Aminocholestane and Aminoandrostane Inhibitors of the SH2 Domain-Containing Inositol 5’-Phosphatase (SHIP)

Abstract

The SH2-containing inositol phosphatase (SHIP) has become an actively researched therapeutic target for a number of disorders, including Alzheimer’s Disease, Graft-vs-Host disease, obesity and cancer. Analogs of the aminosteroid SHIP inhibitor 3α-aminocholestane (3AC) have been synthesized and tested. Analogs with improved water solubility have been identified. Deletion of the C17 alkyl group from the cholestane skeleton improves water solubility, however these compounds inhibit both SHIP1 and SHIP2. Enzyme kinetics imply that these molecules are competitive inhibitors of SHIP, binding at a site near where the substrate binds to the phosphatase. A model of the binding of the inhibitors within the active site of SHIP1 is proposed to explain the structure activity studies. Overall this work provides more water soluble aminosteroid pan-SHIP1/2 inhibitors that can be used for future studies of SHIP activity.

Graphical Abstract

Analogs of the aminosteroid SHIP inhibitor 3α-aminocholestane (3AC) have been synthesized and tested. Deletion of the C17 alkyl chain improves water solubility, however these analogs inhibit both SHIP1 and SHIP2. Enzyme kinetics imply that these aminosteroids are competitive inhibitors, and a model of the inhibitors within the active site of SHIP is proposed.

Introduction

The SH2 domain-containing inositol 5’-phosphatase (SHIP) is an active participant in the PI3K signaling pathway, cleaving the 5’-phosphate from phosphatidylinositol 3,4,5-triphosphate (PI(3,4,5)P₃) to generate phosphatidylinositol 3,4-bisphosphate (PI(3,4)P₂) (Figure 1). Both PI(3,4,5)P₃ and PI(3,4)P₂ are important second messengers in cell signaling, with PI(3,4,5)P₃ levels being known to influence cell division through AKT/mTOR signaling,^[1] while PI(3,4)P₂ levels can also influence apoptosis by promoting Akt phosphorylation at an alternate site^[2] and through a pathway involving TAPP adaptors.^[3] Dysregulation of inositol kinases and inositol phosphatases like SHIP is often associated with a number of diseases and disorders, including cancer,^[4] diabetes,^[5] and Crohn’s Disease.^[6]

Figure 1. SHIP, the PI3K pathway and 3α-Aminocholestane Hydrochloride (3AC, 1).

Two major isoforms of SHIP have been identified: SHIP1 and SHIP2, which are similar (~48% identity in amino acid sequence as determined by BLAST^[7] analysis). Comparison of the phosphatase domains of SHIP1 and SHIP2 shows an even higher ~64% identity of amino acid sequence. Expression of SHIP1 is confined to cells with a hematolymphoid lineage, primarily blood cells, mesenchymal stem cells and microglia in the brain.^[8] SHIP2 is expressed in all cells,^[9] with especially high levels being found in astrocytes.^[10] While SHIP1 and SHIP2 share a high rate of amino acid conservation^[11] they differ significantly in their expression and the binding kinetics of their SH2 domains. For example, the SH2 domain of SHIP2 associates and disassociates from its ligand proteins very slowly. As a result, determining the equilibrium dissociation constant (K_D) with certain peptides is very difficult. Meanwhile, the SH2 domain of SHIP1 quickly associates and disassociates with its ligand proteins.^[12] Differences in cellular expression and binding kinetics may explain the varying roles these two homologs play in cellular pathology. For example, SHIP2 acts as a negative regulator of the insulin-signaling pathway. SHIP1, on the other hand, functions as an important negative controller in immunoreceptor signaling^[13] and hematopoietic progenitor cell proliferation/survival,^[11] and as an inducer of cellular apoptosis.^[14]

More recently the SHIP1 gene (INPP5D) was identified as a risk gene for Alzheimer’s disease (AD) in several large genome-wide association studies.^[15] This has led to a number of studies on the role SHIP1 plays in AD pathology.^{[8, 16]} Interestingly, a number of other studies have shown that the level of expression of SHIP2 also correlates with Aβ load in AD patients.^{[10, 17]} Further studies on the role of SHIP2 in AD determined that downregulation of SHIP2 led to lower levels of PI(3,4)P₂, and this reduced hyperphosphorylation of tau, resulting in an improvement in memory impairment in transgenic Alzheimer’s mice.^[18]

