Simplified immunosuppressive and neuroprotective agents based on gracilin A

Simplified Immunosuppressive and Neuroprotective Agents Based on Gracilin A Mikail E. Abbasov,1 Rebeca Alvariño,2 Christian M. Chaheine,1 Eva Alonso,2 Jon A. Sánchez,2 Michael L. Conner,1 Amparo Alfonso,2 Marcel Jaspars,3 Luis M. Botana,2, ★ Daniel Romo 1 , ★ The architecture and bioactivity of natural products frequently serves as an embarkation point for exploration of biologically-relevant chemical space. Total synthesis followed by derivative synthesis has historically enabled a deeper understanding of structure-activity relationships. However, synthetic strategies toward a natural product are not always guided by hypotheses regarding structural features required for bioactivity. Herein, we describe an approach to natural product total synthesis that we term ‘pharmacophore-directed retrosynthesis.’ A hypothesized, pharmacophore of a natural product is selected as an early synthetic target and this dictates the retrosynthetic analysis. In an ideal application, sequential increases in structural complexity of this minimal structure enables development of an SAR profile throughout the course of the total synthesis effort. This approach enables the identification of simpler congeners retaining bioactivity at a much earlier stage of a synthetic effort as demonstrated herein for the spongiane diterpenoid, gracilin A, leading to simplified derivatives with potent neuroprotective and immunosuppressive activity. Complementary to diverted total synthesis and a subset of function-oriented synthesis, we anticipate that adoption of 1 Department of Chemistry and Biochemistry, Baylor University, One Bear Place #97348, Waco, TX 76798, United States. 2Departamento de Farmacología, Facultad de Veterinaria, Universidad de Santiago de Compostela, Lugo 27003, Spain. 3Marine Biodiscovery Centre, Department of Chemistry, University of Aberdeen, Meston Walk, Aberdeen AB24 3UE, Scotland, United Kingdom. ★e–mail: [email protected], [email protected] 2 pharmacophore-directed retrosynthesis, in conjunction with efficiency considerations (e.g. atom economy, protecting-group free), will expedite the exploration of natural products as drug leads for human disease through systematic mining of their information–rich content. The impactful and enduring role that natural products have played in improving the quality and duration of life for both humans and animals cannot be overstated. For example, rapamycin and its congeners have received FDA approval for various ailments and this natural product continues to provide insights into basic cell biology.1 Synthetic chemists are at the forefront of harvesting the full potential of natural products through synthetic efforts including classic total synthesis. Toward this goal, strategies to synthesize natural products have evolved significantly in recent years as more emphasis is placed on biological function.2 In Danishefsky's ‘diverted total synthesis’ (DTS), a synthetic sequence is developed from simple building blocks employing classical retrosynthetic analysis and then various advanced intermediates previously employed in the synthesis effort are diverted toward simplified derivatives for biological analysis (Fig. 1a).3 In a strategy not necessarily directed toward total synthesis, Wender's ‘function-oriented synthesis’ seeks to develop hypotheses regarding pharmacophores based on structural and computational analysis of distinct natural products and typically larger SAR data sets enabling the design and synthesis of simplified derivatives bearing a common pharmacophore (Fig. 1b).4 Alternatively, Schreiber’s ‘diversity oriented synthesis’ (DOS) of natural product-like libraries5 and the derived ‘bioactivity-oriented