Debra S. Perlow

A super active cyclic hexapeptide analog of somatostatin

The cyclic hexapeptide, cyclo (Pro-Phe-D-Trp-Lys-Thr-Phe), I, has been shown to have the biological properties of somatostatin. We now report structure-activity studies which optimize the potency of this cyclic hexapeptide series with the synthesis of cyclo (N-Me-Ala-Tyr-D-Trp-Lys-Val-Phe), II, which is 50-100 times more potent than somatostatin for the inhibition of insulin, glucagon and growth hormone release. The hydroxyl group of tyrosine is seen to lend a 10-fold enhancement to the potency. Potency also is found to be correlated with hydrophobicity. II is found to improve the control of postprandial hyperglycemia in diabetic animals when given in combination with insulin. The analog is found to be quite stable in the blood and in the gastrointestinal tract, but the bioavailability after oral administration is only 1-3%. The biological properties and long duration of II should allow clinical evaluation of the inhibition of glucagon release as an adjunct to insulin in the treatment of patients with diabetes.

Methyl-substitution of an iminohydantoin spiropiperidine β-secretase (BACE-1) inhibitor has a profound effect on its potency

The IC50 of a beta-secretase (BACE-1) lead compound was improved ∼200-fold from 11 μM to 55 nM through the addition of a single methyl group. Computational chemistry, small molecule NMR, and protein crystallography capabilities were used to compare the solution conformation of the ligand under varying pH conditions to its conformation when bound in the active site. Chemical modification then explored available binding pockets adjacent to the ligand. A strategically placed methyl group not only maintained the required pKa of the piperidine nitrogen and filled a small hydrophobic pocket, but more importantly, stabilized the conformation best suited for optimized binding to the receptor.

Synthesis of New Peptides Based on Models of Receptor-Bound Conformation

About three dozen small peptides (mostly 2–40 residues) have been identified in the nervous system (Iversen 1983,1984; Krieger 1983). The neuropeptides mediate a wide range of endocrine, motor, and behavioral responses (Snyder 1980; Krieger 1983). These substances control such diverse functions as growth, reproduction, digestion, metabolism, and the dynamics of the cardiovascular system. Immunostaining techniques have been used to visualize and map the distribution of peptides, in particular neuronal pathways. Many of these substances have also been shown to occur in peripheral regions, such as the gastrointestinal tract. The role of many of these agents is largely unknown, but their potential utility in therapy is great.

Conformationally constrained o-tolylpiperazine camphorsulfonamide oxytocin antagonists. Structural modifications that provide high receptor affinity and suggest a bioactive conformation.

A series of new o-tolylpiperazine camphorsulfonamide OT antagonists is described. Analogs containing conformationally constrained 1-acylamino-2-propyl substituents at the camphor C2 endo position exhibit high affinity for OT and AVP-V1a receptors or high affinity and selectivity for OT receptors, depending on functionalities present in the acyl group. Determination of the preferred conformation of potency-enhancing 1-acylamino-2-propyl substituents using molecular mechanics energy calculations and X-ray crystallography, along with topological similarities to a conformationally constrained cyclic hexapeptide OT antagonist, suggests a receptor-bound conformation for this series of non-peptide OT antagonists.

Design of Renin Inhibitors Containing Conformationally Restricted Mimetics of the P1-P1′ and P1 through P2′ Sites

The clinical efficacy of converting enzyme inhibitors1 for reducing blood pressure in a large percentage of hypertensive patients has aroused considerable interest in developing agents that interrupt the renin-angiotensin system at other points, for example by blockade of the angiotensin II receptor2 or by inhibition of the aspartic proteinase, renin. Substrate based design of renin inhibitors in which the scissile P1-P1′ dipeptide is replaced with a non-hydrolyzable group, often a mimetic of a tetrahedral transition state for amide bond hydrolysis, has provided a useful approach for obtaining a variety of inhibitor structure types,3 many with Ki’s of better than 10−9 M. A clinically useful renin inhibitor, however, has not yet emerged due to poor pharmacokinetics (i.e., metabolism or rapid clearance) or poor oral absorption, problems often encountered with peptidic drug targets. An example of this is seen with 1, a “tetrapeptide” inhibitor4 that spans the P4 through P3′ sites of the renin substrate and which utilizes the statine analog, ACHPA5, as a P1-P1′ dipeptide replacement (Figure 1). Although 1 is quite potent in vitro, very low levels of drug are found in the blood after oral administration to the rhesus monkey at 50 mg/kg, and the half life after intravenous administration is short (< 1 h). Rapid biliary excretion of intact drug has been demonstrated for a number of other renin inhibitors.6

