Synthesis of 4(5)-phenylimidazole-based analogues of sphingosine-1-phosphate and FTY720: discovery of potent S1P1 receptor agonists

The novel immunosuppressant FTY720 has been demonstrated to elicit immunomodulating effects via interaction with the G-protein coupled receptor S1P(1). FTY720 induced agonism at the S1P(3) receptor, however, has been shown to result in mild bradycardia, a minor side-effect of initial FTY720 therapy. This report describes the synthesis of several potent 4(5)-phenylimidazole-based S1P(1) receptor agonists that are accompanied by poor agonist activity at S1P(3). For instance, compound 20 displayed an EC(50)=4.7+/-1.3 nM at the S1P(1) receptor and EC(50)=780+/-1.3 nM at the S1P(3) receptor using a [gamma-(35)S]GTP-binding assay as compared to phospho-FTY720 (S1P(1): EC(50)=1.3+/-1.3nM, S1P(3): EC(50)=2.0+/-2.4 nM).     

Synthesis of para-alkyl aryl amide analogues of sphingosine-1-phosphate: discovery of potent S1P receptor agonists

Sphingosine-1-phosphate (S1P) is a biologically active lysophospholipid with the capacity to induce a broad range of cellular responses via its interaction with the S1P family of G-protein coupled receptors. This report describes the synthesis of several potent S1P receptor agonists. For instance, compound 9c displayed an EC(50)=8.6 nM at the S1P(1) receptor using a [gamma-35S]GTP binding assay as compared to an EC(50)=4.5 nM for the endogenous ligand. We also report the effects associated with introduction of a phenyl ring between the 'linker' and 'lipophilic tail' regions of the analogues, for example total loss of activity at S1P(2) and increased agonism at S1P(5).     

Structure-activity studies of new melanocortin peptides containing an aromatic amino acid at the N-terminal position

Cyclic melanotropin peptides, designed with an aromatic amino acid substitution at the N-terminal position of the MT-II-type scaffold, were prepared by solid-phase peptide synthesis and evaluated for their ability to bind to and activate human melanocortin-1, -3, -4, and -5 receptors. The structure-activity studies of these MT-II analogues have identified a selective antagonist at the hMC4R (H-Phe-c[Asp-Pro-d-Nal(2')-Arg-Trp-Gly-Lys]-NH(2), pA(2)=8.7), a selective partial agonist at the hMC4R (H-d-Nal(2')-c[Asp-Pro-d-Phe-Arg-Trp-Gly-Lys]-NH(2), IC(50)=11nM, EC(50)=56nM), and a selective partial agonist at the hMC3R (H-d-Phe-c[Asp-Pro-d-Phe-Arg-Trp-Lys]-NH(2), IC(50)=3.7nM, EC(50)=4.9nM). Aromatic amino acid substitution at the N-terminus in conjuction with the expansion of the 23-membered cyclic lactam MT-II scaffold to a 26-membered scaffold by addition of a Gly residue in position 10 leads to melanotropin peptides with enhanced receptor selectivity.     

N-Alkylidenearylcarboxamides as new potent and selective CB(2) cannabinoid receptor agonists with good oral bioavailability

A novel series of N-alkylidenearylcarboxamides 4, a CB(2) receptor agonist, were synthesized and evaluated for activity against the human CB(2) receptor. In a previous paper, we reported that sulfonamide derivative 1 acted as a potent CB(2) receptor agonist (IC(50)=65 nM, EC(50)=19 nM, E(max)=90%). However, compound 1 also exhibited poor metabolic stability in human liver microsomes. During the structural modification of 1, we found that a novel series of N-alkylidenearylcarboxamide, 4-1, had a moderate affinity for the CB(2) receptor (IC(50)=260 nM, EC(50)=86 nM, E(max)=100%) and good metabolic stability in human liver microsomes. We explored its analogues to discover compounds with a high affinity for the CB(2) receptor and with good oral bioavailability. Among them, compounds 4-9 and 4-27 had high affinities for the human CB(2) receptor (CB(2) IC(50)=13 nM and 1.2 nM) and a high selectivity for CB(2) (CB(1) IC(50)/CB(2) IC(50)=270 and 1600); furthermore, significant plasma levels were observed following oral administration in rats (C(max)=233 ng/mL and 148 ng/mL, respectively, after a dose of 10 mg/kg). Furthermore, compound 4-9 had good oral bioavailability (F=52%, 3mg/kg).     