synthesis’ (BIOS) by Waldmann6 seeks to synthesize collections of compounds based on structural features found in natural products. Finally, Myers recently developed a convergent building block strategy for rapid access to a diverse array of structurally related scaffolds such as macrolides related to erythromycin for the discovery of novel antibiotics7 that was subsequently termed ‘analogue-oriented synthesis’ by Vanderwal.8 The simultaneous alignment of total synthesis efforts with structure-function relationship studies has not been fully realized to the extent possible and in particular with novel natural products for which minimal SAR information exists. Several truncated natural products,9 which were found to possess similar bioactivity to the parent natural product, are known (e.g. eribulin mesylate from halichondrin10), 3 however these derivatives were typically identified following completion of a total synthesis.11 In an example from our laboratory, a des-methyl, des-amino variant of the protein translation initiation inhibitor pateamine A was designed and synthesized following our total synthesis and found to have nearly equipotent activity to the natural product.12 This led to a retrospective question of whether such a derivative may have been accessed in route to the natural product and led us to consider the following question. Can the total synthesis of natural products, in particular with limited SAR or unknown or unconfirmed cellular targets, be more closely aligned to proposed biological activity during the retrosynthetic planning stages? Herein, we describe a type of retrosynthetic analysis that seeks to more closely align total synthesis efforts with concurrent biological studies. The strategy opens the potential to identify simplified versions of the natural product with similar potency or potentially new functions in the course of a total synthesis effort. We term this strategy ‘pharmacophore-directed retrosynthesis’ (PDR) to emphasize the importance of considering hypothesized pharmacophores at the retrosynthetic planning stage of a total synthesis effort. While this approach increases the challenges of natural product total synthesis beyond important, contemporary goals including atom-economy,13 step and redox efficiency,14 and protecting group avoidance,15 it has the potential to greatly accelerate harvesting of the vast information content of natural products for basic cell biology and medicine. In PDR, we build on Wender’s notion of bringing function to the forefront of a synthetic endeavor, cf. function-oriented synthesis, but employ the logic of retrosynthesis16 to target simplified intermediates that importantly possess the proposed minimal structural features required for bioactivity, or pharmacophore, in route to the natural product.17 In applying PDR, a key first step is the identification of a proposed pharmacophore that may be based on (i) structural analysis with chemical intuition; (ii) existing SAR from isolated natural product congeners; (iii) activity of structurally related compounds; or (iv) anticipated reactivity. A retrosynthesis is then devised that ensures the proposed pharmacophore is present in multiple intermediates, with increasing complexity, ultimately leading to the natural product. We selected a member of the spongiane diterpenoid family, gracilin A (1),18 to initiate assessment of the utility of PDR toward exploring the recently described immunosuppressive19 and neuroprotective 4 activity20 given the limited SAR information and no prior synthetic work.21 In the retrosynthetic analysis, as complexity is increased toward the natural product, several intermediates possessing the proposed pharmacophore are specifically targeted thus enabling SAR to be gathered as the