Synthesis and physicochemical characterization of (6S)-5-formiminotetrahydrofolate; a reference standard for metabolomics.

The one-carbon carrier of the formate oxidation level derived from the interaction of tetrahydrofolate and formiminoglutamate, which has been tentatively identified as 5-formiminoltetrahydrofolate, has been prepared by chemical synthesis. Treatment of a solution of (6S)-tetrahydrofolate in aqueous base with excess ethyl formimidate in the presence of anti-oxidant under anaerobic conditions afforded a gummy solid which, based on mass spectral analysis, conformed to a monoformimino derivative of tetrahydrofolate. Further physicochemical characterization by validated methods strongly suggested that the product of chemical synthesis was identical to the enzymatically produced material and that it was, in fact, (6S)-5-formiminotetrahydrofolate. Conditions and handling methods toward maintaining the integrity of this highly sensitive compound were identified and are described, as is analytical methodology, useful for research studies using it.

Identification of the first trans-(3R,4R)- dimethyl-4-(3-hydroxyphenyl)piperidine derivative to possess highly potent and selective opioid kappa receptor antagonist activity.

A structurally novel opioid kappa receptor selective ligand has been identified. This compound, (3R)-7-hydroxy-N-((1S)-1-[[(3R,4R)-4-(3-hydroxyphenyl)-3,4-dimethyl-1-piperidinyl]methyl]-2-methylpropyl)-1,2,3,4-tetrahydro-3-isoquinolinecarboxamide (JDTic, 10) demonstrated high affinity for the kappa receptor in the binding assay (kappa K(i) = 0.3 nM) and highly potent and selective kappa antagonism in the [(35)S]GTP-gamma-S assay using cloned opioid receptors (kappa K(i) = 0.006 nM, mu/kappa ratio = 570, delta/kappa ratio > 16600).

Factors influencing agonist potency and selectivity for the opioid delta receptor are revealed in structure-activity relationship studies of the 4-[(N-substituted-4-piperidinyl)arylamino]-N,N-diethylbenzamides.

A study of the effect of transposition of the internal nitrogen atom for the adjacent benzylic carbon atom in delta-selective agonists such as BW373U86 (1) and SNC-80 (2) has been undertaken. It was shown that high-affinity, fully efficacious, and delta opioid receptor-selective compounds can be obtained from this transposition. In addition to the N,N-diethylamido group needed as the delta address, the structural features identified to promote delta receptor affinity in the set of compounds studied included a cis relative stereochemistry between the 3- and 4-substituents in the piperidine ring, a trans-crotyl or allyl substituent on the basic nitrogen, the lack of a 2-methyl group in the piperidine ring, and either no substitution or hydroxyl substitution in the aryl ring not substituted with the N,N-diethylamido group. Structural features found to be important for mu affinity include hydroxyl substitution in the aryl ring, the presence of a 2-methyl group in a cis relative relationship to the 4-amino group as well as N-substituents such as cyclopropylmethyl. It was also determined that mu receptor affinity could be increased while maintaining delta receptor affinity, especially when hydroxyl-substituted compounds are considered. Additionally, it was discovered that the somewhat lower mu/delta selectivities observed for the piperidine compounds relative to the piperazine-based ligands appear to arise as a consequence of the carbon-nitrogen transposition which imparts an overall lower delta and higher mu affinity to the piperidine-based ligands. This higher affinity for the mu receptor, apparently intrinsic to the piperidine-based compounds, suggests that ligands of this class will more easily be converted to mu/delta combination agonists compared to the piperazine ligands such as 1. This is particularly important since analogues of 1, which show both mu- and delta-type activity, are now recognized as important for their strong analgesia and cross-canceling of many of the side effects found in agonists operating exclusively from either the delta or mu opioid receptor.

4-[(8-Alkyl-8-azabicyclo[3.2.1]octyl-3-yl)-3-arylanilino]-N,N-diethylbenzamides: high affinity, selective ligands for the delta opioid receptor illustrate factors important to antagonist activity.