In order to explore the biological effects of inhibiting or activating the SHIP enzyme a number of SHIP antagonists have been developed (Figure 2).^[19] These include selective SHIP1 inhibitors like the aminosteroid 3AC (1) and compounds that inhibit both SHIP1 and SHIP2 (pan-SHIP1/2 inhibitors) including quinolines like 2^[20] and tryptamines like 3.^[21] Selective SHIP2 inhibitors like the substituted pyridine 4,^{[19c, 22]} have also been developed. Most recently tetrahydroquinolines like 5^[19a] and oxazyl pyrroles like 6^[19a] have been disclosed. As both SHIP paralogs have been implicated as therapeutic targets, it is useful to have access to selective inhibitors of both enzymes and the pan-SHIP1/2 inhibitors, as some diseased cells may compensate for the inhibition of one paralog by overexpressing the other. This “paralog compensation” behavior has been observed in some cancer model systems with 3AC (1), for example.^[23]

Figure 2. Known SHIP Inhibitors.

The first useful small molecule SHIP1 inhibitor was the aminosteroid 3α-aminocholestane (1).^[24] This molecule showed an IC₅₀ for SHIP1 of ~10 μM. The activity of 1 has also been evaluated with regards to SHIP2 and PTEN, the other enzymes that hydrolyze the phosphates on PI(3,4,5)P₃. Studies showed that 1 is at least 4-fold selective for SHIP1 over SHIP2,^[19a] and does not inhibit PTEN.^[15] Treatment of mice with 3AC demonstrated in vivo bioactivity comparable to genetic phenotypes observed in SHIP knockout mice.^[24–25] While the initial bioactivity studies were promising, 3AC was at times problematic to solubilize, as the hydrochloride salt was nearly insoluble in DMSO and required the use of ethanol and Klucel (a type of hydroxypropylcellulose^[26]) to solubilize the molecule. Synthetic studies were therefore initiated on the steroid core to attempt to identify a similar molecule with improved water solubility. We also hoped to better understand how aminosteroids interacts with the SHIP enzyme, i.e. are aminosteroids competitive or allosteric inhibitors, and investigate what structural features of the inhibitor are responsible for the selective inhibition of SHIP1.

Results and Discussion

In order to secure a supply of 3α-aminocholestane 1 we undertook a synthesis of this molecule from the 5α–cholestan–3β–ol 7 (Scheme 1). The alcohol of 7 was converted into the corresponding α–azide 8 via mesylate formation and displacement with NaN₃, which resulted in inversion of the C3 stereochemistry. This S_N2 reaction was very selective, and none of the β-azide was detected. Reduction of the α–azide 8 to the α–amine was accomplished using LiAlH₄. The α–amine was then converted to the hydrochloride salt 1. Synthesis of the β–aminosteroid 11 began by converting the alcohol 2 into the α–iodide 9.^[27] Nucleophilic substitution via an S_N² mechanism with NaN₃ in DMSO followed by reduction of the β-azide 9 using LiAH₄ provided β–amine hydrochloride 11. Both formation of the iodide and the azide displacement were stereoselective for inversion, and none of the other diastereomers was detected. Functionalization of the amine was then explored. Transformation of (1) to the methanesulfonamide 12 and acetamide 13 was easily accomplished by reaction with methanesulfonyl chloride and acetic anhydride respectively. Reduction of the acetamide with LiAlH₄ then provided the ethylamine 14. The dimethylamino analog 15 was also prepared utilizing reductive amination conditions with excess formaldehyde. While undertaking the modifications of the amine (1) we quickly realized that the lipophilicity of the inhibitors was increasing, with many of these structures being more hydrophobic than recommended by common measures of drug-likeness.^[28] An alternative approach would be needed to access analogs with improved solubility in DMSO and/or water. This was addressed in two ways: 1) to use an alternate, less hydrophobic steroid scaffold as a starting material and 2) incorporate more polar groups on the steroid core. Both approaches were explored as it was unknown which sections of the steroid would be necessary to maintain SHIP inhibition and also the tolerance of functional groups outside the C3-amine was unclear.