total synthesis progresses (e.g. 8 ® 7 ® 6 ® 1, Fig. 1d). It should be noted that several hypotheses regarding the pharmacophore of a particular natural product could be posited for PDR leading to alternate retrosynthetic strategies. The gracilins, including the rare nor–diterpene gracilin A,18 were originally isolated and characterized from the Mediterranean sponge Spongionella gracilis.22 These diterpenes are structurally unique owing to the unusual diacetoxy furanose found in most members. The cytotoxic activity of gracilins B and G–I isolated from Spongionella pulchella against a diverse panel of 12 human cancer cell lines has been reported, but these compounds did not progress further in preclinical evaluation.23 The absolute configuration of gracilin A was never established, while its relative configuration was based on X–ray analysis of the keto derivative of 9,11–dihydrogracilin A (3).24 Gracilin A was reported to be a potent inhibitor of phospholipase A2 (PLA2) with a 69% inactivation efficiency.25 We previously reported that gracilin A was mildly cytotoxic against K562 and PBMC cells, with IC50 values of 0.6 and 0.8 µM respectively, and inhibited EGF–R by 70% at 100 µM concentration.22 We also recently posited that gracilin A mimics the effects of cyclosporin A (CsA) through interaction with cyclophilin A (CypA)19 and also improves Alzheimer’s Disease (AD) hallmarks in vitro and in vivo.20, 26, 27 These results prompted the current study to employ PDR to unravel and ideally differentiate the structural requirements for immunosuppressive and neuroprotective effects observed with gracilin A. 5 Figure 1 | ‘Pharmacophore–Directed Retrosynthesis’ (PDR) applied to gracilin A and comparison to other synthetic strategies harvesting the rich information content of natural products. a, Strategies toward simplified, bioactive small molecules using natural products as starting points including diversity-oriented synthesis (DOS) directed toward libraries of natural product-inspired compounds. b, PDR seeks to identify or hypothesize the pharmacophore of a natural product, dictating the retrosynthesis by ensuring these features are Danishefsky's Diverted Total Synthesis (DTS) Total Synthesis Bioactive Natural Product Target Building Blocks Advanced Intermediate Wender's Function-Oriented Synthesis (FOS) Me O H H Me Me Me 9,11–dihydrogracilin A (3) H Me O H H Me Me Me gracilin A (1)gracilin L (2) MeMe Me H H O Me HO OAc OAc OAc OAc OAc OAc O H H Me Me Me 6 OAc OAc O H H 8 OAc OAc O 9 OAc OAc Structural Complexity | SAR Profile | Total Synthesis Progress FG2FG2 FG1 FG3 FG1 Simplified, Equipotent Natural Product Derivative FG2 FG1 FG3FG3 Bioactive Natural Product I Bioactive Natural Product II 'Minimal Structural Feature' Required for Bioactivity (known or hypothesized) Present work: Pharmacophore-Directed Retrosynthesis (PDR) Bioactive natural product target FG2 FG1 FG3 1 FG1 FG3 FG1 FG3 FG1 FG3 Hypothetical 'minimal structural feature' FG1 FG1 FG3 PDR 7 b Me O H H Me Me Me dendrillin (4) HOOAc OAc Me Me Me O H HO O OAc H Me tetrahydroaplysulphurin-1 (5) FG3 Me O H H Me Me Me OAc OAc Schreiber's Diversity-Oriented Synthesis (DOS) Forward Synthetic Analysis Simple Building Blocks FG2 FG1 FG3 simple complex and diverse FG1 FG1 FG3 FG1 FG3 FG1 FG3 c FG1 d FOS DTS DOS Simplified structures bearing 'minimal structural feature' FG1 FG1 FG1 FG2 FG2 FG2 1 5 11 15 6 8 10 16 13 & Other Possible Hypotheses O H H O O 6 present in multiple intermediates toward the natural product target. c, Naturally–occurring spongiane diterpenoids bearing a common bisacetoxy furanose moiety (red) selected as the hypothetical pharmacophore. d, PDR applied to gracilin A (1) leads to increasingly complex intermediates (i.e. 8, 7, 6) throughout the course