The tropane derived compounds, 4-[(8-alkyl-8-azabicyclo[3.2.1]octyl-3-yl)-3-arylanilino]-N,N-d iethylbenzamides (5a-d), were synthesized and found to have high affinity and selectivity for the delta receptor. Compounds 5a-d are structurally similar to the full agonist (-)-RTI-5989-54 (3); yet, efficacy studies for compounds in this series (5a-d) reveal greatly diminished agonist activity as well as antagonism not found in piperidine-based compounds like 3.

QSAR analysis of Delta(8)-THC analogues: relationship of side-chain conformation to cannabinoid receptor affinity and pharmacological potency.

A novel quantitative structure-activity relationship (QSAR) for the side-chain region of Delta(8)-tetrahydrocannabinol (Delta(8)-THC) analogues is reported. A series of 36 side-chain-substituted Delta(8)-THCs with a wide range of pharmacological potency and CB1 receptor affinity was investigated using computational molecular modeling and QSAR analyses. The conformational mobility of each compound's side chain was characterized using a quenched molecular dynamics approach. The QSAR techniques included a modified active analogue approach (MAA), multiple linear regression analyses (MLR), and comparative molecular field analysis (CoMFA) studies. All three approaches yielded consistent results. The MAA approach applied to a set of alkene/alkyne pairs identified the most active conformers as those with conformational mobility constrained within an approximately 8 A radius. MLR analyses (restricted to 15 hydrocarbon side-chain analogues) identified two variables describing side-chain length and terminus position that were able to fit the pharmacological data for receptor affinity with a correlation coefficient for pK(D) of 0.82. While chain length was found to be directly related to receptor affinity, the angle made by the side chain from its attachment point to its terminus (angle defined by C3-C1'-side-chain terminus carbon, see Figure 1) was found to be inversely related to affinity. These results suggest that increased side-chain length and increased side-chain ability to wrap around the ring system are predicted to increase affinity. Therefore, the side chain's conformational mobility must not restrict the chain straight away from the ring system but must allow the chain to wrap back around toward the ring system. Finally, the CoMFA analyses involved all 36 analogues; they also provided data to support the hypothesis that for optimum affinity and potency the side chain must have conformational freedom that allows its terminus to fold back and come into proximity with the phenolic ring.

Optically pure (-)-4-[(N-allyl-3-methyl-4-piperidinyl)phenyl-amino]-N,N-diethylbenzami de displays selective binding and full agonist activity for the delta opioid receptor.

The optical isomers of 4-[(N-allyl-3-methyl-4-piperidinyl)phenylamino]-N,N-diethylbenzamide+ ++ (3) have been prepared and tested in both binding and functional assays. The data show that (-)-3 is responsible for the delta opioid activity demonstrated by the racemic material. This compound displays a binding affinity of 5.5 nM for the delta opioid receptor as well as a 470-fold delta versus mu selectivity. Importantly, (-)-3 is a full agonist at the delta receptor in comparison with SNC-80 (2). Taken together, the data suggest that (-)-3 behaves more like the prototypical delta agonists, BW373U86 or SNC-80, and less like the peptidomimetic compound SL-3111 (5).

Asymmetric synthesis of 9-alkyl-2-benzyl-6,7-benzomorphans: characterization as novel sigma receptor ligands.

A convenient enantioselective synthesis of (1R,5R,9R)- and (1S,5S, 9S)-9-alkyl-2-benzyl-6,7-benzomorphans (2a-c) which starts with naphthaldehyde is described. These compounds were designed to gain additional information on the structure-sigma binding relationship of the 6,7-benzomorphan class of sigma ligands. In contrast to pentazocine and most 6,7-benzomorphans, the (1R,5R,9R)-isomers of 2a-c showed greater affinity for the sigma(1) receptor than the (1S, 5S,9S)-isomers. Despite reversal of enantioselectivity at the sigma(1) sites, moderate affinity and enantioselectivity at the sigma(2) sites [greater affinity for (1R,5R,9R)-isomers than (1S,5S, 9S)-isomers] were maintained. A comparison of the binding affinities of 2a-c to the more conformationally flexible trans-2-alkyl-1-benzaminoethyl-1,2-dihydronaphthalenes (10a-c) suggested that the relatively rigid structure of 2a-c played an important part in their sigma(1) binding properties. These compounds, particularly (1R,5R,9R)-2-benzyl-9-methyl-6,7-benzomorphan [(-)-2a], which has a K(i) value of 0.96 nM, will be useful in further characterization of the sigma(1) receptor.

