A topographically and conformationally constrained, spin-labeled, alpha-amino acid: crystallographic characterization in peptides.

2,2,6,6-Tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC) is a topographically and conformationally restricted, nitroxide containing, C(alpha)-tetrasubstituted alpha-amino acid. Here, we describe the molecular and crystal structures, as determined by X-ray diffraction analyses, of a TOAC terminally protected derivative, the cyclic dipeptide c(TOAC)(2).1,1,1,3,3,3-hexafluoropropan-2-ol (HFIP) solvate, and five TOAC-containing, terminally protected, linear peptides ranging in length from tetra- to hepta-peptides. Incipient and fully developed, regular or distorted 3(10)-helical structures are formed by the linear peptides. A detailed discussion on the average geometry and preferred conformation for the TOAC piperidine ring is also reported. The X-ray diffraction structure of an intramolecularly cyclized side product resulting from a C-activated TOAC residue has also been determined.

Crystal structure of biphalin sulfate: a multireceptor opioid peptide.

Biphalin is a dimeric opioid peptide, composed of two tetrapeptides connected 'tail-to-tail', that exhibits a high affinity for all three opioid receptor types (i.e. mu, delta and kappa). This study presents the X-ray crystal structure of biphalin sulfate and compares it to other opioids that interact with the same biological targets. Both halves of the molecule have a folded backbone conformation but differ significantly from one another. Residues 1-4 in biphalin, which compare well with the delta selective opioid peptide DADLE, fold into a random coil. Residues 5-8, which can be fit to the mu selective peptide D-TIPP-NH2, exhibit a fairly normal type III' beta bend. Biphalin also exhibits structural similarities with two naltrexone analogs, naltrexonazine and norbinaltorphamine, that are specific to mu and kappa receptor sites.

Methyl analogues of the experimental Alzheimer drug phenserine: synthesis and structure/activity relationships for acetyl- and butyrylcholinesterase inhibitory action.

With the goal of developing potential Alzheimer's pharmacotherapeutics, we have synthesized a series of novel analogues of the potent anticholinesterases phenserine (2) and physostigmine (1). These derivatives contain methyl (3, 4, 6), dimethyl (5, 7, 8, 10, 11) and trimethyl (14) substituents in each position of the phenyl group of the phenylcarbamoyl moieties, and with N-methyl and 6-methyl substituents (12, 13, 31, 33). We also quantified the inhibitory action of these compounds against human acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). An analysis of the structure/anticholinesterase activity relationship of the described compounds, together with molecular modeling, confirmed the catalytic triad mechanism of the binding of this class of carabamate analogues within AChE and BChE and defined structural requirements for their differential inhibition.

Synthesis and biological evaluation of tropane-like 1-[2-[bis(4-fluorophenyl)methoxy]ethyl]-4-(3-phenylpropyl)piperazine (GBR 12909) analogues.

We have prepared azabicyclo[3.2.1] derivatives (C-3-substituted tropanes) that bind with high affinity to the dopamine transporter and inhibit dopamine reuptake. Within the series, 3-[2-[bis-(4-fluorophenyl)methoxy]ethylidene]-8-methyl-8-azabicyclo[3.2.1]octane (8) was found to have the highest affinity and selectivity for the dopamine transporter. These azabicyclo[3.2.1] (bridged piperidine) series of compounds differ from the well-known benztropines by a 2-carbon spacer between C-3 and a diarylmethoxy moiety. Interestingly, these new compounds demonstrated a much lower affinity for the muscarinic-1 site, at least a 100-fold decrease compared to benztropine. Replacing N-methyl with N-phenylpropyl in two of the compounds resulted in a 3-10-fold increase in binding affinity for the dopamine transporter. However, those compounds lost selectivity for the dopamine transporter over the serotonin transporter. Replacement of the ether oxygen in the diarylmethoxy moiety with a nitrogen atom gave relatively inactive amines, indicating the important role which is played by the ether oxygen in transporter binding. Reduction of the C-3 double bond in 8 gave 3 alpha-substituted tropanes, as shown by X-ray crystallographic analyses of 11, 12, and 19. The 3 alpha-substituted tropanes had lower affinity and less selectivity than the comparable unsaturated ligands.

Chemistry of the diazeniumdiolates. I. Structural and spectral characteristics of the [N(O)NO]- functional group.

