Alkyl lysophosphatidic acid and fluoromethylene phosphonate analogs as metabolically-stabilized agonists for LPA receptors

We describe an efficient method for the synthesis of alkyl lysophosphatidic acid (LPA) analogs as well as alkyl LPA mono- and difluoromethylene phosphonate analogs. Each alkyl LPA analog was evaluated for subtype-specific LPA receptor agonist activity using a cell migration assay for LPA(1) activation in cancer cells and an intracellular calcium mobilization assay for LPA(2) and LPA(3) activation. Alkyl LPAs induced pronounced cell migration activity with equivalent or higher potency than sn-1-oleoyl LPA, while the alkyl LPA fluoromethylene phosphonates proved to be less potent agonists in this assay. However, each alkyl LPA analog activated Ca(2+) release by activation of LPA(2) and LPA(3) receptors. Interestingly, the absolute configuration of the sn-2 hydroxyl group of the alkyl LPA analogs was not recognized by any of the three LPA receptors. The use of alkyl LPA analogs further expands the scope of structure-activity studies, which will better define LPA-LPA receptor interactions.     

Development of selective agonists and antagonists of P2Y receptors

Although elucidation of the medicinal chemistry of agonists and antagonists of the P2Y receptors has lagged behind that of many other members of group A G protein-coupled receptors, detailed qualitative and quantitative structure–activity relationships (SARs) were recently constructed for several of the subtypes. Agonists selective for P2Y₁, P2Y₂, and P2Y₆ receptors and nucleotide antagonists selective for P2Y₁ and P2Y₁₂ receptors are now known. Selective nonnucleotide antagonists were reported for P2Y₁, P2Y₂, P2Y₆, P2Y₁₁, P2Y₁₂, and P2Y₁₃ receptors. At the P2Y₁ and P2Y₁₂ receptors, nucleotide agonists (5′-diphosphate derivatives) were converted into antagonists of nanomolar affinity by altering the phosphate moieties, with a focus particularly on the ribose conformation and substitution pattern. Nucleotide analogues with conformationally constrained ribose-like rings were introduced as selective receptor probes for P2Y₁ and P2Y₆ receptors. Screening chemically diverse compound libraries has begun to yield new lead compounds for the development of P2Y receptor antagonists, such as competitive P2Y₁₂ receptor antagonists with antithrombotic activity. Selective agonists for the P2Y₄, P2Y₁₁, and P2Y₁₃ receptors and selective antagonists for P2Y₄ and P2Y₁₄ receptors have not yet been identified. The P2Y₁₄ receptor appears to be the most restrictive of the class with respect to modification of the nucleobase, ribose, and phosphate moieties. The continuing process of ligand design for the P2Y receptors will aid in the identification of new clinical targets.     

Tetrahydroisoquinolines as subtype selective estrogen agonists/antagonists

Two series of 6-hydroxy and 7-hydroxy tetrahydroisoquinolines were prepared. Evaluating a range of C-1, C-4, and N-substituents led to the discovery of ER alpha and ER beta selective analogs.     

Structure-activity relationships of lysophosphatidic acid analogs

The physiologic effects of lysophosphatidic acid (LPA) remain poorly understood. Our ignorance is due in part to lack of medicinal chemistry focussed on this pleiotropic lipid mediator. Beginning with commercially available phospholipids tested on whole cells or tissues and continuing with synthetic analogs tested at recombinant LPA receptors, the features of the LPA pharmacophore have become visible. An active LPA mimetic has a long aliphatic chain terminating in a phosphate monoester; bulky substitutions at the second carbon (relative to the phosphate) are tolerated poorly and a dissociable proton near the phosphate group seems required for optimal activity. These requirements are met by substituting ethanolamine for the glyceryl group in LPA. Substitutions at the second carbon of the N-acyl ethanolamide phosphoric acid (NAEPA) result in highly active agonists, including some receptor type selective compounds, if the substituent is small (e.g. methyl, methylene amino, methylene hydroxy). However, bulky hydrophobic substituents lead to compounds with decreased agonist, or even antagonist, activities. Examination of naturally occurring plant lipids led to the discovery of another LPA receptor antagonist, di-octyl glyceryl pyrophosphate. An unexplained result obtained in testing the LPA mimetics is the strong stereoselectivity exhibited by some responses (e.g. calcium mobilization) and the lack of stereoselectivity of other responses (e.g. platelet aggregation).     