(+/-)-4-[(N-allyl-cis-3-methyl-4-piperidinyl)phenylamino]-N,N-diethylbenzamide displays selective binding for the delta opioid receptor.

Racemic 4-[(N-allyl-cis-3-methyl-4-piperidinyl)phenylamino]-N,N-diethylbenzam ide (3a) was synthesized and found to have good affinity and selectivity for the delta receptor. These compounds can be viewed as an analog of BW373U86 and SNC-80 where an internal piperazine nitrogen has been transposed with a benzylic carbon. Functionally, 3a behaves as an agonist at the delta receptor with no measurable stimulation of either the mu or kappa receptor subtypes and was found to be devoid of any measurable amount of antagonist activity for any opioid receptor. A comparison of 3a to SNC-80 and DPDPE in the [35S]GTPgammaS functional assay suggests that 3a may be more like the peptide DPDPE.

Identification of an opioid kappa receptor subtype-selective N-substituent for (+)-(3R,4R)-dimethyl-4-(3-hydroxyphenyl)piperidine.

A three-component library of compounds was prepared in parallel using multiple simultaneous solution-phase synthetic methodology. The compounds were biased toward opioid receptor antagonist activity by incorporating (+)-(3R,4R)-dimethyl-4-(3-hydroxyphenyl)piperidine (a potent, nonselective opioid pure antagonist) as one of the monomers. The other two monomers, which included N-substituted or unsubstituted Boc-protected amino acids and a range of substituted aryl carboxylic acids, were selected to add chemical diversity. Screening of these compounds in competitive binding experiments with the kappa opioid receptor selective ligand [3H]U69,593 led to the discovery of a novel kappa opioid receptor selective ligand, N-¿(2'S)-[3-(4-hydroxyphenyl)propanamido]-3'-methylbutyl¿-(3R, 4R)-dimethyl-4-(3-hydroxyphenyl)piperidine (8, RTI-5989-29). Additional structure-activity relationship studies suggested that 8 possesses lipophilic and hydrogen-bonding sites that are important to its opioid receptor potency and selectivity. These sites appear to exist predominantly within the kappa receptor since the selectivity arises from a 530-fold loss of affinity of 8 for the mu receptor and an 18-fold increase in affinity for the kappa receptor relative to the mu-selective ligand, (+)-N-[trans-4-phenyl-2-butenyl]-(3R, 4R)-dimethyl-4-(3-hydroxyphenyl)piperidine (5a). The degree of selectivity observed in the radioligand binding experiments was not observed in the functional assay. According to its ability to inhibit agonist stimulated binding of [35S]GTPgammaS at all three opioid receptors, compound 8 behaves as a mu/kappa opioid receptor pure antagonist with negligible affinity for the delta receptor.

N-Substituted 9beta-methyl-5-(3-hydroxyphenyl)morphans are opioid receptor pure antagonists.

The inhibition of radioligand binding and [35S]GTPgammaS functional assay data for N-methyl- and N-phenethyl-9beta-methyl-5-(3-hydroxyphenyl)morphans (5b and 5c) show that these compounds are pure antagonists at the micro, delta, and kappa opioid receptors. Since 5b and 5c have the 5-(3-hydroxyphenyl) group locked in a conformation comparable to an equatorial group of a piperidine chair conformation, this information provides very strong evidence that opioid antagonists can interact with opioid receptors in this conformation. In addition, it suggests that the trans-3, 4-dimethyl-4-(3-hydroxyphenyl)piperidine class of antagonist operates via a phenyl equatorial piperidine chair conformation. Importantly, the close relationship between the 4-(3-hydroxyphenyl)piperidines and 5-(3-hydroxyphenyl)morphan antagonists shows that the latter class of compound provides a rigid platform on which to build a novel series of opioid antagonists.

Investigation of the N-substituent conformation governing potency and mu receptor subtype-selectivity in (+)-(3R, 4R)-dimethyl-4-(3-hydroxyphenyl)piperidine opioid antagonists.