Ions of structure X[N(O)NO]-, examples of which have seen increasing use as probes for studying the biology of nitric oxide (NO) over the past decade, have a varied chemical history spanning nearly two centuries. Nevertheless, they have not been widely appreciated for their physicochemical similarities. Here we begin a series of systematic inquiries into the fundamental chemistry of such compounds aimed at identifying both the characteristics that justify considering them as a group and the factors that contribute to observed differences in their physicochemical properties. In the present paper, X-ray structures in which X is SO3- (1), O- (2), Ph (3), and Et2N (5), as well as that of the gem-disubstituted carbon derivative CH2[N(O)NO]2-(2) (4), are compared. All their O-N-N-O systems are essentially planar, with cis oxygens and an N-N linkage exhibiting considerable double-bond character. The ultraviolet spectrum of the isolated chromophore consists of a relatively intense ( approximately 6-10 mM(-1) x cm(-1) per [N(O)NO]- group) absorption at 248-250 nm (for 2 and 5) that is red shifted by through-space Stark interactions (e.g., by approximately 10 nm in 1 and 4) as well as by conjugative interaction with X (lambda(max) = 284 nm for 3). Infrared and Raman spectra for the widely used pharmacological probe 5 were determined, with analysis of vibrational modes being aided by comparison with the spectra of the [15N(O)15NO]- isotopomer and density functional theory calculations at the B3LYP/6-311++G** level. To address confusion that has arisen in the literature resulting from rather widespread use of differing trivial designations for this class of compounds, a unifying nomenclature system is recommended in which compounds containing the [N(O)NO]- moiety are named as diazeniumdiolates. It is hoped that these and other efforts to understand and predict the physicochemical similarities and differences among different members of the diazeniumdiolate class will aid in reaping their full potential in the area of rational drug design.

Efficient asymmetric synthesis of biologically important tryptophan analogues via a palladium-mediated heteroannulation reaction.

A novel and concise synthesis of optically active tryptophan derivatives was developed via a palladium-catalyzed heteroannulation reaction of substituted o-iodoanilines with an internal alkyne. The required internal alkyne 14a or 25 was prepared in greater than 96% de via alkylation of the Schöllkopf chiral auxiliary 19 employing diphenyl phosphate as the leaving group. The Schöllkopf chiral auxiliary was chosen here for the preparation of L-tryptophans would be available from D-valine while the D-isomers required for natural product total synthesis would originate from the inexpensive L-valine (300-g scale). Applications of the palladium-catalyzed heteroannulation reaction were extended to the first asymmetric synthesis of L-isotryptophan 38 and L-benz[f]tryptophan 39. More importantly, the optically pure 6-methoxy-D-tryptophan 62 was prepared by this protocol on a large scale (>300 g). This should permit entry into many ring-A oxygenated indole alkaloids when coupled with the asymmetric Pictet-Spengler reaction. In addition, an improved total synthesis of tryprostatin A (9a) was accomplished in 43% overall yield employing this palladium-mediated process.

Rational design, synthesis, and biological evaluation of rigid pyrrolidone analogues as potential inhibitors of prostate cancer cell growth.

In view of its role in tumor promotion and signal transduction, protein kinase C (PKC) has proven to be an exciting target for cancer therapy. With the aid of molecular modeling, we rationally designed and stereoselectively synthesized a new class of rigidified pyrrolidone-based PKC activators. Pyrrolidone 15 was found to exhibit reasonable affinity for PKCdelta, with lower affinity for the other isozymes tested. Pyrrolidone 2 causes the dose-dependent induction of apoptosis in LNCaP prostate cancer cells. This apoptotic effect could be markedly potentiated by the use of LNCaP cells overexpressing the PKCalpha or delta isozymes.

Pharmacological and behavioral analysis of the effects of some bivalent ligand-based monoamine reuptake inhibitors.

Novel piperidine-based bivalent ligands were prepared in enantiomerically pure form and evaluated for their ability to inhibit reuptake of dopamine (DA), serotonin (5-HT), and norepinephrine (NE) into rat brain nerve endings (synaptosomes). In this study, we have succeeded in using (1) the length of the linking chain connecting the two piperidine-based monomer units and (2) the absolute configuration of the piperidine monomer as a means to tailor activity and selectivity at the three monoamine transporters tested. In this series, the bivalent ligand 16, comprised of two (+)-trans-piperidine units linked by a pentamethylene spacer, exhibits a combination of high DA transporter (DAT) and 5-HT transporter (SERT) activity (K(i) = 39 nM and 7 nM, respectively). Piperidine 16 is capable of reducing cocaine's locomotor effects in mice while not having any effect on locomotion when tested alone. Additionally, compound 16 (1-10 mg/kg) does not substitute for cocaine in drug discrimination studies in rats. However, the analogous bivalent ligand 15 comprised of two (-)-trans-piperidine units, which is SERT selective, was less effective in antagonizing cocaine's locomotor stimulant activity. The piperidine-based bivalent inhibitors described herein constitute a new class of monoamine reuptake inhibitors that exhibit varying levels of monoamine transporter activity and selectivity, and these ligands may serve as lead candidates in the discovery of new therapeutics to treat a range of neurological disorders including cocaine addiction.