Highly selective and potent agonists of sphingosine-1-phosphate 1 (S1P1) receptor

Novel series of sphingosine-1-phosphate (S1P) receptor agonists were developed through a systematic SAR aimed to achieve high selectivity for a single member of the S1P family of receptors, S1P1. The optimized structure represents a highly S1P1-selective and efficacious agonist: S1P1/S1P2, S1P1/S1P3, S1P1/S1P4>10,000-fold, S1P1/S1P5>600-fold, while EC50 (S1P1) <0.2 nM. In vivo experiments are consistent with S1P1 receptor agonism alone being sufficient for achieving desired lymphocyte-lowering effect.     

Phosphorylation of IRS1 at serine 307 and serine 312 in response to insulin in human adipocytes

Feedback control in insulin signaling involves serine phosphorylation of insulin receptor substrate-1 (IRS1). By analyzing the insulin-induced phosphorylation of IRS1 at serine 307, serine 312, and tyrosine in the same primary human adipocytes, we now report that negative feedback phosphorylation of serine 312 (corresponding to murine serine 307) required relatively high concentrations of insulin (EC(50)=3 nM) for a long time (t(1/2) ca. 30 min) and reduced the steady-state tyrosine phosphorylation, without affecting the cellular concentration, of IRS1. In contrast, positive feedback phosphorylation of serine 307 was a rapid (t(1/2) ca. 2 min) event at physiological concentrations of insulin (EC(50)=0.2 nM).     

Development of an orexin-2 receptor selective agonist, [Ala(11), D-Leu(15)]orexin-B

Investigation of L-alanine and D-amino acid replacement of orexin-B revealed that three L-leucine residues at the positions of 11, 14, and 15 in orexin-B were important to show selectivity for the orexin-2 receptor (OX(2)) over the orexin-1 receptor (OX(1)). L-Alanine substitution at position 11 and D-leucine substitution at positions 14 and 15 maintained the potency of orexin-B to mobilize [Ca(2+)](i) in CHO cells expressing the OX(2), while their potency for the OX(1) was significantly reduced. In combined substitutions, we identified that [Ala(11), D-Leu(15)]orexin-B showed a 400-fold selectivity for the OX(2) (EC(50)=0.13nM) over OX(1) (EC(50)=52nM). [Ala(11), D-Leu(15)]orexin-B is a beneficial tool for addressing the functional roles of the OX(2).     

Sphingosine-1-phosphate rapidly induces Rho-dependent neurite retraction: action through a specific cell surface receptor.

Sphingosine-1-phosphate (S1P) is a bioactive lysosphingolipid implicated in mitogenesis and cytoskeletal remodelling, but its mechanism of action is poorly understood. We report here that in N1E-115 neuronal cells, S1P mimics the G protein-coupled receptor agonist lysophosphatidic acid (LPA) in rapidly inducing neurite retraction and soma rounding, a process driven by Rho-dependent contraction of the actin cytoskeleton. S1P is approximately 100-fold more potent than LPA in evoking these shape changes, with an EC50 as low as 1.5 nM. Microinjection of S1P has no effect, neither has addition of sphingosine or ceramide. As with LPA, S1P action is inhibited by suramin and subject to homologous desensitization; however, the responses to S1P and LPA do not show cross-desensitization. We conclude that S1P activates its own high affinity receptor to trigger Rho-regutated cytoskeletal events. Thus, S1P and LPA may belong to an emerging family of bioactive lysophospholipids that act through distinct G protein-coupled receptors to mediate similar actions.     

Sulfonamide derivatives as new potent and selective CB2 cannabinoid receptor agonists

A novel series of sulfonamide derivatives 3, the CB(2) receptor agonists, was synthesized and evaluated for activity against the human CB(2) receptor. We first identified sulfonamide 3a, which was obtained by random screening of our in-house chemical library as a moderately active (CB(2) IC(50)=340nM) CB(2) receptor agonist. We then attempted to test its analogues to identify compounds with a high affinity for the CB(2) receptor. One of these, compound 3f, exhibited high affinity for the human CB(2) receptor (IC(50)=16nM) and high selectivity for CB(2) over CB(1) (CB(1) IC(50)/CB(2)IC(50)=106), and behaved as a full CB(2) receptor agonist in the [(35)S]GTPgammaS binding assay (CB(2) EC(50)=7.2nM, E(max)=100%).     