A study of the binding site requirements associated with the N-substituent of (+)-(3R,4R)-dimethyl-4-(3-hydroxyphenyl)piperidine (4) derivatives was undertaken using a set of rigid vs flexible N-substituents. The study showed that compounds 7-9 bearing the trans-cinnamyl N-substituent most closely reproduced the potency at the opioid receptor of the flexible N-propylphenyl or N-propylcyclohexyl analogues previously reported. Neither the N-substituted cis-cinnamyl nor the cis-phenylcyclopropylmethyl compounds 10 and 11, respectively, showed high affinity for the opioid receptor. However, the N-trans-phenylcyclopropylmethyl compound 12 closely approximated the affinity of compounds 7-9. Additionally, we found that free rotation of the phenyl ring is necessary for high affinity binding and mu receptor subtype selectivity as the planar N-substituted thianaphthylmethyl and benzofuranylmethyl compounds 13 and 14 had significantly lower binding affinities. Altogether, these findings suggest that the high binding affinity, selectivity, and antagonist potency of N-propylphenyl or N-propylcyclohexyl analogues of (+)-(3R, 4R)-dimethyl-4-(3-hydroxyphenyl)piperidine (4) are achieved via a conformation wherein the connecting chain of the N-substituents is extended away from piperidine nitrogen with the appended ring system rotated out-of-plane relative to the connecting chain atoms. This conformation is quite similar to that observed in the solid state for 5, as determined by single crystal X-ray analysis. Additionally, it was found that, unlike naltrexone, N-substituents bearing secondary carbons attached directly to the piperidine nitrogen of 4 suffer dramatic losses of potency vs analogues not substituted in this manner. Using a functional assay which measured stimulation or inhibition of [35S]GTP-gamma-S binding, we show that the trans-cinnamyl analogues of (+)-(3R, 4R)-dimethyl-4-(3-hydroxyphenyl)piperidine (4) retain opioid pure antagonist activity and possess picomolar antagonist potency at the mu receptor.

N-substituted octahydro-4a-(3-hydroxyphenyl)-10a-methyl-benzo[g]isoquinolines are opioid receptor pure antagonists.

N-Methyl- and N-phenylethyl-(+/-)-1,2,3,4,4a,5,10,10a- octahydro-4a-(3-hydroxyphenyl)-10a-methyl-benzo[g]isoquinolines (4 and 5, respectively) were found to be pure opioid antagonists. These compounds were shown to share many of the characteristics identified with the N-methyl- and N-phenylethyl trans-3,4-dimethyl-4-(3-hydroxyphenyl)piperidine (1 and 2, respectively) including N-substituent mediated potency and a lack of N-substituent mediated antagonism. These data suggest that compounds 4 and 5 and the N-substituted trans-3,4-dimethyl-4-(3-hydroxyphenyl)piperidines (1 and 2) may interact with opioid receptors similarly.

Synthesis and transporter binding properties of 2,3-diphenyltropane stereoisomers. Comparison to 3beta-phenyltropane-2beta-carboxylic acid esters.

2beta,3beta-Diphenyl-(5), 2alpha,3alpha-diphenyl-(6), and 2alpha,3beta-diphenyltropane (3) as well as 2,3-diphenyltrop-2-ene (4) were prepared in racemic form and assayed for inhibition of radioligand binding at the dopamine (DA), serotonin (5-HT), and norepinephrine (NE) transporters. Among all three transporters, compounds 4-6 bound the DA transporter with the highest affinity. The 2beta,3beta-diphenyltropane (5) bound the DA transporter with an IC50 value (28 nM) almost identical to that of 3beta-phenyltropane-2beta-carboxylic acid methyl ester (WIN 35,065-2) and has much greater selectivity relative to binding to the serotonin transporter. A comparison of the radioligand data from this study to radioligand data obtained on other WIN 35,065-2 analogs suggests that hydrophobicity of the C-2 substituent of some analogs of the WIN 35,065-2 class may be an important contributing factor to binding at the DA transporter.

Effects of sphingosine stereoisomers on P-glycoprotein phosphorylation and vinblastine accumulation in multidrug-resistant MCF-7 cells.

