A-286501 was orally effective to reduce nociception in animal models of acute (thermal), inflammatory (formalin and carrageenan), and neuropathic (L5/L6 nerve ligation and streptozotocin-induced diabetic) pain. A-286501 was particularly potent (ED50=1 micromol/kg, p.o.) to reduce carrageenan-induced inflammatory thermal hyperalgesia as compared to its analgesic actions in other pain models (acute and neuropathic) and its ability to alter hemodynamic function and motor performance. A-286501 was also effective to reduce carrageenan-induced paw edema and myeloperoxidase activity, a measure of neutrophil influx (ED50=10 micromol/kg, p.o.), in the injured paw. The anti-nociceptive effects of A-286501 in the L5/L6 nerve injury model of neuropathic pain (ED50=20 micromol/kg, p.o.) were not blocked by the opioid antagonist naloxone, but were blocked by the ADO receptor antagonist, theophylline. Following repeated administration, A-286501 showed less potential to produce tolerance as compared to morphine. Thus, A-286501 is a structurally novel AK inhibitor that effectively attenuates nociception by a non-opioid, non-non-steroidal anti-inflammatory drug ADO, receptor mediated mechanism.