of total synthesis from key intermediate 9. The cyclophilins (Cyp) are highly conserved peptidyl-prolyl, cis-trans isomerases (PPIase) involved in protein folding and trafficking28, 29, 30 and are found in multiple cellular compartments.31CypA, the cytoplasmic isoform, has several roles in cell metabolism and energy homeostasis 32 with an enhanced expression in inflammation and cancer.33 The use of small molecules that selectively block the inflammation-related functions of CypA, is an important pharmacological strategy leading to effective immunosuppressive agents.33 On the other hand, Cyclophilin D (CypD), the mitochondrial isoform, translocates to form the mitochondrial permeability transition pore (mPTP) and its activity correlates with the mitochondrial dysfunction observed in AD leading to neuronal death.34, CypD inhibitors devoid of immunosuppressive activity through lack of binding to CypA but possessing the desired mitochondrial effect and appropriate BBB permeability could provide an approach to address AD. Herein, we demonstrate a proof-of-principle study of PDR through application to the spongiane diterpene, gracilin A, that has led to useful lead compounds for both immunosuppression and neuroprotection. This study revealed simplified gracilin A derivatives with high affinity for CypA and others that demonstrated significant selectivity for CypD over CypA demonstrating their potential as neuroprotective agents devoid of immunosuppressive effects. Results and discussion Pharmacophore-directed retrosynthesis applied to gracilin A. The bis-acetoxy furanose moiety was selected as the pharmacophore of gracilin A based on several lines of evidence. Studies of the reactivity of macfarlandin E with lysine derivatives, which possesses a 1,4-dicarbonyl masked as a bis-acyloxy furanose, provided evidence for pyrrole formation through a Paal-Knorr process35 and mounting evidence suggests this is possible with a number of other bis-acetoxy furanose-containing natural products.36 Furthermore, a computational study demonstrated the potential of the bis-acyloxy 7 furanoses of gracilin A and aplysulphurin-1 to bind divalent cations such as Ca2+ pointing to the potential importance of this moiety for bioactivity.37 Finally, it is interesting to note that several bioactive members of the spongiane diterpene family possess a bis-acyloxy furanose including the structurally related gracilin L (2),22 9, 11 dihydrogracilin A (3), dendrillin (4),38 and tetrahydroaplysulphurin-1 (5).39 These considerations guided application of PDR to gracilin A and imposed a requirement that multiple intermediates along the synthetic route would bear or be converted to the proposed pharmacophore, namely the bis-acetoxy furanose (e.g. derivatives 6-8, Fig. 1d). In this way, structure-activity relationship (SAR) studies could be conducted throughout the course of the total synthesis. We recognized that keto lactone 9, accessible in gram quantities through our recently described Diels-Alder-lactonization organocascade,40 would serve as a key intermediate to study the importance of the C8-exocyclic alkene (e.g. derivative 6), the C9-appended cyclohexyl moiety (e.g. derivative 7). The importance of the bicyclic core of gracilin A would be ascertained by synthesis of the highly simplified monocyclic furanose 8 (Fig. 1d). Sequential synthesis of increasingly complex gracilin A derivatives. We recognized that in applying PDR, synthetic progress with concurrent biological assays of intermediates could best be achieved in stages based on increasing complexity of intermediates in the synthetic sequence as outlined in Fig. 2a-d. A final stage of diverted total synthesis could enable further refinement of the SAR profile through ‘gap filling’ with particular