To investigate the role of protein kinase C (PKC) in the regulation of multidrug resistance and P-glycoprotein (P-gp) phosphorylation, the natural isomer of sphingosine (SPH), D-erythro sphingosine (De SPH), and its three unnatural stereoisomers were synthesized. The SPH isomers showed similar potencies as inhibitors of in vitro PKC activity and phorbol binding, with IC50 values of approximately 50 microM in both assays. Treatment of multidrug-resistant MCF-7ADR cells with SPH stereoisomers increased vinblastine (VLB) accumulation up to 6-fold at 50 microM but did not alter VLB accumulation in drug-sensitive MCF-7 wild-type (WT) cells or accumulation of 5-fluorouracil in either cell line. Phorbol dibutyrate treatment of MCF-7ADR cells increased phosphorylation of P-gp, and this increase was inhibited by prior treatment with SPH stereoisomers. Treatment of MCF-7ADR cells with SPH stereoisomers decreased basal phosphorylation of the P-gp, suggesting inhibition of PKC-mediated phosphorylation of P-gp. Most drugs that are known to reverse multidrug resistance, including several PKC inhibitors, have been shown to directly interact with P-gp and inhibit drug binding. SPH stereoisomers did not inhibit specific binding of [3H] VLB to MCF-7ADR cell membranes or [3H]azidopine photoaffinity labeling of P-gp or alter P-gp ATPase activity. These results suggest that SPH isomers are not substrates of P-gp and suggest that modulation of VLB accumulation by SPH stereoisomers is associated with inhibition of PKC-mediated phosphorylation of P-gp.

Synthesis, ligand binding, and quantitative structure-activity relationship study of 3 beta-(4'-substituted phenyl)-2 beta-heterocyclic tropanes: evidence for an electrostatic interaction at the 2 beta-position.

A set of 3 beta-(4'-substituted phenyl)-2 beta-heterocyclic tropanes was designed, synthesized, and characterized. We discovered that these compounds can function as bioisosteric replacements for the corresponding WIN 35,065-2 analogs which possess a 2 beta-carbomethoxy group. Several of the compounds showed high affinity and selectivity for the dopamine transporter (DAT) relative to the serotonin and norepinephrine transporters. From the structure-activity relationship study, the 3 beta-(4'-chlorophenyl)-2 beta-(3'-phenylisoxazol-5-yl)tropane (5d) emerged as the most potent and selective compound. The binding data for 2 beta-heterocyclic tropanes were found to show a high correlation with molecular electrostatic potential (MEP) minima near one of the heteroatoms in the 2 beta-substituents. In contrast, low correlations were found for other MEP minima near the 2 beta-substituent as well as for calculated log P or substituent volume. These quantitative structure-activity relationship studies are consistent with an electrostatic contribution to the binding potency of these WIN 35,065-2 analogs at the DAT.

Structure-activity analysis of anandamide analogs: relationship to a cannabinoid pharmacophore.

Anandamides are endogenous fatty acid ethanolamides that have been shown to bind to the cannabinoid receptor and possess cannabimimetic activity yet are structurally dissimilar from the classical cannabinoids found in Cannabis sativa. We have employed molecular dynamics studies of a variety of anandamides to characterize their conformational mobility and determine whether there are pharmacophoric similarities with delta 9-THC. We have found that a looped conformation of these arachidonyl compounds is energetically favorable and that a structural correlation between this low-energy conformation and the classical cannabinoids can be obtained with the superposition of (1) the oxygen of the carboxyamide with the pyran oxygen in delta 9-THC, (2) the hydroxyl group of the ethanol with the phenolic hydroxyl group of delta 9-THC, (3) the five terminal carbons and the pentyl side chain of delta9-THC, and (4) the polyolefin loop overlaying with the cannabinoid tricyclic ring. The shape similarity is extended to show that other fatty acid ethanolamides that possess varying degrees of unsaturation also vary in their conformational mobility, which affects their ability to overlay with delta 9-THC as described above. Within this series of compounds, the most potent analog, the tetraene (arachidonyl) analog (i.e., anandamide itself), was determined to have restricted conformational mobility that favored an optimal pharmacophore overlay with delta9-THC. Eight pharmacologically active anandamide analogs are shown to have similar conformational mobility and pharmacophore alignments that are conformationally accessible. Furthermore, when these compounds are aligned to delta 9-THC according to the proposed pharmacophore overlay, their potencies are predicted by a quantitative model of cannabinoid structure--activity relationships based solely on classical and nonclassical cannabinoids with a reasonable degree of accuracy. The ability to incorporate the pharmacological potency of these anandamides into the cannabinoid pharmacophore model is also shown to support the relevance of the proposed pharmacophore model.

