Cyclopropyl- and Methyl-Containing Inhibitors of Neuronal Nitric Oxide Synthase

Nitric oxide (NO) has a wide variety of functions in the body, the most well-studied of which are its regulatory action on smooth muscle relaxation, cytotoxic activity in the immune system, neurotransmission, brain development and protection, and maintenance of synaptic plasticity.1 The production of NO is mediated via the activity of nitric oxide synthase (NOS),2, 3 a family of homodimeric heme-containing monooxygenases, which metabolizes L-arginine and O2 to L-citrulline and NO.4 A considerable body of research has shown that overproduction of cerebral NO by the neuronal isoform of NOS (nNOS) is a general pathological phenomenon for various neurological disorders such as Parkinson’s,5 Alzheimer’s,6 Huntington’s,7 headaches,8 and cerebral palsy.5–10 This has led to the search for and development of selective inhibitors of nNOS over endothelial NOS (eNOS), the isozyme that is responsible for the regulation of blood pressure, and inducible NOS (iNOS), the isozyme that is critical for immune responses, as therapeutic agents for the treatment of neurological disorders.11 In our on-going effort to develop novel nNOS inhibitors, we recently reported a series of cis-3,4-pyrrolidine-based inhibitors.12–14 In this family, several inhibitors have shown remarkable neuroprotective properties in a preclinical rabbit model for cerebral palsy.13 Compound 1as an example, not only is neuroprotective but is the most selective nNOS inhibitor over eNOS and iNOS yet reported.14 Despite its promising inhibitory activity, further application of 1 has been impeded by several of its structural characteristics. First, the flexible m-fluorophenyl ethanamino tail produces multiple rotatable bonds in the inhibitor. In addition, the benzylic position of the m-fluorophenyl ring is highly susceptible to metabolic oxidation.15 Also, the two positive charges of 1 at physiological pH, derived from the two secondary amino groups, decreases the chance of 1 to penetrate the blood-brain barrier (BBB).13 These considerations prompted the development of new pyrrolidine nNOS inhibitors with a potentially more desirable pharmacokinetic and pharmacodynamic profile. Different strategies have been applied to modify the chemical structure of 1.16–20 Herein we describe the design and synthesis of a new series of inhibitors (2), with a structurally constrained cyclopropyl ring inserted in the position adjacent to the amino group of the ethanamino tail. Conformational restriction using cyclopropyl fragments is a strategy that has been widely used in modern drug design to create novel inhibitors for a variety of enzymes.21–23 Introduction of the cyclopropyl group (2) can potentially enhance inhibitory activity by stabilizing and thereby reducing the energetic penalty in binding to the enzyme active site and thus improve selectivity.24, 25 In addition, the insertion of a cyclopropyl fragment can block the potential metabolic oxidation at the benzylic position of the m-fluorophenyl ring.15 Furthermore, the electron-withdrawing character of the cyclopropyl ring decreases the basicity of the adjacent amino group. The calculated pKa value of the amino group in the lipophilic tail of 2 is ~7.4, which is significantly lower than 8.9 in 1.26 As a result, pseudo-monocationic molecule 2 may have improved BBB permeability.15 Here we report structure-activity relationship studies on these cyclopropyl containing inhibitors to determine if enhanced potency and selectivity can be attained prior to consideration of pharmacokinetic property assessment.