targeted derivatives to answer more specific questions building on information gathered during the initial stages. As applied to gracilin A, we first targeted the simple bis-acetoxy furanose 8 as the minimal pharmacophore that was readily obtained through a twostep oxidation/hydrogenation sequence to deliver the racemic syn-substituted acetoxy furanose (±)-8a along with the anti-diastereomer (±)-8b (dr 1.7:1). In a second stage, derivatives devoid of the cyclohexyl substituent and exocyclic alkene were targeted, namely bicyclic bis-acetoxy furanose 7a,b (Fig. 2b). The bicyclic lactone endo-15a (3:1, dr; 94% ee) was obtained from a Diels-Alderlactonization organocascade employing diene 12 and acryloyl chloride (11) with tetramisole as Lewis base promoter. The required endo-diastereomer 15a could be isolated in 58% yield and subsequent reduction with LiAlH4 followed by desilylation delivered the ketodiol 16. Swern oxidation led to a 8 dialdehyde which was directly subjected to acid-promoted acetylation to give the unstable keto bisacetoxy furanoses 17 as a mixture of syn/anti diastereomers. A subsequent reduction with NaBH4 and dehydration with SOCl2 delivered the bicyclic bis-acetoxy furanoses 7a,b as a mixture of alkene regioisomers. The low yields obtained in this and subsequent 4-step sequences leading to the bisacetoxy furanoses were primarily a result of incomplete or non-simultaneous Swern oxidation of the diols leading to regioisomeric mono-acetoxy furanoses. We next targeted derivatives devoid of the exocyclic alkene but bearing the cyclohexyl substituent (Fig. 3c). Desilylation of endo-Diels-Alder adduct 15a, gave bicyclic lactone (–)-9 which was subjected to an allylzinc reagent derived from cyclohexenyl choride 19 employing conditions we previously employed in our synthesis of spongiolactone.41 This delivered the tricyclic adducts 21a as a 1:1 mixture of diastereomers at the generated quaternary carbon but with high facial selectivity leading to a single epimer at the tertiary alcohol center. The contra-steric addition of the allylzinc reagent to the concave face of the ketone (–)-9 was unexpected but was verified by extensive NMR studies (see ESI for details) and, in the case of addition of the gem-dimethyl cyclohexenyl zinc reagent derived from (±)-19, was further verified by single crystal X-ray analysis of adduct (–)-21b. However, this diastereoselectivity is inconsequential since the tertiary alcohol in (–)-21b is subsequently dehydrated. Hydrogenation reduced the cyclohexene of (–)-21b and a 3-step process delivered the hydroxy bisacetoxy furanose 22 as a 1:1 mixture of diastereomers. Dehydration with Martin’s sulfurane gave the alkenes 23 as a mixture of 4 diastereomers due to the alkene regioisomers produced. The four diastereomers were separable by preparative chiral HPLC enabling biological analysis of each stereoisomer. Gaps in the SAR profile were also back-filled following initial assays described below. This entailed application of aspects of diverted total synthesis to synthesize derivatives devoid of the cyclohexyl moiety (i.e. 24), changes in the oxidation state and substituents of the tetrahydrofuran, and the complexity of the cyclohexyl moiety (i.e. 28, Fig. 2d). 