Synthesis and sigma binding properties of 2'-substituted 5,9 alpha-dimethyl-6,7-benzomorphans.

The synthesis and sigma 1 and sigma 2 binding properties of several (+)- and (-)-2-benzyl- and 2-dimethylallyl-2'-substituted-5,9 alpha-dimethyl-6,7-benzomorphans (3 and 4) are presented. In agreement with previously reported binding data for 2-substituted 5,9 alpha-dimethyl-2'-hydroxy-6,7-benzomorphans (N-substituted-N-normetazocine), all (1S,5S,9S)-(+)-isomers showed higher affinity for the sigma 1 site than the corresponding (1R,5R,9R)-(-)-isomers. Replacement of the 2'-hydroxy group of (+)-2-benzyl-5,9 alpha-dimethyl-2'-hydroxy-6,7-benzomorphan [(+)-1f] with a 2'-NH2 and 2'-N(CH3)2 [(+)-3b and (+)-3c, respectively] had only a small effect on the sigma 1 Ki values. Changing the 2'-hydroxy group of (+)-1f to an H, F, Cl, Br, I, NHAc, or NHSO2CH3 resulted in a 5-fold or greater loss in potency. In contrast, replacement of the 2'-hydroxy group of (+)-2-(dimethylallyl)-5,9 alpha-dimethyl-2'-hydroxy-6,7-benzomorphan [(+)-1b, (+)-pentazocine] with a 2'-H or 2'-F group resulted in a 2-fold increase in potency. Conversion of (+)-1f to its 2'-desoxy analogue (+)-2d resulted in a 27.5-fold loss in affinity. This suggests that (+)-1f and other N-substituted benzomorphan analogues may be binding to single sigma 1 receptors in a different way or to different sigma 1 receptors. (-)-Pentazocine [(-)-1b] and its 2'-fluoro analogue, (-)-2-(dimethylallyl)-5,9 alpha-dimethyl-2'-fluoro-6,7-benzomorphan [(-)-4a] showed the highest potency for the sigma 2 binding site.

Synthesis and sigma binding properties of 1'- and 3'-halo- and 1',3'-dihalo-N-normetazocine analogues.

The synthesis and sigma 1 and sigma 2 binding properties of several 1'- and 3'-halo- and 1',3'-dihalo-substituted analogues of (+)-N-benzyl- and (+)- and (-)-N-dimethylallyl-N-normetazocine are presented. Structure-activity relationship analyses of the binding data showed that halogen substitution at the 1'-position of these N-substituted N-normetazocine analogues had little effect on sigma 1 binding affinity, whereas 3'-halo substitution as well as 1',3'-dihalo substitution resulted in a reduction of affinity. sigma 2 affinity was increased by the presence of a 3'-bromo substituent in this series of (+)-N-substituted N-normetazocines.

(+)-cis-N-(para-, meta-, and ortho-substituted benzyl)-N-normetazocines: synthesis and binding affinity at the [3H]-(+)-pentazocine-labeled (sigma 1) site and quantitative structure-affinity relationship studies.

sigma 1 receptor ligands have potential pharmacological significance as antipsychotic drugs, as tools in the study of drug-induced motor function disorders, and as radiopharmaceutical imaging agents for the noninvasive imaging of malignant tumors in human subjects. A series of substituted N-benzyl-N-normetazocines were synthesized and their binding affinity at the sigma 1 receptor evaluated in order to examine the details of the structure--affinity relationships (SAR) of a previously determined high-affinity lead compound, (+)-cis-N-benzyl-N-normetazocine (Ki = 0.67 nM). Variation in the benzyl substituents of these compounds produced a 1590-fold range in affinity at the sigma 1 receptor from the unsubstituted benzyl analog to the lowest affinity p-tert-butylbenzyl analog (Ki = 1066 nM). The nanomolar binding affinity for the sigma 1 receptor of (+)-cis-N-(4-fluorobenzyl)-N-normetzocine suggests that this analog may be a useful PET imaging agent.