Characterization of Tachykinin Receptors by Ligand Binding Studies and by Utilization of Conformationally Restricted Tachykinin Analogues

Tachykinins are a family of peptides that share the carboxyl-terminal sequence Phe-X-Gly-Leu-Met-NH2 and that cause contraction of various smooth muscle systems. Substance P (Chang and Leeman, 1970) and the newly discovered neurokinin A (Maggio et al., 1983; Minamino et al., 1984a; Kimura et al., 1983) and neurokinin B* (Kimura et al., 1983; Kangawa et al., 1983) are mammalian tachykinins. Several nonmammalian tachykinins have been isolated, which include eledoisin, physalaemin, kassinin, and phyllomedusin (for a review, see Erspamer and Melchiorri, 1983). The amino acid sequences of these peptides are shown in Fig. 1.

Synthesis of Protected Lactam-Bridged Dipeptides

Lactam-bridged dipeptides are useful tools for the introduction of conformational constraint in higher peptides. General methods have been devised for the synthesis of dipeptides having five-, six-, and seven-membered ring constraints (1,2). This chapter will focus on four synthetic paths from protected chiral a-amino acids to lactams that involve intramolecular alkylation, intermolecular alkylation, intramolecular acylation, and condensation with formaldehyde for a one carbon unit insertion.

Potent and Selective Oxytocin Antagonists Obtained by Chemical Modification of a Streptomyces Silvensis Derived Cyclic Hexapeptide and by Total Synthesis

Oxytocin (OT) is a neurohypophyseal hormone which has an important function in parturition.1 There is considerable evidence that the uterotonic action of OT and its stimulation of uterine prostaglandin release combine to initiate labor.2,3 Additionally, OT mediates the postpartum function of contracting the mammary myoepithelium to elicit milk letdown4 and has also recently been implicated as a key element in preterm labor.5,6 Attempts to further delineate these OT connected events have provided the impetus to discover agents which interact selectively and competitively at the OT receptor. Such compounds are invaluable in determining the physiological and pathophysiological role of OT and its close structurally related hormone, arginine vasopressin (AVP). Additional interest derives from the prospect of their use as novel therapeutic agents.

Interaction of Mouse Submaxillary Gland Renin with a Statine-Containing Subnanomolar, Competitive Inhibitor

The interaction between mouse submaxillary gland renin and a statine-containing, iodinated substrate analog inhibitor was studied. The compound, 1 (Boc-His-Pro-Phe-(4-iodo)-Phe-Sta-Leu-Phe-NH2, Sta = (3S,4S)-4-amino-3-hydroxy-6-methyl-heptanoic acid), a statine-containing analog of the renin substrate octapeptide, was a competitive inhibitor of cleavage of synthetic tetradecapeptide renin substrate by mouse submaxillary gland renin, with a Ki of 6.2 x 10(-10) M (pH 7.2, 37 degrees C). Titration of the partial quenching of the tryptophan fluorescence of the enzyme by 1 revealed tight binding with a dissociation constant less than 3 nM and a binding stoichiometry of one mole 1 per mole enzyme. The time course of tight binding of 1 to mouse renin appeared to be fast, with kON greater than or equal to 1.3 x 10(6) s-1 M-1. The UV difference spectrum generated upon binding of 1 to mouse renin had two prominent features: a strong, broad band that had a minimum at 242 nm with delta epsilon (242) = -19,500 cm-1 M-1, and a triplet of enhanced bands centered at 286 nm with delta epsilon (286) about +1100 cm-1 M-1. The strong, broad, negative band was similar to the difference between the UV absorbance of 1 in methanol and in 0.1 M citrate phosphate pH 7.2. A structure-activity correlation for analogs of 1 showed some moieties of 1 that are important for potent inhibition of mouse renin. The inhibition data for these compounds versus human kidney renin suggested that the solution of the crystal structure of 1 bound to mouse renin will provide useful information for the design of inhibitors of human kidney renin.