Identification of potent lysophosphatidic acid receptor 5 (LPA5) antagonists as potential analgesic agents

Lysophosphatidic acid (LPA) plays an important role in a variety of cellular functions. In particular, LPA5 receptor is highly expressed in spinal cord and dorsal root ganglion, which are associated with pain. This fact prompted us to hypothesize that LPA5 antagonists show analgesic effects. To search for potent LPA5 antagonists with blood brain barrier (BBB) permeability, we conducted high throughput screening (HTS). In HTS campaign, we found a 2H-isoquinoline-1-one scaffold showing antagonistic activity against LPA5 and synthesized a series of 2H-isoquinoline-1-one derivatives and evaluated their LPA5 activities. Among these compounds, compound 7e showed potent LPA5 activity with an IC₅₀ value of 0.12?μM, and acceptable BBB permeability. Furthermore, it showed effective analgesic effect in a chronic constriction injury rat model. Therefore, 7e may have a potential as novel pain therapeutic approach.     

Identification of lysophospholipid receptors in human platelets: the relation of two agonists, lysophosphatidic acid and sphingosine 1-phosphate

Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (Sph-1-P) are known as structurally related bio-active lipids activating platelets through their respective receptors. Although the receptors for LPA and Sph-1-P have been recently identified in various cells, the identification and characterization of ones in platelets have been reported only preliminarily. In this report, we first investigated the distinct modes of LPA and Sph-1-P actions in platelet activation and found that LPA functioned as a much stronger agonist than Sph-1-P, and high concentrations of Sph-1-P specifically desensitized LPA-induced intracellular Ca(2+) mobilization. In order to identify the responsible receptors underlying these observations, we analyzed the LPA and Sph-1-P receptors which might be expressed in human platelets, by RT-PCR. We found for the first time that Edg2, 4, 6 and 7 mRNA are expressed in human platelets.     

Phosphatase-resistant analogues of lysophosphatidic acid: agonists promote healing, antagonists and autotaxin inhibitors treat cancer

Isoform-selective agonists and antagonists of the lysophosphatidic acid (LPA) G protein-coupled receptors (GPCRs) have important potential applications in cell biology and therapy. LPA GPCRs regulate cancer cell proliferation, invasion, angiogenesis, and also biochemical resistance to chemotherapy- and radiotherapy-induced apoptosis. LPA and its analogues also are feedback inhibitors of the enzyme lysophospholipase D (lysoPLD, a.k.a., autotaxin, ATX), a central regulator of invasion and metastasis. For cancer therapy, the optimal therapeutic profile would be a metabolically-stabilized, pan-LPA receptor antagonist that also inhibited lysoPLD. For protection of gastrointestinal mucosa and lymphocytes, LPA agonists would be desirable to minimize or reverse radiation or chemical-induced injury. Analogues of lysophosphatidic acid (LPA) that are chemically modified to be less susceptible to phospholipases and phosphatases show activity as long-lived receptor-specific agonists and antagonists for LPA receptors, as well as inhibitors for the lysoPLD activity of ATX.     

Synthesis and evaluation of isoxazole derivatives as lysophosphatidic acid (LPA) antagonists

A series of isoxazole derivatives were synthesized and their antagonistic activities against LPA stimulation on both LPA(1)/CHO cells and rHSC cells were evaluated. Among them, 3-(4-[4-[1-(2-chloro-cyclopent-1-enyl)-ethoxycarbonylamino]-isoxazol-3- y]]-benzylsulfanyl)-propionic acid (34) showed the most potent activities.     

Phosphorothioate analogs of sn-2 radyl lysophosphatidic acid (LPA): Metabolically stabilized LPA receptor agonists

We describe an efficient synthesis of metabolically stabilized sn-2 radyl phosphorothioate analogs of lysophosphatidic acid (LPA), and the determination of the agonist activity of each analog for the six LPA receptors (LPA_1–6) using a recently developed TGFα shedding assay. In general, the sn-2 radyl OMPT analogs showed similar agonist activities to the previous 1-oleoyl-2-O-methyl-glycerophosphothioate (sn-1 OMPT) analogs for LPA_1–6 receptors. In most cases, the sn-2 radyl-OMPT analogs were more potent agonists than LPA itself. Most importantly, sn-2 alkyl OMPT analogs were very potent LPA₅ and LPA₆ agonists. The availability of sn-2 radyl OPMT analogs further refines the structure–activity relationships for ligand–receptor interactions for this class of GPCRs.     

