Peptide synthesis catalyzed by an antibody containing a binding site for variable amino acids.

Monoclonal antibodies, induced with a phosphonate diester hapten, catalyzed the coupling of p-nitrophenyl esters of N-acetyl valine, leucine, and phenylalanine with tryptophan amide to form the corresponding dipeptides. All possible stereoisomeric combinations of the ester and amide substrates were coupled at comparable rates. The antibodies did not catalyze the hydrolysis of the dipeptide product nor hydrolysis or racemization of the activated esters. The yields of the dipeptides ranged from 44 to 94 percent. The antibodies were capable of multiple turnovers at rates that exceeded the rate of spontaneous ester hydrolysis. This achievement suggests routes toward creating a small number of antibody catalysts for polypeptide syntheses.

The carboxyl terminus heptapeptide of the R2 subunit of mammalian ribonucleotide reductase inhibits enzyme activity and can be used to purify the R1 subunit.

The heptapeptide, FTLDADF, identical in sequence to the last seven amino acid residues of the carboxyl terminus of the R2 subunit of mouse ribonucleotide reductase (RR), and its N alpha-acetyl derivative both inhibit calf thymus RR. The N alpha-acetyl derivative is considerably more potent, displaying a K1 of 20 microM. The same K1 was found for N-AcFTLDADF inhibition of a reconstituted ribonucleotide reductase from calf thymus R1 and mouse R2, indicating that the C-termini of calf R2 and mouse R2 might be identical. Our results, taken together with previous results of others on inhibition of viral RR, suggest that inhibition of RRs by peptides mimicking the C-terminus of R2 may be a general phenomenon. In addition, we have shown that an affinity column, FTLDADF-Sepharose 4B, can be used to prepare approximately 95% pure calf thymus R1, devoid of contamination with R2, in a very simple procedure that should be generally applicable to R1 purification from many sources.

Approaches to vasodilating/beta-adrenergic blocking agents: examples of the dihydrolutidine type.

The aminohydroxypropoxy moiety has been incorporated into the dihydrolutidine class of vasodilators. In the spontaneously hypertensive rat, one of these, (S)-4-[2-methyl-4-[3-(tert-butylamino)-2-hydroxypropoxy]phenyl]-3,5-dicarboethoxy-1,4-dihydrolutidine (4c), exhibited antihypertensive activity on the order of the standard 4-[2-(trifluoromethyl)phenyl]-3,5-dicarboethoxy-1,4-dihydrolutidine (2a). This antihypertensive activity could not be explained in terms of a vasodilating effect, as determined in the dog. In this latter model, 2a decreased both mean arterial and hindlimb perfusion pressures.

Synthesis of thyrotropin-releasing hormone analogues with selective central nervous system effects.

Thyrotropin-releasing hormone (TRH) analogues which show relative selectivity for action in the central nervous system have been recognized. Practical syntheses for three of these TRH analogues which show the greatest selectivity, less than Aad-His-Tzl-NH2 (5), less than Glu-His-Pip-OMe (2), and less than Aad-His-Pro-NH2 (6), are described. The first two were prepared by solution methods of peptide synthesis. Compound 6 was prepared by the solid-phase method. Problems of histidine racemization, facile diketopiperazine formation, and instability of acylated thiazolidine carboxylic acid derivatives under acidic conditions have been minimized in order to attain optimal yields. Physical properties such as pK, NMR shifts, and circular dichroism have been examined as they might relate to biological activity and peptide conformation.

Heterocyclic analogues of the antihypertensive beta-adrenergic blocking agent (S)-2-[3-(ter-butylamino)-2-hydroxypropoxy]-3-cyanopyridine.

The synthesis of a series of isoelectronic analogues of (S)-2-[3-(tert-butylamino)-2-hydroxypropoxy]-3-cyanopyridine (1) are described; included in this group are examples of thiazole, isothiazole, thiadiazole, pyrazine, and the structurally related naphthyridines. All of the compounds are similar to 1 in that they contain a cyano group ortho to the aminohydroxypropoxy side chain and meta to the nitrogen heteroatom. In addition, several related examples, having additional nuclear substituents and/or groups other than CN in the position adjacent to the aminohydroxypropoxy group, were prepared, and beta-adrenoceptor antagonist activity and vasodilating potency were determined. Three compounds, thiazole 2 and isothiazoles 3 and 27, effectively lowered mean arterial pressure in the SH rat at 5 mg/kg. Compounds 2, 3, and 27 increased iliac blood flow and exhibited beta-adrenergic blocking properties in the dog.