The secondary amine/nitric oxide complex ion R(2)N[N(O)NO](-) as nucleophile and leaving group in S9N)Ar reactions.

Ions of structure R(2)N[N(O)NO](-) and their alkylation products have seen increasing use as nitric oxide (NO)-generating agents for biomedical research applications. Here we show that such diazeniumdiolate anions can readily displace halide from a variety of electrophilic aza- or nitroaromatic substrates to form O(2)-arylated derivatives of structure R(2)N-N(O)=N-OAr. The site of arylation and the cis arrangement of the oxygens were confirmed by X-ray crystallography. Displacement by various nucleophiles showed R(2)N[N(O)NO](-) to be a reasonably good leaving group, with rate constants for displacement by hydroxide, methoxide, and isopropylamine that were between those of chloride and fluoride in the S(N)Ar reactions we surveyed. The Meisenheimer intermediate could be spectrally observed. These O(2)-aryl diazeniumdiolates proved capable of reacting with the nucleophilic sulfur of the HIV-1 p7 nucleocapsid protein's zinc finger assembly to eject the zinc, disrupting a structural motif critical to viral replication and suggesting possible utility in the drug discovery realm.

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.

Design, synthesis, and monoamine transporter binding site affinities of methoxy derivatives of indatraline.

A series of methoxy-containing derivatives of indatraline 13a-f and 17 were synthesized, and their binding affinities for the dopamine, serotonin, and norepinephrine transporter binding sites were determined. Introduction of a methoxy group to indatraline affected its affinity and selectivity greatly. Except for the 4-methoxy derivative 13a,which had the same high affinity at the dopamine transporter binding site as indatraline, the other methoxy-containing analogues (13b-f and 17) exhibited lower affinity than indatraline for the three transporter binding sites. However, some of the analogues were more selective than indatraline, and the 6-methoxy derivative 13c displayed the highest affinity for both the serotonin and norepinephrine transporters. This compound retained reasonable affinity for the dopamine transporter and is a promising template for the development of a long-acting inhibitor of monoamine transporters. Such inhibitors have potential as medications for treatment, as a substitution medication, or for prevention of the abuse of methamphetamine-like stimulants.

Chemical synthesis and pharmacology of 6- and 7-hydroxylated 2-carbomethoxy-3-(p-tolyl)tropanes: antagonism of cocaine's locomotor stimulant effects.

In our efforts to identify molecules that might act as cocaine antagonists or cocaine partial agonists, efforts were made to further capitalize on our earlier finding regarding the ability of a 7-methoxylated pseudococaine analogue to act as a weak cocaine functional antagonist. Herein, a series of the 6- and 7-hydroxylated WIN analogues possessing a boat or chair conformation of the tropane ring were prepared and tested for their ability to displace [(3)H]mazindol binding and to inhibit high-affinity monoamine uptake into rat brain nerve endings. These 6- and 7-hydroxylated WIN analogues were readily prepared by use of a classical Willstätter synthesis to construct an appropriately functionalized tropane ring followed by use of a Suzuki coupling reaction to introduce the aryl group at position 3. Reduction of the resulting tropene by use of SmI(2) or by catalytic hydrogenation followed by deprotection delivered the final target compounds. Some of these compounds were found to retain considerable affinity as inhibitors of the dopamine transporter (DAT) and the norepinephrine transporter (NET), but they were less potent inhibitors of the serotonin transporter (SERT). None of the compounds of the present series revealed any substantial potency difference in [(3)H]mazindol binding versus [(3)H]DA uptake, and failed to show "cocaine antagonism" when tested for their ability to prevent cocaine's inhibition of DA transport. However, one of these hydroxylated WIN analogues, namely 12b, which possesses nanomolar potency at the DAT and NET and micromolar potency at the SERT, when tested in vivo, was found capable of attenuating cocaine's locomotor activity (AD(50) = 94 mg/kg). Taken together, this work provides further support for our hypothesis that drugs that lack the ability to inhibit transport by all three monoaminergic transporters may exhibit "partial" cocaine-like properties, but act as cocaine antagonists. Consequently, it may prove valuable to examine the behavioral activity of other 6- and 7-substituted tropanes in animal behavioral paradigms in the search for a cocaine medication.