Neurotensin Stimulates Inositol Phospholipid Metabolism and Calcium Mobilization in Murine Neuroblastoma Clone N1E-115

Murine neuroblastoma cells (clone N1E-115) possess neurotensin receptors that mediate cyclic GMP synthesis. Because of the hypothesized relationship between phospholipid metabolism, intracellular Ca2+, and cyclic GMP synthesis, we determined with these cells the effects of neurotensin on 32P labeling of phospholipids, release of inositol phosphates, and intracellular Ca2+ (as determined with the use of Quin-2, a fluorescent probe sensitive to free Ca2+ levels). Neurotensin stimulated incorporation of 32P into phospholipids, especially phosphatidylinositol and phosphatidate. Neurotensin also stimulated the release of [3H]inositol phosphates with an EC50 of about 1 nM. Mean basal Ca2+ concentration in these cells was 134 nM and this level was increased in a rapid and dose-dependent manner by neurotensin, with an EC50 of 4 nM. Since the EC50 for neurotensin in stimulating cyclic GMP synthesis is 1.5 nM and the KD for binding of [3H]neurotensin at 0 degrees C is 11 nM, all these different effects appear to be shared proximal consequences of neurotensin receptor activation.     

Synthesis and pharmacological evaluation of second-generation phosphatidic acid derivatives as lysophosphatidic acid receptor ligands

Short-chain phosphatidic acid derivatives, dioctanoyl glycerol pyrophosphate (DGPP 8:0, 1) and phosphatidic acid 8:0 (PA 8:0, 2), were previously identified as subtype-selective LPA(1) and LPA(3) receptor antagonists. Recently, we reported that the replacement of the phosphate headgroup by thiophosphate in a series of fatty alcohol phosphates (FAP) improves agonist as well as antagonist activities at LPA GPCR. Here, we report the synthesis of stereoisomers of PA 8:0 analogs and their biological evaluation at LPA GPCR, PPARgamma, and ATX. The results indicate that LPA receptors stereoselectively interact with glycerol backbone modified ligands. We observed entirely stereospecific responses by dioctyl PA 8:0 compounds, in which (R)-isomers were found to be agonists and (S)-isomers were antagonists of LPA GPCR. From this series, we identified compound 13b as the most potent LPA(3) receptor subtype-selective agonist (EC(50)=3 nM), and 8b as a potent and selective LPA(3) receptor antagonist (K(i)=5 nM) and inhibitor of ATX (IC(50)=600 nM). Serinediamide phosphate 19b was identified as an LPA(3) receptor specific antagonist with no effect on LPA(1), LPA(2), and PPARgamma.     

Synthesis and SAR studies of chiral non-racemic dexoxadrol analogues as uncompetitive NMDA receptor antagonists

A series of chiral non-racemic dexoxadrol analogues with various substituents in position 4 of the piperidine ring was synthesized and pharmacologically evaluated. Only the enantiomers having (S)-configuration at the 2-position of the piperidine ring and 4-position of the dioxolane ring were considered. Key steps in the synthesis were an imino-Diels-Alder reaction of enantiomerically pure imine (S)-13, which had been obtained from d-mannitol, with Danishefsky's Diene 14 and the replacement of the p-methoxybenzyl protective group with a Cbz-group. It was shown that (S,S)-configuration of the ring junction (position 2 of the piperidine ring and position 4 of the dioxolane ring) and axial orientation of the C-4-substituent ((4S)-configuration) are crucial for high NMDA receptor affinity. 2-(2,2-Diphenyl-1,3-dioxolan-4-yl)piperidines with a hydroxy moiety ((S,S,S)-5, K(i)=28nM), a fluorine atom ((S,S,S)-6, WMS-2539, K(i)=7nM) and two fluorine atoms ((S,S)-7, K(i)=48nM) in position 4 represent the most potent NMDA antagonists with high selectivity against σ(1) and σ(2) receptors and the polyamine binding site of the NMDA receptor. The NMDA receptor affinities of the new ligands were correlated with their electrostatic potentials, calculated gas phase proton affinities (negative enthalpies of deprotonation) and dipole moments. According to these calculations decreasing proton affinity and increasing dipole moment are correlated with decreasing NMDA receptor affinity.     