Piperazinylpyrazines with central serotoninmimetic activity.

A series of 2-(1-piperazinyl)pyrazines was synthesized and evaluated for central serotonin-like activity. The most interesting member of the series, 6-chloro-2(1-piperazinyl)pyrazine (3a), had pharmacological properties characteristic of potent central serotoninmimetic activity and only weak peripheral serotoninmimetic action. Structural similarities between 3a and serotonin are discussed.

Beta 1-selective adrenoceptor antagonists: examples of the 2-[4-[3-(substituted-amino)-2-hydroxypropoxy]phenyl]imidazole class.

A series of 2-[4-[3-(substituted-amino)-2-hydroxypropoxy]phenyl]imidazoles is described. The compounds were investigated in vitro for beta-adrenoceptor antagonism, and several examples were found to be selective for the beta 1-adrenoceptor. The structure--activity relationship exhibited by this series of compounds is discussed. (S)-2-[p-[3-[[2-(3,4-dimethoxyphenyl)ethyl]amino]-2-hydroxypropoxy]phenyl]-4-(2 -thienyl)imidazole [(S)-13] was over 100 times more selective than atenolol for the beta 1-adrenergic receptor and has been selected for in-depth studies.

A deuterium isotope effect on the inhibition of gastric secretion by N,N-dimethyl-N'-[2-(diisopropylamino)ethyl]-N'-(4,6-dimethyl-2-pyridyl)urea. Synthesis of metabolites.

The use of isotopic substitution to retard the oxidative metabolism of the gastric antisecretory agent N,N-dimethyl-N'-[2-(diisopropylamino)ethyl]-N'-(4,6-dimethyl-2-pyridyl)urea (1) and improve its antisecretory potency was examined. The pyridine ring methyl hydrogens of 1 were replaced with either deuterium or fluorine. The hexadeuterated analogue (12) was found to be approximately 2.1 times more potent than the protio form (1) as an inhibitor of gastric acid secretion stimulated by gastrin tetrapeptide. The hexafluoro analogue (11) was 0.4 times as potent as 1. A useful pyridine ring synthesis was developed to prepare the metabolites of 1, 10a (4-hydroxymethyl) and 10b (6-hydroxymethyl), and the hexafluoro analogue 11. These syntheses involved the condensation of 1,3-diketones with an appropriately N-substituted amidinoacetate.

An approach to peripheral vasodilator-beta-adrenergic blocking agents.

The syntheses of 2-phenyl- and 2-pyridyl-4-trifluoromethylimidazoles having a 3-tert-butylamino-2-hydroxypropoxy moiety attached to the aryl or heteroaryl substituent are described. Structure--activity relationships based on results from an evaluation of these compounds for antihypertensive, vasodilating, and beta-adrenergic blocking activities are discussed.

Synthesis and antihypertensive activity of some ester progenitors of methyldopa.

A variety of esters of methyldopa was synthesized with the objective of obtaining derivatives that would be more efficiently absorbed from the gastrointestinal tract than the free amino acid and would undergo conversion to methyldopa readily in the blood or target tissues. Two of the esters, alpha-pivaloyloxyethyl (4u) and alpha-succinimidoethyl (4w), were found to be more potent antihypertensive agents than methyldopa in animal models and were selected for further study in man. The amino esters were prepared by three different methods, including direct esterification of methyldopa without the use of N- or O-protecting groups.

Synthesis of potent cyclic hexapeptide NK-1 antagonists. Use of a minilibrary in transforming a peptidal somatostatin receptor ligand into an NK-1 receptor ligand via a polyvalent peptidomimetic.

The endogenous peptides somatostatin (SRIF) and substance P comprise very different structures. Although both bind G-protein-coupled receptors, the SRIF receptors (SSTR 1-5) recognize SRIF and related peptides which retain its beta-turn such as the potent cyclic hexapeptide SRIF agonist L-363,301 (6a), but not substance P. Conversely the NK-1 receptor binds substance P but not the above ligands. In contrast, the beta-D-glucosides 1 and 2, designed to mimic the beta-turn of 6a, bind both receptors. This observation led us to attempt the conversion of 6a into the first potent, selective cyclic hexapeptide ligand for the NK-1 receptor. To this end, we combined design with a minilibrary approach. The goal was accomplished with surprising ease, leading to the NK-1 receptor antagonist 9 (IC50 2.0 +/- 0.4 nM). This demonstrates that peptidomimetics, incorporating in this case the promiscuous beta-D-glucose scaffold, can provide valuable clues about receptor similarities not revealed by their endogenous ligands. In addition, this work suggests that the use of libraries and rational design need not be mutually exclusive approaches to lead discovery.

beta-Adrenergic blocking agents with acute antihypertensive activity.