Binding of agonists and antagonists to muscarinic receptors

The binding of one irreversible and two reversible radioactive antagonists to muscarinic receptors in synaptosome preparations of rat cerebral cortex has been studied. The ligands all bind to the same receptor pool and directly and competitively yield self-consistent binding constants closely similar to those obtained by pharmacological methods on intact smooth muscle. The binding process for antagonists seems to be a simple mass action-determined process with a Hill slope of 1.0. The quantitative correlations strongly support the view that the receptor studied by ligand binding corresponds to the receptor studied by pharmacological methods. Inhibition of antagonist binding by most agonists shows a reduced Hill slope which also applies to direct binding studies of [³H] acetylcholine. Mechanisms that might account for the behavior of agonists are discussed but do not conclusively point to any single mechanism.     

Quinuclidines as selective agonists for alpha-7 nicotinic acetylcholine receptors

The alpha7 subtype of the neuronal nicotinic acetylcholine receptors (nAChRs) was targeted for the design of selective agonists deriving from the quinuclidine scaffold. Arylidene groups at the 3-position and N-methyl quinuclidine were found to be selective agonists with EC(50)s of 1.5 and 40 microM, respectively.     

Discovery of novel N-acylsulfonamide analogs as potent and selective EP3 receptor antagonists

A series of novel N-acylsulfonamide analogs were synthesized and evaluated for their binding affinity and antagonist activity for the EP3 receptor subtype. Representative compounds were also evaluated for their inhibitory effect on PGE₂-induced uterine contraction in pregnant rats. Among those tested, a series of N-acylbenzenesulfonamide analogs were found to be more potent than the corresponding carboxylic acid analogs in both the in vitro and in vivo evaluations. The structure activity relationships (SAR) are also discussed.     

New retinoid chemotypes: 9-cis-retinoic acid analogs with hydrophobic rings derived from terpenes as selective RAR agonists

A series of 9-cis-retinoic acid analogs modified at the hydrophobic ring with a (bi)cyclohexenyl moiety derived from natural terpenes has been stereoselectively prepared using a Suzuki cross-coupling as key step. Transient transactivation studies indicate that modification of the hydrophobic ring impacts dramatically on RXR-binding and transactivation, with most retinoids being inactive on RXRbeta, while preserving their RAR pan-agonist profile. Furthermore, only the RARgamma subtype was capable of enantiomeric discrimination with some pairs of enantiomeric terpene-retinoids.     

Design of subtype selective melatonin receptor agonists and antagonists.

Studies of the physiological actions of melatonin have been hindered by the lack of specific, potent and subtype selective agonists and antagonists. In the present study, we describe the utility of a melanophore cell line from Xenopus laevis for exploring structure-activity relationships among novel melatonin analogues and report a novel MT2-selective agonist (IIK7) and MT2-selective receptor antagonist (K185). IIK7 is a potent melatonin receptor agonist in the melanophore model, and in NIH3T3 cells expressing human mt1 and MT2 receptor subtypes. In radioligand binding experiments IIK7 is 90-fold selective for the MT2 subtype. K185 is devoid of agonist activity, but acts as a competitive melatonin antagonist in melanophores. A low concentration (10(-9) M) antagonizes melatonin inhibition of forskolin stimulation of cyclic AMP in NIH3T3 cells expressing human MT2 receptors, but has no effect in cells expressing mt1 receptors. In binding assays, K185 is 140-fold selective for the MT2 subtype.     

Thermic response of selective muscarinic agonists and antagonists in rat

Effect of some selective agonists and antagonists of cholinergic M receptor subtypes on rectal temperature was investigated in rats at an ambient temperature of 25 degrees +/- 2 degrees C. Centrally administered acetylcholine (ACh) induced transient hypothermia, whereas the muscarinic M1 receptor agonists, arecholine (ip) and McN-A-343 (McN) (icv), induced sustained and dose-related hypothermia. However, the nonspecific muscarinic receptor agonist, oxotremorine, and physostigmine, induced hypothermia at a lower dose and hyperthermia, accompanied by tremors, at higher doses. The muscarinic M2 receptor agonist, carbachol (icv) also produced a dose-related dual effect, hyperthermia and hypothermia being induced by the lower and higher doses, respectively. The M1 receptor antagonists, scopolamine (ip) and pirenzepine (icv), induced hyperthermia, whereas the M2 receptor antagonists, gallamine (icv) and AF-DX 116 (AFDX) (ip), produced hypothermia. The hypothermic effects of ACh. arecholine, McN, physostigmine, oxotremorine and carbachol were attenuated by scopolamine and pirenzepine. However, although scopolamine also inhibited the hyperthermic and tremorogenic effects of the higher dose of oxotremorine, it had a synergistic effect with the hyperthermia-inducing higher dose of physostigmine. AFDX attenuated the hyperthermic effect of the lower dose of carbachol, indicating that it was M2 receptor-mediated. Hemicholinium, an ACh synthesis inhibitor, had a transient hypothermic effect followed by slight hyperthermia. However, it markedly antagonized the hypothermic effects of gallamine and AFDX, indicating that their effects were dependent upon the availability of neuronal ACh. The results indicate that cholinergic hypothermia is a function of central muscarinic M1 receptors, with the M2 receptors serving as automodulators.(ABSTRACT TRUNCATED AT 250 WORDS)     

