α-Methylated simplified resiniferatoxin (sRTX) thiourea analogues as potent and stereospecific TRPV1 antagonists.

A series of α-methylated analogues of the potent sRTX thiourea antagonists were investigated as rTRPV1 ligands in order to examine the effect of α-methylation on receptor activity. The SAR analysis indicated that activity was stereospecific with the (R)-configuration of the newly formed chiral center providing high binding affinity and potent antagonism while the configuration of the C-region was not significant.

GPR119 agonists: a promising approach for T2DM treatment? A SWOT analysis of GPR119.

Ever since its advent as a promising therapeutic target for type 2 diabetes mellitus (T2DM), G-protein-coupled receptor 119 (GPR119) has received much interest from the pharmaceutical industry. This interest peaked in June 2010, when Sanofi-Aventis agreed to pay Metabolex (Cymabay Therapeutics) US$375 million for MBX-2982, which was a representative orally active GPR119 agonist. However, Sanofi-Aventis opted to terminate the deal in May 2011 and another leading GPR119 agonist, GSK1292263, had a loss of efficacy during its clinical trial. In this review, I discuss the pros and cons of GPR119 through a strengths, weaknesses, opportunities, and threats (SWOT) analysis and propose development strategies for the eventual success of a GPR119 agonist development program.

N-4-t-Butylbenzyl 2-(4-methylsulfonylaminophenyl) propanamide TRPV1 antagonists: Structure-activity relationships in the A-region.

Structure-activity relationships for the A-region in a series of N-4-t-butylbenzyl 2-(4-methylsulfonylaminophenyl) propanamides as TRPV1 antagonists have been investigated. Among them, the 3-fluoro analogue 54 showed high binding affinity and potent antagonism for both rTRPV1 and hTRPV1 in CHO cells. Its stereospecific activity was demonstrated with marked selectivity for the (S)-configuration (54S versus 54R). A docking study of 54S with our hTRPV1 homology model highlighted crucial hydrogen bonds between the ligand and the receptor contributing to its potency.

Stereospecific high-affinity TRPV1 antagonists: chiral N-(2-benzyl-3-pivaloyloxypropyl) 2-[4-(methylsulfonylamino)phenyl]propionamide analogues.

Previously, we reported the thiourea antagonists 2a and 2b as potent and high affinity TRPV1 antagonists. For further optimization of the lead compounds, a series of their amide and alpha-substituted amide surrogates were investigated and novel chiral N-(2-benzyl-3-pivaloyloxypropyl) 2-[4-(methylsulfonylamino)phenyl]propionamide analogues were characterized as potent and stereospecific rTRPV1 antagonists. In particular, compounds 72 and 73 displayed high binding affinities, with K i values of 4.12 and 1.83 nM and potent antagonism with K i values of 0.58 and 5.2 nM, respectively, in rTRPV1/CHO cells. These values are comparable or more potent than those of 5-iodoRTX under the same assay conditions. A distinctive binding model that includes two hydrophobic pockets is proposed for this series of compounds based on docking studies of 57 and 72 with a homology model of the TM3/4 region of TRPV1.

Alpha-substituted N-(4-tert-butylbenzyl)-N'-[4-(methylsulfonylamino)benzyl]thiourea analogues as potent and stereospecific TRPV1 antagonists.

A series of alpha-substituted N-(4-tert-butylbenzyl)-N'-[4-(methylsulfonylamino)benzyl]thiourea analogues have been investigated as TRPV1 receptor antagonists. alpha-Methyl substituted analogues showed potent and stereospecific antagonism to the action of capsaicin on rat TRPV1 heterologously expressed in Chinese hamster ovary cells. In particular, compounds 14 and 18, which possess the R-configuration, exhibited excellent potencies (respectively, K(i)=41 and 39.2 nM and K(i(ant))=4.5 and 37 nM).

Examination of acylated 4-aminopiperidine-4-carboxylic acid residues in the phosphotyrosyl+1 position of Grb2 SH2 domain-binding tripeptides.