9 Figure 2 | Pharmacophore-directed retrosynthesis applied to gracilin A: a, Synthesis of a highly simplified analogue bearing the proposed pharmacophore. b, Assembly of the cis–fused, 6,5–bicyclic core 15a of gracilin A through an enantioselective, organocatalytic Diels–Alder/lactonization cascade and manipulation to a simplified bicyclic analog. c, Annulation of the natural and a simplified cyclohexyl moiety. (inset: ORTEP representation of the single crystal X-ray structure of (–)-21b) d, SAR profile gap filling: Alternate oxidation states of the original bis-acetoxy furanose and an enantiomeric series. O OAc OAc (±)-8a O H H O OAc OAc H H Me Me Me O OAc OAc O O H H OAc (–)-24 23 (4 diastereomers) 23a: 10S,Δ9,11; 23b: 10R,Δ9,11 23c: 10S,Δ8,9; 23d: 10R,Δ8,9 HPLC sep'n. 1) H2, Pd(OH)2, EtOH 23 ºC, 15 h 2) LiAlH4, THF, 0 ºC, 1 h 3) (COCl)2, DMSO, Et3N CH2Cl2, –78→ 23 ºC, 3 h H H Me (–)-26 O O (–)-21a 10 10 4 11 8 (–)-27a (60%, 2 steps, >19:1 dr) OH TBSO Cl N S N Ph O TBSO O H H O endo (-)-15a (58%, 94% ee) TBSO O H H O + (3.7 : 1) [gram scale] 14 exo (+)-15b (19%, 94% ee) 21b (–)-15a H H OH OH (–)-16 2) SOCl2, pyridine 0 → 23 ºC, 3 h (5%, 4 steps) (–)-15a 1) Pb(OAc)4, AcOH, 23 oC, 20 h H H Me O OAc Core Structure Synthesis O O H H O Me Cl R R O H H O OH Me R R TBAF, AcOH, THF 0 → 23 ºC, 4 h (–)-9 Zno, LiCl, BrCH2CH2Br THF, 0 → 23 ºC 20: R = H, Δ1,2 (–)-21a: R = H ( (88%, 2 steps, >19:1 dr) 21b: R = Me, Δ1,2 (82%, 1:1 dr) (±)-18: R = H or (±)-19: R = Me O O H H O (+)-9 O H H OAc Me Me Me (–)-21b (R = Me) 4 (–)-15a 29 (2 diastereomers) 29a: 10S 29b: 10R HPLC sep'n. (+)-15a d. SAR Profile Gap Filling Me O H H Me Me Me gracilin A (1) OAc OAc c. Derivatives Devoid of Alkylidene & a Simplified Cyclohexyl Moiety b. Derivatives Devoid of Cyclohexyl Moiety & Alkylidene a. Highly Simplified Proposed Pharmacophore Enantiomeric Series OH TBSO 12 O Cl N S N Ph 11 13 (1.0 equiv) O H H OAc OH Me MeMe OAc 22 2) H2, 5% Rh-Al2O3, EtOAc, 23 oC, 2 h (94%, 2 steps, 1.7:1 syn/anti) + 1) NaBH4, MeOH 0 ºC, 1 h 1) (COCl)2, DMSO, Et3N, CH2Cl2, –78→0 ºC, 2 h 2) TBAF, AcOH THF, 0 oC, 1 h (76%, 2 steps) (98%) 4) NaOAc, Ac2O, H2SO4 AcOH, 0 → 23 ºC, 48 h (37%, 4 steps, 1:1 dr) PhH, 80 oC, 1 h (97%, 1:1 dr, 1.3:1 rr) 2) NaOAc, Ac2O, H2SO4, AcOH, 0 → 23 oC, 24 h H H O OAc OAc 17 O (+)-28 O H H O Me Me Me 10 2,6-lutidine CH2Cl2 23 ºC, 18 h (77%, 3.7:1 dr) O 1) LiAlH4, THF 0 ºC, 1 h N O MeO S O O NEt3 1) DIBAl-H, CH2Cl2 –78 ºC, 3 h 2) TBAF, AcOH, THF 0 → 23 ºC, 4 h (81%, 2 steps, 4:1 dr) 3) Ac2O, DMAP, pyridine 23 ºC, 4 h (98%, >19:1 dr) Martin sulfurane (25) CHCl3, 60 ºC, 1 h (73%, >19:1 rr) 1) DIBAl-H (1.2 equiv) CH2Cl2, –78 ºC, 4 h 2) Ac2O, DMAP, py. 23 ºC, 14 h 1) DBU, CH2Cl2 40 ºC, 24 h (90%) 1) 20b, Zno, LiCl, BrCH2CH2Br THF, 0 → 23 ºC, 14 h (78%, 1:1 dr) 1) DIBAl-H, CH2Cl2 –78 ºC, 4 h 2) Ac2O, DMAP, py. 23 ºC, 14 h (79%, 2 steps, 1:1 dr, >19:1 rr) (63% over 2 steps) 7a,b 2) TBAF, AcOH, THF 0 → 23 ºC, 4 h (96%) O OAc OAc (±)-8b 2) H2, Pd(OH)2, EtOH 23 ºC, 15 h 3) Martin sulfurane CHCl3, 60 ºC, 1 h H2, Pd(OH)2, EtOH 23 ºC, 15 h (+)-27b (99%, 2 steps) H H Me OAc OAc 1) LiAlH4, THF 0 → 23 ºC, 1.5 h 2) Ac2O, DMAP, py. 23 ºC, 24 h (–)-26 11 8 9 1 2 16 importantly while also displaying selectivity for CypD vs CypA inhibition. These gracilin derivatives serve as lead compounds for neurodegenerative diseases and other CypD-mediated diseases including atherosclerosis or autoimmune diseases.49, 50 Although an ideal application of PDR will generally be challenging to implement, in particular when reactive functionality precludes a completely linear synthetic strategy as dictated by PDR and as in the present case of the gracilins, we expect that pharmacophore hypotheses brought into retrosynthetic planning will enable greater SAR information to be gathered in route to a natural product. It is also anticipated that application of PDR will provide an avenue for hypothesis-driven, natural product total synthesis efforts while simultaneously accelerating the exploration of natural product chemical space through total synthesis efforts premised on PDR. Methods Gram-scale Diels-Alder-Lactonization Organocascade providing g -lactones (–)-15a and (+)-15b, the bicyclic core of gracilin A derivatives. To an oven-dried, 250-mL round-bottomed flask equipped with a magnetic stir bar was