Modification of the pharmacological profile of the vasodilating/beta-adrenergic blocking agent 2-[4-[3-(tert-butylamino)-2-hydroxypropoxy]phenyl]-4-(trifluoromethyl)imidazole (1) has been investigated. Introduction of selected substitutents onto the imidazole ring, in place of the trifluoromethyl group, has yielded highly cardioselective beta-adrenergic blocking agents such as 7, 17, and 18. The placement of alkyl or chloro groups onto the aryl ring of 1, as illustrated by 33, has produced a class of compounds characterized as antihypertensive beta-adrenergic blocking agents. In these examples, the acute antihypertensive activity does not appear to be due to either vasodilating or beta 2-agonist properties.

Novel ligands lacking a positive charge for the delta- and mu-opioid receptors.

Recently we reported using minilibraries to replace Lys(9) [somatostatin (SRIF) numbering] of the potent somatostatin agonist L-363,301 (c[-Pro-Phe-D-Trp-Lys-Thr-Phe-]) to generate the potent neurokinin receptor (NK-1) antagonist c[-Pro-Phe-D-Trp-p-F-Phe-Thr-Phe-]. This novel cyclic hexapeptide did not bind the SRIF receptor. Thus, a single mutation converted L-363,301, a SRIF agonist with potency ca. 2-8 times the potency of SRIF in laboratory animals,(24) into a selective NK-1 receptor antagonist with an IC(50) of 2 nM in vitro. During the screening of the same libraries for ligands of the delta-opioid receptor, we identified four compounds (1-4) which represent a new class of delta-opioid antagonists, some of which were also NK-1 receptor antagonists. The most potent delta-opioid antagonist, c[-Pro-1-Nal-D-Trp-Tyr-Thr-Phe-] (2), showed a K(e) value of 128 nM in the mouse vas deferens assay and a delta-receptor binding affinity constant of 152 nM in the rat brain membrane binding assay. These results are of interest because they represent a novel class of delta-opioid antagonists and, like two previously reported delta-opioid antagonists, they lack a positive charge. To examine further the requirement for a positive charge in the delta-opioid ligands, we prepared two analogues of the beta-casomorphin-derived mixed mu-agonist/delta-antagonist, H-Dmt-c[-D-Orn-2-Nal-D-Pro-Gly-] (7), in which we eliminated the positive charge either through formylation of the primary amino group (5) or by the deletion of this N-terminal amino group (6). These latter compounds proved to be delta-opioid antagonists with K(e) values in the 16-120 nM range, as well as fairly potent mu-opioid antagonists (K(e) approximately 200 nM). These six compounds provide the most convincing evidence to date that there is no requirement for a positive charge in mu- and delta-opioid receptor antagonists. In addition, cyclic hexapeptide 4 lacks a phenolic hydroxyl group. Taken together, these data suggest that the prevailing assumptions about delta- and mu-opioid receptor binding need revision and that the receptors for these opioid ligands have much in common with the NK-1 and somatostatin receptors.

Inhibitors of gastric acid secretion: antisecretory 2-pyridylurea derivatives.

A series of aminoalkyl-substituted pyridylureas has been prepared and evaluated as inhibitors of gastric acid secretion. N,N-Dimethyl-N'-[2-(diisopropylamino)ethyl]-N'-(4,6-dimethyl-2-pyridyl)urea (8g) was the most potent example of the class. Comparison of this compound with cimetidine showed it to be equipotent in dogs stimulated with gastrin tetrapeptide but approximately half as potent in dogs stimulated with histamine. Inhibition of secretion does not appear to result from antagonism of the histamine H2 receptor, since the compounds show only weak inhibition of the H2 receptor in vitro.

Modulation of receptor and receptor subtype affinities using diastereomeric and enantiomeric monosaccharide scaffolds as a means to structural and biological diversity. A new route to ether synthesis.