Synthesis and biological evaluation of 1-azabicyclo-[3.2.1]octanes: new dopamine transporter inhibitors.

The synthesis and biological activity of a series of 6-substituted 1-azabicyclo[3.2.1]octanes are described. 1-Azabicyclo[3.2.1]octanes represent a new class of compounds that exhibit monoamine transporter inhibitory activity highly dependent on the overall topology and the absolute stereochemistry of the molecule.

Novel conformationally constrained tropane analogues by 6-endo-trig radical cyclization and stille coupling - switch of activity toward the serotonin and/or norepinephrine transporter.

A novel class of tricyclic tropane analogues has been synthesized by making use of radical cyclization technology in combination with the Stille coupling reaction. As hybrids between tropanes and quinuclidines, these tropaquinuclidines represent a significant structural departure from many of the other classes of tropane ligands synthesized to date. This structure class is characterized by the boat conformation of the tropane ring and the orientation of the additional bridge (and therefore of the nitrogen lone pair) together with the unusual placement of the aromatic moiety. All compounds were tested for their ability to inhibit monoamine reuptake under identical conditions. The ability to inhibit reuptake of dopamine in comparison to cocaine is generally decreased in this series but for one compound. (1S,3R, 6S)-(Z)-9-(thienylmethylene)-7-azatricyclo[4.3.1.0(3, 7)]decane-2beta-carboxylic acid methyl ester (5h) exhibits reasonable activity at the dopamine transporter (DAT) (K(i) = 268 nM) and good activity at the norepinephrine transporter (NET) (K(i) = 26 nM). The potency and selectivity shown by some of these ligands for the NET, serotonine transporter (SERT), or NET/SERT is striking, particularly in view of the displacement of the aromatic ring in this series from its usual position at C-3 in the WIN analogues. Thus, (1S,3R,6S)-(Z)-9-(4-biphenylylmethylene)-7-azatricyclo[4.3.1 . 0(3,7)]decane-2beta-carboxylic acid methyl ester (5a) is a selective inhibitor of norepinephrine reuptake (K(i) = 12 nM). Its p-methoxy analogue 5c is a mixed inhibitor of norepinephrine and serotonin reuptake (K(i) = 187 nM at the NET and 56 nM at the SERT). The most active and selective compound we found in the present series is compound 8b [(1S,3R,6S)-2-(acetoxymethyl)-(Z)-9-(3, 4-dichlorophenylmethylene)-7-azatricyclo[4.3.1.0(3,7)]decane ]. This compound is a potent (K(i) = 1.6 nM) and selective inhibitor of serotonin reuptake into rat midbrain synaptosomes. Its selectivity is about 400-fold over the NET and about 1000-fold over the DAT. The results of this study further demonstrate the possibility of tuning the selectivity of tropane analogues toward the SERT or NET binding site. The ligands disclosed herein provide additional pharmacological tools of use in attempting to correlate structure and transporter selectivity with in vivo studies of behavioral outcomes.

Further SAR studies of piperidine-based analogues of cocaine. 2. Potent dopamine and serotonin reuptake inhibitors.