A 4-aminopiperidine-4-carboxylic acid residue was placed in the pTyr+1 position of a Grb2 SH2 domain-binding peptide to form a general platform, which was then acylated with a variety of groups to yield a library of compounds designed to explore potential binding interactions, with protein features lying below the betaD strand. The highest affinities were obtained using phenylethyl carbamate and phenylbutyrylamide functionalities.

Novel potent antagonists of transient receptor potential channel, vanilloid subfamily member 1: structure-activity relationship of 1,3-diarylalkyl thioureas possessing new vanilloid equivalents.

Recently, 1,3-diarylalkyl thioureas have merged as one of the promising nonvanilloid TRPV1 antagonists possessing excellent therapeutic potential in pain regulation. In this paper, the full structure-activity relationship for TRPV1 antagonism of a novel series of 1,3-diarylalky thioureas is reported. Exploration of the structure-activity relationship, by systemically modulating three essential pharmacophoric regions, led to six examples of 1,3-dibenzyl thioureas, which exhibit Ca(2+) uptake inhibition in rat DRG neuron with IC(50) between 10 and 100 nM.

Design and synthesis of 4-(alpha-hydroxymalonyl)phenylalanine as a new phosphotyrosyl mimetic and its use in growth factor receptor bound 2 src-homology 2 (Grb2 SH2) domain-binding peptides.

A new phosphotyrosyl mimetic 4-(alpha-hydroxymalonyl)phenylalanine and its incorporation into a Grb2 SH2 domain-binding tripeptide are presented. In whole-cell studies using malonyl ethyl ester prodrug derivatives, it was observed that the 4-(alpha-hydroxymalonyl)phenylalanyl-containing peptide exhibited greater efficacy than the nonhydroxylated 4-(malonyl)phenylalanyl-containing congener in blocking the association of Grb2 with activated erbB-2 tyrosine kinase. These results are consistent with de-esterification and at least partial intracellular decarboxylation.

Synthesis of tripeptides as potent Yersinia protein tyrosine phosphatase inhibitors.

We report the synthesis of a series of monoanionic phosphotyrosyl (pTyr) mimetic-containing tripeptides based on 'Fmoc-Glu(OBn)-Xxx-Leu-amide' (where Xxx = pTyr mimetic) and their N-terminally modified derivatives. The inhibitory potencies of compounds were tested against YopH and human PTP1B enzymes. Several compounds exhibited noteworthy activity against both YopH and PTP1B. Among the N-terminally modified analogues, 5-methylindole derivative 30 was found to be the best moiety to replace base-labile Fmoc group. A mode of binding with YopH is proposed for tripeptides 21, 30, and 31.

Analysis of structure-activity relationships for the 'B-region' of N-(4-t-butylbenzyl)-N'-[4-(methylsulfonylamino)benzyl]-thiourea analogues as TRPV1 antagonists.

The structure-activity relationships for the 'B-region' of N-(4-t-butylbenzyl)-N'-[4-(methylsulfonylamino)benzyl]thiourea analogues have been investigated as TRPV1 receptor antagonists. A docking model of potent antagonist 2 with the sensor region of TRPV1 is proposed.

Analysis of structure-activity relationships for the 'A-region' of N-(4-t-butylbenzyl)-N'-[4-(methylsulfonylamino)benzyl]thiourea analogues as TRPV1 antagonists.

The structure-activity relationships for the 'A-region' of N-(4-t-butylbenzyl)-N'-[4-(methylsulfonylamino)benzyl]thiourea analogues have been investigated as TRPV1 receptor antagonists. The 2-halogen analogues showed enhanced antagonism compared to the prototype antagonist.

Examination of phosphoryl-mimicking functionalities within a macrocyclic Grb2 SH2 domain-binding platform.

Reported herein are the design, synthesis, and Grb2 SH2 domain-binding affinities of several phosphoryl-mimicking groups displayed within the context of a conformationally constrained macrocyclic platform. With use of surface plasmon resonance techniques, single-digit nanomolar affinities were exhibited by phosphonic acid and malonyl-containing diacidic phosphoryl mimetics (for 4h and 4g, K(D) = 1.47 and 3.62 nM, respectively). Analogues containing monoacidic phosphoryl mimetics provided affinities of K(D) = 16-67 nM. Neutral phosphoryl-mimicking groups did not show appreciable binding.