We show that carbohydrates constitute an attractive source of readily available, stereochemically defined scaffolds for the facile attachment of side chains contained in genetically encoded and other amino acids. beta-D- and beta-L-glucose, L-mannose, and the 6-deoxy-6-N-analogue of beta-D-glucose have been employed to synthesize peptidomimetics that bind the SRIF receptors on AtT-20 mouse pituitary cells, five cloned human receptor subtypes (hSSTRs), and the NK-1 receptor. The affinity profile of various sugar-based ligands at the hSSTRs is compared with that of SRIF. Compound 19 bound hSSTR4 with a Ki of 100 nM. Subtle structural changes affect affinities. Evidence is presented that suggests that one compound (8) binds both the AtT-20 cell receptors and the five hSSTRs via a unique mode. The SARs of the glycosides at SRIF receptors differ markedly from those at the NK-1 receptor. For example a 4-benzyl substituent is important for SRIF receptor binding, but the 4-desbenzyl analogue 27 was highly potent (IC50 of 27 nM) at the NK-1 receptor. A new, nonbasic method for the synthesis of base-sensitive ethers from primary and secondary alcohols is also described.

Synthesis and stereospecific antipsychotic activity of (-)-1-cyclopropylmethyl-4-(3-trifluoromethylthio-5H-dibenzo[a,d]cyclohepten-5-ylidene)piperidine.

The synthesis and resolution of 3-iodocyproheptadine [(+/-)-5a] and 1-cyclopropylmethyl-4-(3-iodo-5H-dibenzo-[a,d]cyclohepten-5-ylidene)piperidine [(+/-)-5b] are described. The resulting atropisomers undergo reaction with trifluoromethylthiocopper to give optically active products without extensive racemization. In this manner, optically pure (+)- and (-)-3-trifluoromethylthiocyproheptadine [(+)-6a and (-)-6a, respectively] and (+)- and (-)-1-cyclopropylmethyl-4-(3-trifluoromethylthio-5H-dibenzo[a,d]cyclohepten-5-ylidene)piperidine [(+)-6b and (-)-6b, respectively] have been prepared. The influence of a chiral europium shift reagent on the proton and fluorine resonance signals as a diagnostic tool for the determination of the optical purities of these atropisomers is discussed. The four compounds, (+)-6a, (-)-6a, (+)-6b, and (-)-6b, were studied in squirrel monkeys for their ability to block conditioned avoidance responding. All of the antiavoidance activity was found to reside solely in the levorotatory compounds (-)-6a and (-)-6b. Further comparison of the enantiomers (-)-6b and (+)-6b showed that the ability to antagonize apomorphine-induced stereotyped behavior is confined to the levorotatory isomer (-)-6b while weak central anticholinergic activity resides solely in the dextrorotatory isomer (+)-6b. Neither (-)-6b has significant peripheral anticholinergic activity.

A second-generation synthesis of polypyrrolinone nonpeptidomimetics: prelude to the synthesis of polypyrrolinones on solid support.

[reaction: see text] A second-generation asymmetric synthesis of polypyrrolinones (3) has been achieved exploiting scalemic alpha-aminolactones (1) as building blocks. Imine formation between an appropriate lactone (1) and aldehyde (2), followed in turn by pyrrolinone ring construction promoted by KHMDS in the presence of 18-crown-6 and modified Swern oxidation furnished pyrrolinone aldehyde 3. This iterative, efficient three-step protocol paves the way for the synthesis of polypyrrolinones on solid support.

Intramolecular hydrogen-bond participation in phosphonylammonium salt formation.

[reaction: see text] A series of phosphonochloridates and phosphonyl dichlorides were prepared, and their reactivity with triethylamine has been investigated using (31)P NMR spectroscopy. Taken together these studies provide evidence that an intramolecular hydrogen-bond is required for phosphonylammonium salt formation to render the phosphorus more electron-deficient.

Design and synthesis of novel scaffolds for drug discovery: hybrids of beta-D-glucose with 1,2,3,4-tetrahydrobenzo[e][1,4]diazepin-5-one, the corresponding 1-oxazepine, and 2- and 4-pyridyldiazepines.

[structure: see text]. We describe the syntheses of novel tricyclic scaffolds that incorporate a fusion of a substituted pyranose ring with the seven-membered rings of 1,2,3,4-tetrahydrobenzo[e][1,4]diazepin-5-one and the corresponding oxazepine and pyridyldiazepine to generate the benzodiazepines, and the related heterocycles. In each instance, the pyranose rings contain three protected hydroxyls, suitable for selective derivatization.

First generation design, synthesis, and evaluation of azepine-based cryptophycin analogues.

[structure: see text] Azepine-based cryptophycin mimics (+)-4 and (+)-5 have been designed and synthesized. Biological evaluation revealed modest in vitro activity against several human tumor cell lines, thereby supporting the utility of novel scaffolds for the design and synthesis of cryptophycin analogues.