Scheme 1. Synthesis of Cholestane Based Aminosteroids.

At first modifications at the C17 position of the steroid were explored, as deletion of the hydrophobic chain at this position should improve the water solubility of these systems substantially (Scheme 2). The ready availability of epiandrosterone 16, which provides functional groups at C17 and C3 of the steroid, simplifies the synthesis of these systems. Conversion of the C3β-alcohol of 16 to the C3α-azide 17 was accomplished via mesylate formation and S_N2 displacement with NaN₃. Reduction of the azide to the amine was accomplished using Staudinger conditions^[29] to avoid reduction of the C17 ketone, with formation of the hydrochloride salt giving 18. Ketone 17 was also used to access other scaffolds with functional groups at both C3 and C17. For example, reduction of the ketone of azido-ketone 17 gave alcohol 19, which was then converted to aminoalcohol 21. Alternatively the azido-alcohol 19 could also be converted to methyl ether 20, and then transformed to amine 22. The C17 ketone could also be completely removed from 16 using the Wolf-Kishner reaction to access steroid 23. Alcohol 23 was then converted to the C3α-amine 24 and the C3β-amine 26 using similar conditions as described for the cholestanes 1 and 11 (Scheme 2).

Scheme 2. Synthesis of Aminosteroids 18, 21, 22, 24, and 26.

The biological activity of these molecules was then evaluated in the malachite green assay^[30] for phosphatase activity (Figure 3). This assay measures the amount of phosphate released from a inositol triphosphate substrate that has C8 alkyl chains (C₈-PI(3,4,5)P₃)^[31] instead of the full length C16 chains. The C8-substrate was used to avoid micelle formation, as substrates with the longer alkyl chains often aggregate when not in the presence of a cell membrane, and the micelles can complicate final readings.^[32] These assays were performed with SHIP enzyme, buffer, and substrate, so no surfactant was added to simulate the presence of a cell membrane. This rules out effects that could be attributed to disruption of membrane dynamics. Both the C3α-aminocholestane 1 and the C3β-cholestane 11 showed significant activity against SHIP1 and SHIP2, although the C3α isomer 1 demonstrated more selectivity for SHIP1. The sulfonamide 12 and the acetamide 13 were inactive, indicating that a free amine was required near the C3 position of the steroid. The ethylamine 14 and the dimethylamino compound 15 were both active against SHIP1. Interestingly, both 14 and 15 showed selective inhibition of SHIP1, with significantly less effect on the SHIP2 enzyme.

Figure 3. Inhibitory activity for the aminosteroids from Schemes 1 and 2.

Inhibition of SHIP1 and SHIP2 in the malachite green assay for phosphatase activity. Compounds were evaluated at 1 mM. Values reported are means of at least 3 measurements and error bars represent standard deviation.

During these evaluations it became apparent that the lipophilicity of the cholestanes was a drawback, as the cholestanes were not soluble in DMSO/water mixtures (see Table S1 in the ESI for calculated water solubility of the tested compounds). The calculated partition coefficient (CLogP) showed a significant drop between the cholestanes (entries 1–6 ) and the androstanes (entries 7–12). This was consistent with the evaluation of the androsterones, as the more hydrophilic compounds were soluble in DMSO/H₂O mixtures (see Table S2 in the SI for solubility measurements). The C17-ketone 18, the C17-alcohol 21 and the C17-methyl ether 22 were poor inhibitors however, and showed little activity against SHIP1 or SHIP2. Removal of the polar oxygen at C17 gave inhibitors with greater potency, like alkanes 24 and 26. Both of these compounds were pan-SHIP1/2 inhibitors, inhibiting both SHIP1 and SHIP2, with compound 26 showing some selectivity for the SHIP2 enzyme.

