pubs.acs.org/jmc Published on Web 09/08/2009 r 2009 American Chemical Society 6012 J. Med. Chem. 2009, 52, 6012–6023 DOI: 10.1021/jm900712n Development and Screening of Water-Soluble Analogues of Progesterone and Allopregnanolone in Models of Brain Injury Christopher J. MacNevin, † Fahim Atif, ‡ Iqbal Sayeed, ‡ Donald G. Stein, ‡ and Dennis C. Liotta* ,† † Department of Chemistry, Emory University, 1515 Dickey Drive, Emerson Building Room 403, Atlanta, Georgia 30322, and ‡ Department of Emergency Medicine Brain Research Laboratory, Emory University School of Medicine, 1365B Clifton Road, Atlanta, Georgia 30322 Received May 26, 2009 Preclinical and clinical research findings have revealed that the hormone progesterone, when acutely administered, can dramatically reduce cerebral edema, inflammation, tissue necrosis, and programmed cell death following traumatic brain injury (TBI). The poor aqueous solubility of progesterone, however, limits its potential use as a therapeutic. Several chemically novel analogues of progesterone and its natural metabolite allopregnanolone have been synthesized and screened using both in vitro and whole animal models of TBI. The new derivatives demonstrated greatly improved solubility and select compounds have shown equivalent effectiveness to progesterone in reducing cerebral edema after TBI. Introduction Traumatic brain injury (TBI a ) is a significant public health problem that affects nearly 1.5 million Americans each year, resulting in approximately 235000 hospitalizations, 80000 cases of long-term disability, and 50000 deaths. 1 An estimated 5.3 million Americans currently require long-term assistance in performing basic activities of daily living as the result of having suffered a TBI, 2 and care related costs for the treat- ment of TBI patients has been estimated to total nearly $60 billion annually. 3 In addition, a recent comprehensive study of Iraq and Afghanistan war veterans reports that 19% of those surveyed had suffered a TBI, making it one of the so-called “signature injuries” found among soldiers returning from duty. 4 Despite several decades of effort from the scientific com- munity, no single pharmacological agent or treatment proto- col has been found that results in consistently improved outcomes following TBI. 5 A recent meta-analysis of studies evaluating the effectiveness of five experimental treatments for TBI (hyperventilation, mannitol, cerebrospinal fluid drai- nage, barbiturates, and corticosteroids) showed that none of the interventions reliably reduced death or disability. 6 The Corticosteroids After Significant Head Injury (CRASH) trial, which originally sought to utilize a population group of 20000, was terminated at just over 10000 patients as it became clear that the treatment group (0.4 g methylprednisolone per h for 48 h) had a higher mortality rate than that of the control group. 7 Hypothermia has shown some favorable effects among brain injured patients. 8 However, clinical results using hypothermia have been variable and the treatment is thought to be potentially harmful to patients over the age of 45. 9 A growing body of evidence indicates that progesterone (1, Scheme 1) may be a promising alternative therapeutic candidate for the treatment of TBI. 10 Progesterone is an endogenous steroid produced by the adrenal glands and the corpus luteum as well as by neurons and glial cells within the central and peripheral nervous systems. 11 Just as progesterone has been associated with a variety of protective roles during gestation, it has also been linked to several different but mutually supportive modes of neuroprotection following TBI, including the attenuation of cerebral edema, 12 the re- duction of lipid peroxidation and oxidative stress, 13 anti- inflammatory effects, 14 reduction of cellular apoptosis, 15 in- hibition of excitotoxicity, 16 and assisting in the repair of myelin. 17 In light of the promising preclinical evidence, a phase II, randomized, placebo-controlled human clinical trial was initiated in order to evaluate the safety and potential efficacy of intravenous progesterone administration for the treatment of acute TBI. 18 The study concluded that administration of progesterone to brain-injured patients was safe based on the finding that no serious adverse events were noted and that the rates of adverse events among treatment and placebo groups were similar. The most significant result from the study however was that the rate of mortality among severely injured patients treated with progesterone was reduced by over 60% relative to the placebo group. In addition, patients in the moderate group showed significantly better functional out- comes at 30 days postinjury. A second single-center trial with severely brain-injured patients in which outcomes were studied over a 6 month period supported the results of the first study. 19 Despite the encouraging patient outcomes observed in these clinical trials, the eventual use of progesterone as a therapeutic is limited due to its poor aqueous solubility. For human use, the hormone had to be individually mixed with the vehicle solution for several hours under temperature controlled con- ditions. The resulting formulation is not stable at room temperature for long periods of time and therefore had to be *To whom correspondence should be addressed. Phone: 404-727- 6602. Fax: 404-712-8679. E-mail: dliotta@emory.edu. a Abbreviations: TBI, traumatic brain injury; Fmoc, 9-fluorenyl- methyl; HCl, hydrochloride; PTSA, p-toluenesulfonic acid; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; IP, in- traperitoneal; SC, subcutaneous; RBF, round-bottom flask.