Synthesis and immunological evaluation of the 4-β-glucoside of HMBPP

HMBPP ((E)-4-hydroxy-3-methyl-2-butenyl pyrophosphate) is a highly potent innate immunogen that stimulates human γδ T cells expressing the Vγ2Vδ2 T cell antigen receptor. To determine if glycoside conjugates of HMBPP retain activity, the 4-β-glucoside and its acetylated homolog were synthesized and tested for their ability to stimulate γδ T cells. The glycoside HMBPP conjugate stimulated human γδ T cells with an EC(50) of 78nM. The tetraacetyl glycoside HMBPP conjugate was also active (EC(50)=360nM). The two isomeric mono-β-glucosides of the parent (E)-2-methylbut-2-ene-1,4-diol, however, were not active. Thus, HMBPP glycosylated at the 4-OH position stimulates γδ T cells as long as the pyrophosphate moiety is present.     

Nucleotide analogues containing 2-oxa-bicyclo[2.2.1]heptane and l-alpha-threofuranosyl ring systems: interactions with P2Y receptors

The ribose moiety of adenine nucleotide 3',5'-bisphosphate antagonists of the P2Y(1) receptor has been successfully substituted with a rigid methanocarba ring system, leading to the conclusion that the North (N) ring conformation is preferred in receptor binding. Similarly, at P2Y(2) and P2Y(4) receptors, nucleotides constrained in the (N) conformation interact equipotently with the corresponding ribosides. We now have synthesized and examined as P2Y receptor ligands nucleotide analogues substituted with two novel ring systems: (1) a (N) locked-carbocyclic (cLNA) derivative containing the oxabicyclo[2.2.1]heptane ring system and (2) l-alpha-threofuranosyl derivatives. We have also compared potencies and preferred conformations of these nucleotides with the known anhydrohexitol-containing P2Y(1) receptor antagonist MRS2283. A cLNA bisphosphate derivative MRS2584 21 displayed a K(i) value of 22.5 nM in binding to the human P2Y(1) receptor, and antagonized the stimulation of PLC by the potent P2Y(1) receptor agonist 2-methylthio-ADP (30 nM) with an IC(50) of 650 nM. The parent cLNA nucleoside bound only weakly to an adenosine receptor (A(3)). Thus, this ring system afforded some P2Y receptor selectivity. A l-alpha-threofuranosyl bisphosphate derivative 9 displayed an IC(50) of 15.3 microM for inhibition of 2-methylthio-ADP-stimulated PLC activity. l-alpha-Threofuranosyl-UTP 13 was a P2Y receptor agonist with a preference for P2Y(2) (EC(50)=9.9 microM) versus P2Y(4) receptors. The P2Y(1) receptor binding modes, including rotational angles, were estimated using molecular modeling and receptor docking.     

Potent P1' biphenylmethyl substituted aggrecanase inhibitors.

A series of cis-1(S)2(R)-amino-2-indanol based compounds with a biphenylmethyl group at the P1' position was found to be potent aggrecanase inhibitors. Both compounds 2j and 2n possessed very high aggrecanase affinity (IC(50)=1.5nM), and showed excellent selectivity over MMP-1 and MMP-9, with moderate selectivity against MMP-2.     

Imine derivatives as new potent and selective CB2 cannabinoid receptor agonists with an analgesic action

In this study, a novel series of CB(2) receptor agonist imine derivatives, 1-6, was synthesized and evaluated for activity against the CB(2) receptor. In a previous paper we reported the synthesis and SARs of thiazole derivative 1, a potent CB(2) receptor agonist, but we had not assessed chemical modifications of the 5-membered heteroring of 1. In the present study, we therefore tried chemically modifying the 5-membered heteroring of 1 in an attempt to further improve binding affinity for the CB(2) receptor. In the course of making the structural modifications, we discovered that a novel pyrazole derivative 6b (CBS0550) had high affinity for the CB(2) receptor (IC(50)=2.9 nM, EC(50)=1.8 nM, E(max)=85%), high selectivity for CB(2) (CB(1) IC(50)/CB(2) IC(50)=1400), and good physicochemical properties (solubility in water: 5.9 mg/100mL at 25 degrees C). Oral administration of 6b to rats at a dose of 10mg/kg resulted in significant plasma concentrations, and orally administered compound 6b significantly reversed mechanical hyperalgesia in the Randall-Selitto model of inflammatory pain in rats.     

Molecular cloning of a human serotonin receptor (S12) with a pharmacological profile resembling that of the 5-HT1D subtype.