In parallel with the studies on androstanes, routes towards adding more polar functional groups to the steroid A ring were also explored. To access these systems the C3 alcohol of dihydrocholesterol 2 was converted to the corresponding ketone 27 using PCC (Scheme 3). Bromination of ketone 27 with pyridine tribromide has been reported to selectively provide the C2α bromide 29.^[33] This is likely due to the propensity of the C3-ketone to prefer enolization towards C2,^[34] facilitating the rapid epimerization of the C2 bromide to avoid a 1,3-diaxial interaction with the C19 methyl group. Displacement of the C2 bromide with NaN₃ then provided the C2α azides 31 and 32, a reaction that likely proceeds through an S_N2 pathway which is followed by rapid epimerization. A similar sequence was performed starting from alcohol 23, with oxidation of the alcohol (to form 28), bromination (to provide 30) and azide formation proceeding in a similar manner to the cholestane system to give ketone 32. The C3 ketone was then reduced with sodium borohydride to provide both diastereomers of the C3 alcohol (33 and 35 for the dihydrocholesterol system, 34 and 36 for the androstanes). The stereochemistry of the aminoalcohols 34 and 36 was further verified by the ¹H NMR coupling constants of the corresponding acetates (see the SI for details). The stereochemistry of the other compounds was verified by NMR comparison (vida supra).

Scheme 3. Synthesis of Aminoalcohols 37–40.

The azidoalcohols 33-36 served as a starting point to access the C2,C3-diamines 45-48. This was accomplished by conversion of the alcohols to the corresponding mesylates followed by displacement with NaN₃ (Scheme 4), which provided the diazides 41-44. The reduction of these diazides with LiAlH₄ followed by hydrochloride salt formation provided the diamines 45-48.

Scheme 4. Synthesis of Diamines 45–48.

Evaluation of the cholesterol analogs 37, 39, 45 and 47 was again hampered by difficulties in solubilization. The lipophilic substituent at C17 led to these compounds having poor water solubility which the polar aminoalcohol or diamine on the A ring could not overcome. Unlike 3AC (1) which was insoluble in DMSO, these molecules were soluble in DMSO up to 20 mM (see Table S2 in the SI), but they were insoluble in water and solubilization in DMSO/water mixtures typically required heating and use of the solution as soon as the mixture cooled to avoid precipitation. Alternatively, the analogs with unsubstituted D rings had much improved water solubility, and readily dissolved in DMSO/water mixtures.

Most of the analogs with aminoalcohols or diamines on the steroid A ring demonstrated significantly improved inhibitory activity against the SHIP enzymes (Figure 4). Moving the amine from the 3-position to the 2-position (as in 37, 38, 39, and 40) was well tolerated, with these analogs showing significant inhibition of SHIP1. Interestingly, the selectivity of the inhibition for SHIP1 imparted from the C17 alkyl chain was lost when an amine was present at the C2 position, and many of these analogs actually showed weak selectivity for inhibition of SHIP2. The selective inhibition of SHIP2 seemed to be more pronounced when the C17 alkyl chain was not present, as 38, 40 and 46 showed at least some selectivity for inhibition of SHIP2. The least effective inhibitors in this series had an α-amine at C2 and a β-amine at C3 (compounds 47 and 48), evidently this arrangement was not favorable to inhibition. The removal of the C17 alkyl chain was quite beneficial to water solubility, with compounds 38, 40 and 46 all being able to be dissolved in DI water at ~5 mM (see Table S2 in the SI). Diamine 46 could be dissolved in DI water at even higher concentrations, but the addition of buffer often resulted in precipitation as the compound was salted out of solution. This could be easily remedied by the use of DMSO/buffer mixtures, and so these were used for the in vitro evaluations.

Figure 4. Inhibitory activity for the aminosteroids from Schemes 3 and 4.

Inhibition of SHIP1 and SHIP2 in the malachite green assay for phosphatase activity. Compounds were evaluated at 1 mM. Values reported are means of at least 3 measurements and error bars represent standard deviation.