Different vanilloid agonists cause different patterns of calcium response in CHO cells heterologously expressing rat TRPV1.

The vanilloid receptor subtype 1 (TRPV1 or VR1) is expressed in nociceptive primary afferents of the C-fiber 'pain' pathway and has attracted considerable attention as a therapeutic target. Here, using rat TRPV1 heterologously expressed in Chinese hamster ovary cells, we show that different agonists show different patterns of modulation of the intracellular Ca2+ concentration, monitored in individual cells by fura-2 Ca2+ imaging. We identified 5 parameters (potency, maximal response, latency of response, variability of latency of response among individual cells, and desensitization) which behaved differently for different compounds. The potencies of the compounds examined ranged from EC50 values of 80 pM to 9 microM. Peak levels of induced [Ca2+]i were observed either higher (RTX) or lower (anandamide) than for capsaicin. Significant latencies of response were observed for some (e.g. RTX) but not other derivatives, with great variation among individual cells in this latency. Marked desensitization after stimulation was detected in some cases (e.g. anandamide, capsaicin); for others, no desensitization was observed. We conclude that structurally diverse vanilloid agonists induce marked diversity in the patterns of Ca2+ response. This diversity of response may provide opportunities for pharmacological exploitation.

Design and synthesis of conformationally constrained Grb2 SH2 domain binding peptides employing alpha-methylphenylalanyl based phosphotyrosyl mimetics.

Previous work has shown that incorporation of either 1-aminocyclohexanecarboxylic acid (Ac6c) or alpha-methyl-p-phosphonophenylalanine ((alpha-Me)Ppp) in the phosphotyrosyl (pTyr) C-proximal position (pY + 1 residue) of Grb2 SH2 domain binding peptides confers high affinity. The tetralin-based (S)-2-amino-6-phosphonotetralin-2-carboxylic acid (Atc(6-PO3H2)) simultaneously presents structural features of both (alpha-Me)Ppp and Ac6c residues. The current study compares the affinity of this tetralin hybrid Atc(6-PO3H2) versus Ac6c and (alpha-Me)Ppp residues when incorporated into the pY + 1 position of a high-affinity Grb2 SH2 domain binding tripeptide platform. The highest binding affinity (KD = 14.8 nM) was exhibited by the (alpha-Me)Ppp-containing parent, with the corresponding Ac6c-containing peptide being nearly 2-fold less potent (KD = 23.8 nM). The lower KD value was attributable primarily to a 50% increase in off-rate. Replacement of the Ac6c residue with the tetralin-based hybrid resulted in a further 4-fold decrease in binding affinity (KD = 97.8 nM), which was the result of a further 6-fold increase in off-rate, offset by an approximate 45% increase in on-rate. Therefore, by incorporation of the key structural components found in (alpha-Me)Ppp into the Ac6c residue, the tetralin hybrid does enhance binding on-rate. However, net binding affinity is decreased due to an associated increase in binding off-rate. Alternatively, global conformational constraint of an (alpha-Me)Ppp-containing peptide by beta-macrocyclization did result in pronounced elevation of binding affinity, which was achieved primarily through a decrease in the binding off-rate. Mathematical fitting using a simple model that assumed a single binding site yielded an effective KD of 2.28 nM. However this did not closely approximate the data obtained. Rather, use of a complex model that assumed two binding sites resulted in a very close fit of data and provided KD values of 97 pM and 72 nM for the separate sites, respectively. Therefore, although local conformational constraint in the pY + 1 residue proved to be deleterious, global conformational constraint through beta-macrocyclization achieved higher affinity. Similar beta-macrocyclization may potentially be extended to SH2 domain systems other than Grb2, where bend geometries are required.