added silyloxydiene alcohol 1251 (2.14 g, 10.0 mmol, 1.0 equiv), (S)-(–)- TM·HCl (2.40 g, 10.0 mmol, 1.0 equiv), 2,6-lutidine (3.5 mL, 30.0 mmol, 3.0 equiv) and anhydrous CH2Cl2 (100 mL, to make the final concentration of silyloxydiene alcohol 0.1 M) at ambient temperature (23 ºC). With vigorous stirring, 11 (1.05 mL, 13.0 mmol, 1.3 equiv) in CH2Cl2 (9.0 mL) was added over a period of 5 h by syringe pump addition. After stirring for an additional 13 h, the reaction mixture was filtered through a short pad of SiO2 and the filtrate was concentrated by rotary evaporation. Purification by automated flash chromatography (5→50% EtOAc/hexanes) afforded bicyclic g-lactones (–)-15a (1.55 g, 58% yield, 94% ee) and (+)-15b (0.51 g, 19% yield, 94% ee). See SI for full characterization of 15a and 15b. Binding experiments: Surface activation, ligand immobilization and binding. A Biacore X SPR biosensor with Control Software and BIAevaluation software version 3.0 from Biacore (GE Healthcare, Uppsala, Sweden) was used to check the binding between gracilin A derivatives and CypA. Sensor surface activation and ligand immobilization were performed by using HBS-EP as running buffer at a flow rate of 5 μL/min and 25 °C. CM5 sensor chips were used as surface where Cyp A was immobilized as ligand. The CM5 chip is a glass slide coated with a thin layer of gold with a matrix of carboxymethylated dextran covalently attached. The CM5 chip was activated using an amine coupling kit. Following manufacture instructions, a mixture (1:1, v/v) of EDC and NHS was applied for 2 min over 17 the sensor chip. After activation, the ligand, 100 μg/mL of active human CypA protein dissolved in sodium acetate 10 mM at pH 4.5 was added to be immobilized over a CM5 sensor chip. Finally, ethanolamine-HCl was injected to deactivate the remaining active esters and to avoid non-specific binding. Next, analytes (CsA as positive control or synthetic compounds) were added to evaluate the binding with CypA. Once analytes were tested and interaction was observed, individual binding curves were analyzed by determining the kinetic constants of analytes-CypA binding, namely, the observed rate constant (Kobs), the association rate constant (Kass), the dissociation rate constant (Kdiss), and the kinetic equilibrium dissociation constant (KD). At equilibrium, by definition, Kdiss/Kass= KD. The pseudofirst-order association rate constants Kobs (s-1) were determined for each compound concentration by using the 1:1 Langmuir association model of BiaEvaluation software (BiaCore, Uppsala, Sweden). Then a representation of Kobs against the corresponding concentration of each compound was done. These plots follow a linear correlation coefficient. From the equation of these representations, Kass, M-1 s-1, gradient of the plot, and Kdiss, s -1, intercept of the plot was obtained. Within these two values, the kinetic equilibrium dissociation constant KD for each analyte-CypA binding was obtained. The duration of the sample injection was 2 min at 10 μL/min flow rate. Next, dissociation of bound molecules in HBS-EP buffer flow was studied. The bound drugs were removed from the chip surface before the next injection by adding 1 M Glycine-HCl at pH 2.5 for 1 min. The association phase was used to quantify the compound-CypA interactions. All experiments were performed four times. Determination of intracellular ROS levels. Intracellular levels of reactive oxygen species (ROS) were determined with carboxy-H2DCFDA (5-(and-6)-carboxy-2′,7′-dichlorodihydrofluorescein diacetate). This dye diffuses through the cellular membrane and is converted by cellular esterases to carboxy-H2DCFH (non-fluorescent). When carboxy-H2DCFH is oxidized by ROS, it becomes fluorescent.52 Cells were seeded at a density