While the malachite green assay proved useful to rapidly identify SHIP inhibitors, it is difficult to compare the potency of the inhibitors since the assay was performed at a high concentration to overcome the lack of sensitivity. The IC₅₀ of several of these analogs against both SHIP1 and SHIP2 was previously measured by our groups^[8] using a fluorescence polarization (FP) assay,^[35] and this data is reproduced in Figure 5A. The FP assay was used for the IC50 determination as it was reported to be more sensitive^[35] than the malachite assay and also provided a secondary verification of the inhibition in an assay with a different, non-colorimetric readout. The most active compound against SHIP1 from Figure 3 (24) along with the most active compounds from Figure 4 (37, 39, and 46) were evaluated in this assay. The most potent inhibitor appeared to be steroid 46, with an IC₅₀ of ~6 μM against both SHIP1 and SHIP2. Attempts were also made to determine the binding mode of the aminosteroid SHIP inhibitors to the enzyme, specifically if the inhibitor was competitive with the PI(3,4,5)P₃ substrate or whether the steroid was binding at an allosteric site. Towards this end, a kinetic analysis of the effects of inhibitor 46 was performed (Figure 5B). The reaction rate was measured at various concentrations of PI(3,4,5)P₃ in the presence of inhibitor 46. The results showed that the apparent value of V is unaffected by the concentration of the inhibitor, as indicated by the similar intercept point on the Y axis. Addition of the inhibitor 46 does appear to increase the K_m (the slope of the measurements), however. This analysis is consistent with the aminosteroid being competitive with the substrate of the reaction, PI(3,4,5)P₃, and therefore the aminosteroids likely bind to the active site of the phosphatase. While the K_i determined from the kinetic studies (32.6 ± 12.6 μM) is higher than expected based on the IC₅₀ determined with the FP assay, this is likely due to the different conditions used in the two assays, which often leads to divergent results being observed.^[36] The assays use different buffers, and are performed with different concentrations of the PIP substrates, so comparison of IC₅₀ and K_i values between them is difficult. The higher concentrations of PIP substrate used in the malachite likely leads to the higher observed K_i in this assay, as the observed inhibitor potency is expected to decrease due to increased substrate competition.

Figure 5. Fluorescence polarization assay evaluation of aminosteroid analogs and Lineweaver-Burk plot of SHIP1 inhibition with 46.

(A) Fluorescence Polarization assay evaluation of aminosteroid analogs. ^and=not determined. No Inhibition of SHIP2 was observed at 100 μM. (B) Lineweaver-Burk plot of SHIP1 inhibition with 46 (activity was measured with the malachite green assay, see the SI for details, K_i = 32.6 ± 12.6 μM).

Given that the enzyme kinetics implicate the aminosteroids as competitive inhibitors, in silico docking studies were undertaken to better understand the binding mode of the inhibitors. While the entire SHIP1 protein has not been structurally characterized, x-ray crystal structures of catalytically active fragments of SHIP1 and SHIP2 have been determined.^[37] These structures usually include the active site and the C2 domains of the protein. Examination of these structures shows that the two proteins have a very similar structure in and around the active site, especially near the key aspartic acid residue that directs the addition of water to the phosphate of the PI(3,4,5)P₃ substrate, leading to the observed hydrolysis activity.^{[4, 38]} One of the SHIP1 structures (PDB:6IBD^[37b]) was utilized in docking studies with 3AC (1) and 46 to try and understand why 3AC has some selectivity for SHIP1 while 46 has no selectivity between the two SHIP paralogs. Docking was performed using Autodock Vina and led to the structure shown in Figure 6A. The C3 amine of both aminosteroids prefers to reside in the metal ion binding pocket. This metal ion (usually Mg²⁺) acts as a Lewis acid that activates the phosphate for cleavage by a water molecule that is directed by an aspartic acid (Asp586) which resides below the metal ion binding site.^[4] This analysis predicts that the C3 amino group will form hydrogen bonds with Asn411 and/or Asp699 (which rest ~3.3 Å from the C2 amine and ~2.5 Å from the C3 amine respectively). Only one of these H-bonds is probably important to the binding of the aminosteroid however, as the dimethylamino analog 15 is active and is only capable of participating in one hydrogen bond. The inhibition observed with 15 and the ethylamino analog 16 implies that there is some flexibility in the structure of the Mg²⁺ binding site where the substituted amines are predicted to rest. In the case of 46, the C2 amino group is in position to form a secondary interaction with Glu452 (which resides ~2.8 Å away), which rests above the inhibitor. This additional interaction could explain the increased potency observed with this molecule. As the inhibitor blocks the metal ion binding site with the ammonium ion, the rest of the steroid rests in a cleft where the inositol would normally bind.

