Synthesis of Nonretinoid Inhibitors of RPE65 for the Potential Treatment of Dry Age-Related Macular Degeneration and Stargardt Disease

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Issue Date
2019-08
Authors
Donovan, Machayla Rose '19
Degree
MS in Pharmaceutical Sciences
Advisor
Cioffi, Christopher L
Committee Members
Hass, Martha A.
Cen, Yana
Journal Title
Journal ISSN
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Abstract
Age-related macular degeneration (AMD) and Stargardt disease involves neurodegeneration of the macula, which is the central part of the retina that is responsible for high visual acuity. Loss of macular tissue leads to loss of central vision and can ultimately lead to complete blindness. Accumulation of lipofuscin in the retina is associated with the pathogenesis of dry (atrophic) AMD and Stargardt disease. Evidence suggests that cytotoxic bisretinoid fluorophores such as N-retinylidene-N-retinylethanolamine (A2E) mediate lipofuscin toxicity in the dry AMD and Stargardt disease retina. It has been hypothesized that reducing retinal all-trans-retinaldehyde concentrations via modulation of the visual cycle may reduce production of A2E and potentially delay or halt the progression of dry AMD and Stargardt disease.
A critical step in the visual cycle is the conversion of all-trans-retinyl ester to 11-cis-retinol by the isomerohydrolase (IMH) RPE65, which is nearly exclusively expressed within the retinal pigment epithelium (RPE). The IMH reaction is the rate-limiting step in the visual cycle, thus making RPE65 a target of interest for modulating the visual cycle. Thus, the identification and characterization of non-retinoid RPE65 inhibitors to test the therapeutic potential of visual cycle modulation for the treatment of dry AMD and Stargardt disease is of high importance.
Our collaborators conducted an in silico high-throughput virtual screen using a computational docking model derived from X-ray crystallographic data of RPE65 active site, which led to the discovery of CU239, a novel non-retinoid inhibitor of RPE65. CU239 is a selective and competitive inhibitor of RPE65 with a KD = 230 nM and an IC50 = 6 \316\274M. Furthermore, CU239 exhibited in vivo target engagement when administered systemically and conferred a partial protection of the retina in the murine light-induced retinal damage (LIRD) assay. Collectively, these data suggest that CU239 serves as a suitable hit molecule for further optimization.
We present herein efforts toward the identification of a more potent analogue of CU239 that also presents improved physicochemical properties for in vivo proof-of-concept efficacy assessment in murine models of AMD upon oral (p.o.) administration. The objective of the studies described herein is identification and detailed characterization of novel, selective, and non-retinoid RPE65 antagonists derived from CU239 that exhibit improved potency and physicochemical properties. To that end, we have designed and synthesized twelve novel analogues that explore the structure-property (SAR) relationships conferred by altering the amide motif of CU239. The synthesis of these analogues involved a concise synthetic campaign that utilized two different peptide coupling conditions with various amines (e.g., HBTU and T3P) starting from an advanced common carboxylic acid intermediate. The aforementioned conditions allowed for the generation of our desired compounds in a parallel manner. Each compound was purified via standard flash chromatography or utilized an automated column chromatography system. Compounds were isolated and purified over silica gel using a hexanes and ethyl acetate (EtOAc) or dichloromethane (CH2Cl2) methanol (CH3OH) eluent system. All intermediates and final compounds synthesized were fully characterized by 1H NMR and LC-MS. Furthermore, the purity of all final amide compounds synthesized for biological activity assessment was determined to be >95% by 1H NMR and HPLC analysis.
Our sample set of CU239 amide analogues explored various amide appendages that project 4-substituted aryl, cycloalkyl, alkyl, and heterocyclic motifs designed to help us gain a better understanding of the nature of the RPE65 binding pocket. We have found that the hydrophobic aryl, cycloalkyl, and alkyl appendages induce significant RPE65 inhibitory activity, with the n-butyl analogue 13 producing the most robust inhibition of RPE65 (87.1%). Conversely, the polar and ionizable heterocyclic piperidine containing analogue was not as well tolerated and produced the lowest % inhibition of RPE65 (16.9%). This trend provides evidence in support of our proposed pharmacophore and binding pose for CU239, which involves the amide carbonyl chelating with the Fe2+ co-factor, the amide appendage projecting into the deep and hydrophobic binding cavity buried within active site that accommodates the fatty acid side chain of all-trans-retinyl-palmitate (the natural substrate), and the urea phenyl ring residing within the \357\201\242-ionone pocket near the opening of the active site. Lastly, we were further encouraged by results for compound 13, as this compound provided the best % inhibition of RPE65 within our sample set while also presenting a significantly lower molecular weight (MW), CLogP, and topological polar surface area (TPSA) relative to CU239. The lower MW, CLogP, and TPSA values observed for 13 are encouraging as they may confer improved physicochemical properties relative to CU239, which is required in order to ultimately generate a potentially orally bioavailable compound for future in vivo testing.
Our efforts in this campaign will assist with the design of the next iterative wave of future analogues to be synthesized and tested. The goal of the next set of analogues will be to build upon compound 13 and to identify compounds that are more potent than CU239 but exhibit more favorable MW, CLogP, and TPSA parameters. The identification of such compounds will then lead to the evaluation of their pharmacokinetic (PK) characteristics in mouse prior to in vivo proof-of-concept target engagement and efficacy testing in mouse models for dry AMD upon oral (p.o.) administration.
Citation
Donovan, M. Synthesis of nonretinoid inhibitors of RPE65 for the potential treatment of dry age-related macular degeneration and Stargardt Disease [thesis]. Ann Arbor (MI): Proquest LLC; 2019. 121 p.
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