The synthesis and monoamine transporter activity of additional members of a series of 3,4-disubstituted piperidines (truncated analogues of the WIN series) are described. All members of this series were prepared from arecoline hydrobromide in optically pure form and were evaluated for their ability to inhibit high affinity uptake of dopamine (DA), serotonin (5-HT) and norepinephrine (NE) into rat brain nerve endings (synaptosomes). Most of the compounds prepared in this series are reasonably potent DAT inhibitors (K(i) values of 4-400 nM) and have selectivity for the 5-HT transporter relative to both the NE transporter (3-9-fold) and to the DAT ( approximately 25-fold). In the present series, (-)-methyl 1-methyl-4beta-(2-naphthyl)piperidine-3beta-carboxylate (6) was found to be the most potent piperidine-based ligand, exhibiting K(i)'s of 21 nM and 7.6 nM at the DAT and 5-HTT, respectively. While the 5-HTT activity of compound 6 is comparable to that of the antidepressant medication fluoxetine, it is less selective. As is apparent from the data presented, the naphthyl substituted piperidines 6-9, which differ in their stereochemistry, show different degrees of selectivity for the three transporters. Consistent with results reported in the literature for the tropane analogues, removal of the methyl group from the nitrogen atom of 9 leads to a further enhancement in 5-HTT activity. To examine the in vivo effects of these piperidines, preliminary behavioral screening was carried out on piperidine 14. Despite its 2.5-fold greater DAT activity compared to cocaine, piperidine 14 was found to be about 2. 5-fold less potent in increasing distance traveled in mice. However, consistent with its DAT activity, piperidine 14 was found to be about 2.5-fold more potent than cocaine in enhancing stereotypic movements. Further studies of these piperidine-based ligands may provide valuable insights into the pharmacological mechanisms underlying the enhancement in distance traveled versus stereotypic movements. The present results have important implications for better understanding the structural motifs required in the design of agents with specific potency and selectivity at monoamine transporters.

Determining the occurrence of a 3(10)-helix and an alpha-helix in two different segments of a lipopeptaibol antibiotic using TOAC, a nitroxide spin-labeled C(alpha)-tetrasubstituted alpha-aminoacid.

Trichogin GA IV is a 11-residue lipopeptaibol antibiotic exhibiting membrane modifying properties. We synthesized step-by-step by solution methods three trichogin analogues, each with a double Aib (alpha-aminoisobutyric acid)-->TOAC (2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid) replacement. The strict similarity in the conformational propensities of Aib and TOAC allowed us to exploit these analogues in a detailed investigation of the conformation of this lipopeptaibol in different organic solvents and in a membrane-mimetic environment using in particular the double spin labeling ESR technique. We conclude that the secondary structure in solution remains essentially unchanged if compared to that previously found in the crystal state for trichogin. More specifically, the N-terminal region of the peptide folds in a 3(10)-helix, while the central and C-terminal regions are mainly alpha-helical. An additional, significant proof for the modest plasticity of the trichogin structure was obtained by an X-ray diffraction analysis of the nOct-[TOAC4,8, Leu-OMe11] analogue. For the three analogues permeability measurements revealed membrane-modifying properties comparable to those of natural trichogin.

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.

Chemistry and pharmacology of the piperidine-based analogues of cocaine. Identification of potent DAT inhibitors lacking the tropane skeleton.

To discover agents that might be useful in the treatment of cocaine abuse, we have chosen to re-explore a class of molecules that was first reported by Clarke et al. in 1973 and that was and shown to lack locomotor stimulatory activity in mice. These compounds are piperidine-3-carboxylic acid esters bearing a 4-chlorophenyl group in position 4, and as such, these structures may be viewed as truncated versions of the WIN series compounds, i.e., they lack the two-carbon bridge of the tropanes. All members of this class were synthesized starting from arecoline hydrobromide and obtained in optically pure form through resolution methods using either (+)- or (-)-dibenzoyltartaric acid. Interestingly, we have found that these piperidines do, in fact, exhibit substantial affinity in both WIN 35, 428 binding at the dopamine transporter and in the inhibition of [3H]dopamine uptake. Of all of the compounds synthesized, the 3-n-propyl derivative (-)-9 was found to be the most potent with a binding affinity of 3 nM. This simple piperidine is thus 33-fold more potent than cocaine in binding affinity and 29-fold more potent in its inhibition of dopamine uptake. Although no efforts have presently been made to "optimize" binding affinity at the DAT, the substantive activity found for the n-propyl derivative (-)-9 is remarkable; the compound is only about 10-fold less active than the best of the high-affinity tropanes of the WIN series. As a further point of interest, it was found that the cis-disubstituted piperidine (-)-3 is only about 2-fold more potent than its trans isomer (+)-11. This result stands in sharp contrast to the data reported for the tropane series, for the epimerization of the substituent at C-2 from beta to alpha has been reported to result in a lowering of activity by 30-200-fold. This smaller spread in binding affinities for the piperidines may reflect the smaller size of these molecules relative to the tropanes, which allows both the cis and the trans isomers to adjust themselves to the binding site on the DAT. Our present demonstration that these piperidine structures do, in fact, possess significant DAT activity, taken together with their reported lack of locomotor activity, provides a compelling argument for exploring this class of molecules further in animal behavioral experiments. The present work thus broadens the scope of structures that may be considered as lead structures in the search for cocaine abuse medications.