Analysis of structure-activity relationships for the 'B-region' of N-(3-acyloxy-2-benzylpropyl)-N(')-[4-(methylsulfonylamino)benzyl]thiourea analogues as vanilloid receptor antagonists: discovery of an N-hydroxythiourea analogue with potent analgesic activity.

The structural modifications on the B-region of the potent and high affinity vanilloid receptor (VR1) lead ligand N-(3-acyloxy-2-benzylpropyl)-N(')-[4-(methylsulfonylamino)benzyl]thiourea were investigated by the replacement of the thiourea with diverse isosteric functional groups. Structure-activity analysis indicated that the A-region in this series was the primary factor in determining the agonistic/antagonistic activities regardless of the B-region. The N(C)-hydroxy thiourea analogues (12, 13) showed excellent analgesic activities in the acetic acid writhing assay compared to the parent thiourea analogues.

Structure-activity relationships of simplified resiniferatoxin analogues with potent VR1 agonism elucidates an active conformation of RTX for VR1 binding.

We previously described a series of N-(3-acyloxy-2-benzylpropyl) homovanillate and N'-(4-hydroxy-3-methoxybenzyl) thiourea derivatives that were potent VR1 agonists with high-affinities and excellent analgesic profiles. The design of these simplified RTX analogues was based on our RTX-derived pharmacophore model which incorporates the 4-hydroxy-3-methoxyphenyl (A-region), C(20)-ester (B-region), orthophenyl (C1-region) and C(3)-keto (C2-region) groups of RTX. For the purpose of optimizing the spatial arrangement of the four principal pharmacophores on the lead agonists (1-4), we have modified the distances in the parent C-region, 3-acyloxy-2-benzylpropyl groups, by lengthening or shortening one carbon to vary the distances between the pharmacophores. We find that two of the amides, 4 and 19, possess EC(50) values <1 nM for induction of calcium influx in the VR1-CHO cells. As observed previously, the structure-activity relations for inhibition of RTX binding to VR1 and for induction of calcium uptake were distinct, presumably reflecting both intrinsic and methodological factors. In order to find the active conformation of VR1 ligands, the energy-minimized conformations of seven selected agonists were determined and the positions of their four pharmacophores were matched with those of five low energy RTX conformations. The rms values for the overlaps in the pharmacophores were calculated and correlated with the measured binding affinities (K(i)) and calcium influx (EC(50)) values. The binding affinities of the agonists correlated best with the RMS values derived from RTX conformation E (r(2)=0.92), predicting a model of the active conformation of RTX and related vanilloids for binding to VR1. Poorer correlation was obtained between any of the conformations and the EC(50) values for calcium influx.

N-[4-(methylsulfonylamino)benzyl]thiourea analogues as vanilloid receptor antagonists: analysis of structure-activity relationships for the "C-Region".

We recently reported that N-(4-t-butylbenzyl)-N'-[4-(methylsulfonylamino)benzyl] thiourea (2) was a high affinity antagonist of the vanilloid receptor with a binding affinity of K(i)=63 nM and an antagonism of K(i)=53.9 nM in rat VR1 heterologously expressed in Chinese hamster ovary (CHO) cells (Mol. Pharmacol. 2002, 62, 947-956). In an effort to further improve binding affinity and antagonistic potency, we have modified the C-region of the lead 4-t-butylbenzyl group with diverse surrogates, such as araalkyl, alkyl, 4-alkynylbenzyl, indanyl, 3,3-diarylpropyl, 4-alkoxybenzyl, 4-substituted piperazine and piperidine. The lipophilic surrogates, arylalkyl and alkyl, conferred modest decreases in binding affinities and antagonistic potencies; the groups having heteroatoms resulted in dramatic decreases. Our findings indicate that 4-t-butylbenzyl is one of the most favorable groups for high receptor binding and potent antagonism to VR1 in this structural series.

N-4-Substituted-benzyl-N'-tert-butylbenzyl thioureas as vanilloid receptor ligands: investigation on the role of methanesulfonamido group in antagonistic activity.