The agonist paradox: agonists and antagonists of acetylcholine receptors and opioid receptors

In contrast to antagonists, agonists tend to induce considerable conformational changes in their receptors, resulting in opening of ion channels, either directly or via secondary messengers. These conformational transformations require great energy expenses. However, the experimentally determined free energies of complexation between agonists and receptors are often relatively smaller than those for the corresponding antagonists. To rationalize this so-called 'agonist paradox', which has not been clarified in the literature, we have developed an alternative model. Our model may help to discriminate between agonists and antagonists of the acetylcholine (ACh) and mu-opioid receptors. For this purpose, a series of ligands (1-18) have been analyzed both in structural terms and with respect to complexation geometry within the anionic binding sites of these two receptor types.     

Identification of N-substituted 8-azatetrahydroquinolone derivatives as selective and orally active M1 and M4 muscarinic acetylcholine receptors agonists

We designed and synthesized N-substituted 8-azatetrahydroquinolone derivatives as selective M₁ and M₄ muscarinic acetylcholine receptors agonists. Optimization of selected derivatives led to the discovery of compound 7 as a highly potent M₁ and M₄ agonist with weak hERG inhibition. Oral administration of compound 7 improved psychosis-like behavior in rats.     

Identification of a phosphothionate analogue of lysophosphatidic acid (LPA) as a selective agonist of the LPA3 receptor

Lysophosphatidic acid (LPA) is a bioactive lysophospholipid mediator that acts through G protein-coupled receptors. Most cell lines in culture express one or more LPA receptors, making it difficult to assign a response to specific LPA receptors. Dissection of the signaling properties of LPA has been hampered by lack of LPA receptor subtype-specific agonists and antagonists. The present study characterizes an ester-linked thiophosphate derivative (1-oleoyl-2-O-methyl-rac-glycerophosphothionate, OMPT) of LPA. OMPT is a functional LPA analogue with potent mitogenic activity in fibroblasts. In contrast to LPA, OMPT does not couple to the pheromone response through the LPA(1) receptor in yeast cells. OMPT induces intracellular calcium increases efficiently in LPA(3) receptor-expressing Sf9 cells but poorly in LPA(2) receptor-expressing cells. Guanosine 5'-O-(3-[(35)S]thio)triphosphate binding assays in mammalian cells showed that LPA exhibits agonistic activity on all three LPA receptor subtypes, whereas OMPT has a potent agonistic effect only on the LPA(3) receptor. In transiently transfected HEK293 cells, OMPT stimulates mitogen-activated protein kinases through the LPA(3) but not the LPA(1) or LPA(2) receptors. Furthermore, OMPT-induced intracellular calcium mobilization in mammalian cells is efficiently inhibited by the LPA(1)/LPA(3) receptor-selective antagonist VPC12249. These results establish that OMPT is an LPA(3)-selective agonist. OMPT binding to the LPA(3) receptor in mammalian cells is sufficient to elicit multiple responses, including activation of G proteins, calcium mobilization, and activation of mitogen-activated protein kinases. Thus OMPT offers a powerful probe for the dissection of LPA signaling events in complex mammalian systems.     

Discovery of new SCH 39166 analogs as potent and selective dopamine D1 receptor antagonists

A series of novel dopamine D₁ antagonists derived from functionalization of the D-ring of SCH 39166 were prepared. A number of these compounds displayed subnanomolar D₁ activity and more than 1000-fold selectivity over D₂. We found C-3 derivatization afforded compounds with superior overall profile in comparison to the C-2 and C-4 derivatization. A number of highly potent D₁ antagonists were discovered which have excellent selectivity over other dopamine receptors and improved PK profile compared to SCH 39166.