We report the molecular cloning of a fragment of human genomic DNA called S12, containing an open reading frame of 1170 nucleotides, which encodes a receptor for serotonin of 390 amino acids. The receptor function of the S12 protein was demonstrated by functional expression in mouse LS12 cells obtained by stable transfection of Ltk- cells, and LM5S12 cells, derived from LM5 cells (Ltk- cells previously transfected with the M5 muscarinic acetylcholine receptor). Adenylyl cyclase studies showed that the S12 receptor is able to mediate inhibition of adenylyl cyclase in response to serotonin in both types of cells. As studied in LM5S12 cells, the S12 receptor did not promote Ca2+ mobilization from internal stores, nor did it significantly modulate the sustained increase in [Ca2+]i elicited by stimulation of the phospholipase C stimulating M5 acetylcholine receptor. The pharmacologic profile of S12 as seen in adenylyl cyclase assays is as follows: (EC50 in nM): serotonin, full agonist (37 nM), 5-carboxamidotryptamine, full agonist (10 nM), sumatriptan, full agonist (50 nM), metergoline, partial agonist (10 nM), methysergide, partial agonist (40 nM), yohimbine, partial agonist (150 nM), metitepin, antagonist (KB = 0.7 to 1.2 nM). We propose that the human S12 serotonin receptor is a receptor of the 5-hydroxytryptamine1D subtype.     

Bimatoprost (Lumigan((R))) is an agonist at the cloned human ocular FP prostaglandin receptor: real-time FLIPR-based intracellular Ca(2+) mobilization studies

Bimatoprost is the ethyl amide derivative of 17-phenyl-trinor prostaglandin F(2alpha). Here, we show that bimatoprost (K(i)=9250+/-846nM) and bimatoprost free acid (17-phenyl-trinor prostaglandin F(2alpha); K(i)=59+/-6nM) bind to the FP receptor and displace [(3)H]-travoprost acid, a selective FP agonist. Bimatoprost (EC(50)=3070+/-1330nM), Lumigan((R)) (bimatoprost 0.03% ophthalmic solution; EC(50)=1150+/-93nM) and bimatoprost acid (EC(50)=15+/-3nM) mobilized intracellular Ca(2+) ([Ca(2+)](i)) in <5s in HEK-293 cells expressing the cloned human ciliary body FP receptor on a fluorometric imaging plate reader (FLIPR). Furthermore, agonist effects of bimatoprost and bimatoprost acid were blocked by AL-8810 (11beta-fluoro-15-epi-15-indanyl prostaglandin F(2alpha); K(i)=0.7-2.1 MicroM), an FP receptor-selective antagonist. Therefore, the prodrug bimatoprost and its hydrolytic product, bimatoprost free acid, bind to and activate the human ocular FP prostaglandin receptor to mobilize [Ca(2+)](i), thus behaving as FP receptor agonists.     

Synthetic FP-prostaglandin-induced contraction of rat uterus smooth muscle in vitro

Numerous synthetic FP-class prostaglandin (PG) analogs stimulated the contraction of isolated non-pregnant female rat uterus in a concentration-dependent manner with the following agonist potencies: bimatoprost acid (17-phenyl-trinor PGF(2alpha); EC(50)=0.68+/-0.06 nM)=cloprostenol (EC(50)=0.73+/-0.01 nM)>travoprost acid (EC(50)=1.3+/-0.07 nM)>latanoprost acid (EC(50)=2.7+/-0.08 nM)>PGF(2alpha) (EC(50)=52+/-11 nM)>unoprostone (UF-021; EC(50)=310+/-101 nM)>S-1033 (EC(50)=610+/-4 nM)>bimatoprost (EC(50)=1130+/-173 nM). The FP-receptor antagonist, AL-8810, antagonized the contractile effects of PGF(2alpha) (K(i)=2.9+/-0.2 microM), travoprost acid (K(i)=0.6+/-0.1 microM) and bimatoprost (K(i)=0.2+/-0.02 microM). Agonist and antagonist potencies for rat uterus contraction by these PGs compared well with their potencies for inducing/blocking functional responses in other systems (r=0.83-0.94) except with bovine iris sphincter (r=0.2; p<0.7). In conclusion, the rat uterus contains functionally active FP-receptors whose activation by a variety of free acid and an amide forms of synthetic PGs leads to the contraction of this tissue and which can be pharmacologically blocked by an FP-receptor antagonist, AL-8810.