A series of N-4-substituted-benzyl-N'-tert-butylbenzyl thioureas were prepared for the study of their agonistic/antagonistic activities to the vanilloid receptor in rat DRG neurons. Their structure-activity relationship reveals that not only the two oxygens and amide hydrogen of sulfonamido group, but also the optimal size of methyl in methanesulfonamido group play an integral role for the antagonistic activity on vanilloid receptor.

Enantioselective synthesis of (S)-3-carboxy-4-((carboxy)difluoromethyl)phenylalanine in protected form and its incorporation into a PTP-1B-directed tripeptide.

Recent findings have shown that, when expressed in the tripeptide platform, 'Fmoc-Glu-Xxx-Leu-amide', the phosphotyrosyl mimetic (pTyr), Xxx=(S)-3-carboxy-4-(carboxymethyl)-Phe, provides higher PTP-1B affinity than that obtained with Xxx=(S)-difluorophosphonomethyl-Phe (F(2)Pmp). This was of note, since difluorophosphonomethyl-containing pTyr mimetics have typically exhibited higher PTP-inhibitory potencies than carboxy-based mimetics, indicating the potential value of 3-carboxy-4-(carboxymethyl)-Phe as a starting point for further analogue development. Therefore, relying on precedence that alpha-fluorination often enhances PTP-1B affinity, (S)-3-carboxy-4-((carboxy)difluoromethyl)-Phe was designed as a PTP-1B-directed pTyr mimetic. Reported herein is the synthesis of this new amino acid analogue through application of commercially available Williams chiral auxiliary. The target was prepared in an orthogonally protected form suitable for peptide synthesis according to Fmoc chemistries and utilization for the synthesis of a PTP-1B-directed tripeptide bearing the sequence indicated above. Biological evaluation with in vitro PTP-1B assays indicated nearly total loss of affinity for this peptide. Evidence is provided that the unexpected deleterious effect of fluorination is probably not related to pK(a) effects.

High-affinity partial agonists of the vanilloid receptor.

The vanilloid receptor VR1 is a polymodal nociceptor sensitive to capsaicin, protons, and heat. Because VR1 represents an attractive therapeutic target for conditions ranging from long-term pain to bladder hyperreflexia, we and other groups have sought to develop novel ligands with enhanced potencies and novel pharmacological properties. Here, we characterize two compounds, N-[2-(3,4-dimethylbenzyl)-3-(pivaloyloxy)propyl]-N'-[4-(methylsulfonylamino)benzyl]thiourea (JYL827) and N-(4-tert-butylbenzyl)-N'-[3-methoxy-4-(methylsulfonylamino)benzyl]thiourea (JYL1511), that function as partial agonists for rat VR1 heterologously expressed in Chinese hamster ovary cells. Both compounds showed substantially enhanced potency, inhibiting [3H] resiniferatoxin binding with Ki values of 29.3 +/- 7.6 and 50.4 +/- 16.5 nM, respectively, compared with 1810 +/- 270 nM for capsaicin. The compounds showed different extents of partial agonism, 6.8 +/- 0.7% and 17.4 +/- 0.6%, respectively, and the expected corresponding degrees of partial antagonism (93.9 +/- 0.9 and 84.1 +/- 3.2%, respectively). Their IC50 values for antagonism of 45Ca2+ uptake in response to capsaicin were 67.3 +/- 24.9 nM and 3.4 +/- 0.5 nM, respectively. Protons, temperature, and protein kinase C all function as coactivators/modulators of rVR1. All enhanced the extent of partial agonism of JYL827 and JYL1511. Thus, at pH 5.5, for example, the extents of partial agonism increased to 54.9 +/- 2.5% and to 90.7 +/- 1.7%, respectively, relative to the response elicited by 300 nM capsaicin. The extents of partial antagonism decreased correspondingly. Compounds such as JYL827 and JYL1511 now permit exploration of the potential utility of partial agonists of rVR1 in animal models. Our results emphasize, moreover, the strong dependence of such partial agonists on other modulators of rVR1 and predict that their biological behavior will depend strongly on biological context.