of 2.5x105 cells/mL in 96-well plates and allowed to attach for 24h. Following treatment with compounds at various concentrations (1 µM®1 nM) and 150 µM H2O2 for 6h, SH-SY5Y cells were washed twice with serum-free culture medium. Then, carboxy-H2DCFDA 20 µM dissolved in serum-free culture medium was added to each well and the cells were incubated for 1h at 37 ºC. After this incubation, the medium with the fluorescent dye was replaced with PBS and the plate was incubated for 30 min at 37 ºC. Fluorescence was read at 527 nm, with an excitation wavelength of 495 nm. All experiments were performed four times. Mitochondrial membrane permeability transition pore (mPTP) measurement. The blockage of mPTP by compounds was determined with the MitoProbe Transition Assay Kit following manufacturer’s instructions. Briefly, SH-SY5Y human neuroblastoma cells were re-suspended in pre-warmed PBS/Ca+2 buffer at a final concentration of 1x106 cells/mL. Cells were loaded with 0.01 µM Calcein-AM and 18 incubated at 37ºC for 15 min. Then, 0.4 mM CoCl2 and compounds at selected concentrations were added and incubated for 15 min at 37ºC. Cyclosporine A (CsA) at 0.2 µM was used as positive control. After this incubation, cells were centrifuged and re-suspended in 100 µL of PBS. Just before analyzing, 1 mMtert-butyl hydroperoxide (TBHP) was added to the samples to induce pore opening. Fluorescence intensity was measured at 488 nm excitation and 517 nm emission wavelengths by flow cytometry using the ImageStreamMKII (Amnis Corporation, Merck-Millipore) and INSPIRE® software. The fluorescence of 10,000 events was analyzed with IDEAS® Application vs 6.0 (Amnis Corporation, Merck-Millipore). Experiments were carried out four times. Acknowledgement. Support from NIH (R37 GM052964 to D.R.), the Robert A. Welch Foundation (AA-1280 to D.R.), FEDER cofunded-grants: from CONSELLERIA DE Cultura, EDUCACION e ordenación Universitaria Xunta de Galicia, 2017 GRC GI-1682 (ED431C 2017/01), from CDTI and Technological Funds, supported by Ministerio de Economía, Industria y Competitividad, AGL2014-58210-R, AGL2016-78728-R (AEI/FEDER, UE)(LB), ISCIII/PI16/01830 (AA) and RTC-2016-5507-2, ITC-20161072, and from European Union POCTEP 0161-Nanoeaters -1-E-1, Interreg AlertoxNet EAPA-317-2016, and H2020 778069EMERTOX (LB) and from the European Union’s Seventh Framework Programme managed by REA–Research Executive Agency (FP7/2007-2013 under grant agreement 312184 PHARMASEA to L.B. and M.J.) is gratefully acknowledged. Drs. Nattamai Bhuvanesh and Joe Reibenspies (Center for X-ray Analysis, TAMU) secured X-ray data and Dr. Bill Russell (Laboratory for Biological Mass Spectrometry, TAMU) provided mass data. Author Contributions. M. E. Abbasov, C. M. Chaheine, and M. Conner synthesized and characterized all gracilin A derivatives described herein. R. Alvariño and J. A. Sánchez performed the neuroprotection and immunosuppression assays and compiled and wrote the assay data, respectively. L. M. Botana, E. Alonso and A. Alfonso designed, analyzed, and wrote the neuroprotection and immunosuppression assay results and data. D. Romo and M. Abbasov analyzed structure-activity relationships and wrote the manuscript with input from all authors. Additional information Full synthetic details for all new, bioactive gracilin derivatives and full characterization including 1H (1D and 2D) and 13C NMR spectra. Further details of the immunosuppressive (Cyp A binding, IL-2 release inhibition and cellular viability) and neuroprotective assays (cellular viability, TMRM assay, ROS release, GSH content, mPTP opening and PPiase activity). 19 Competing financial interests There authors declare no competing financial interests. References 1. 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