Oxytocin (OT) is a neurohypophyseal hormone which has been ascribed a pivotal role in parturition. Evidence has accumulated to support the concept that the uterotonic action of OT and its stimulation of uterine prostaglandin release are events which combine to initiate labor. Moreover, OT mediates the postpartum function of contracting the mammary myoepithelium to elicit milk letdown and has also recently been implicated as a key element in preterm labor. Since the breakthrough synthesis of OT three decades ago, considerable research has been devoted to the design of antagonists of this peptide. Interest in such compounds derives from the prospect of their use as novel therapeutic agents for the prevention of premature birth. While many promising in vivo antagonists of OT have been discovered over the years, the tactics for achieving this objective have generally been limited to modifications of the native OT and the closely related arginine vasopressin (AVP) structures. We recently reported the discovery of an entirely new structural class of OT antagonist, derived from a microbial source, and represented by 3. Despite the attractive OT/AVP antagonist properties of this compound (Table I), more potent and selective analogues with improved aqueous solubility suitable for intravenous administration are required in order for compounds of this structural type to have practical utility. This communication represents our initial disclosure on chemoselective and regioselective transformations carried out on 3 with the aim of optimizing its physicochemical and pharmacological profile. Key modifications included oxidation and dehydrogenation of the piperazic acid (Piz) residues, introduction of a (dimethylamino)methyl group using Eschenmoser's salt, formation of a thioamide moiety with Lawesson's reagent, and desulfurization with Raney nickel. These transformations led to several analogues with improved OT receptor-binding potency, OT/AVP selectivity, and/or aqueous solubility. For example, compound 8, the (dimethylamino)methyl derivative, is more potent than 3, shows enhanced OT vs AVP selectivity, and has significantly higher aqueous solubility (2.7 mg/mL vs 0.068 mg/mL at pH 7.3 as its acetate salt). Reaction of 3 with Lawesson's reagent afforded thioamide 9, a potent and selective OT receptor ligand. In contrast, desulfurization of 9 with Raney nickel yielded 10, which had drastically reduced OT and AVP receptor affinities. Our results illustrate how subtle structural modifications can have dramatic effects on the ability of these compounds to bind to the oxytocin receptor, presumably through effects on conformation and/or hydrogen-bonding potential. Compound 8 has been characterized as a functional OT antagonist similar to 3 in the blockade of OT-stimulated rat uterine contractions in vitro and in vivo, and shows no agonist activity in stimulating phosphatidyl inositol turnover or uterine contractions. These initial findings, together with results from totally synthetic analogues, have provided compounds of greater utility as research tools and have set the stage for the development of therapeutic agents.