Figure 6. Molecular Modeling of the Aminosteroids 1 and 46 in the SHIP1 Active Site.

(A) Structure of SHIP1 (in green, PDB:6IBD) docked with compound 1 (orange) and compound 46 (purple)_. (B) Ligplot rendering of nearby residues and predicted H-bonds for compound 1. (C) Ligplot rendering of nearby residues and predicted H-bonds for compound 46.

While the model in Figure 6A is consistent with the observed inhibition, it does not explain why many of the cholestane inhibitors show selectivity for SHIP1 over SHIP2, while the androstanes tend to be unselective inhibitors of both SHIP1 and SHIP2. Comparison of the amino acid sequences of both enzyme paralogs shows that while most of the amino acids near the active site are nearly identical, there are significant differences in the P4IM (P4 Interacting Motif)^[37a] loop region, which has been defined as residues 650–675 on the SHIP1 enzyme. In SHIP2 this section of the enzyme appears to be flexible and has been proposed to fold over the PI(3,4,5)P₃ substrate and push the substrate deeper into the active site, facilitating hydrolysis of the 5’-phosphate on the inositol.^[38] The P4IM loop of SHIP1 does not appear to be as flexible, however. This is supported by the work of Bradshaw and co-workers, who obtained ~50 x-ray structures of the SHIP active site bound to fragment molecules.^[37b] All of these crystal structures are quite similar and show the P4IM loop in the more “closed” position, nearly identical to that shown in the 6IBD structure. This propensity of the SHIP1 loop to remain in a more rigid “closed position” places the amino acid chain in close proximity to the C17 alkyl chain in the docked structure of the aminosteroids in Figure 6A. Specifically Lys664 is positioned near this section of the steroid. As this residue is a more polar arginine in SHIP2, this may be a key determinant in the selectivity of the aminosteroid SHIP inhibitors. Alternatively, the P4IM loop may not come into contact with the C17 chain, as a comparison of the crystal structure of SHIP2 and SHIP1 shows that the SHIP2 loop is not in a similar position to interact with the C17 sidechain (see Figure S1 in the supporting information). Future work on the mutagenesis of the P4IM loop may be useful in determining the exact amino acid or aminos acids that are responsible for this SHIP1 selectivity of the cholestanes.

This model also seems to predict that it may be interesting to further modify the aminosteroid structure to improve the binding potency. Modifications at the end of the C17 sidechain to interact with the polar amine of the Lys664 or Arg644 may provide another point of contact. Alternatively, the C5-C6 positions also seem to be oriented towards the enzyme, which may provide other positions where contacts with the enzyme can be made. Given that steroids with unsaturation at C5 and C6 are widely available, this may be attainable from these other sources.

This work has resulted in the discovery of more soluble pan-SHIP1/2 inhibitors. While the loss of paralog selectivity in the inhibition of SHIP may at first seem disconcerting, in some cases this is beneficial as it proactively prevents paralog compensation, where cells utilize SHIP2 when SHIP1 is selectively inhibited. This type of paralog compensation was observed in some cancer model systems previously.^[23] Compound 46 was shown to increase the microglial phagocytosis of dead neurons in a number of microglial cell lines, while the selective inhibitor 1 was much less effective,^[8] which may be attributed to the inhibition of both SHIP paralogs. Compound 46 was also shown to cross the blood brain barrier in mice, and dosing mice with 46 increased the phagocytic capacity of their microglia for both Aβ1–42 and dead neurons, which might be a method to slow progression of AD.^[8] Further studies in other AD disease model systems are ongoing.

Conclusion

Approximately 20 analogs of aminosteroid SHIP inhibitors have been synthesized and evaluated for inhibition of SHIP1 and SHIP2. The steroid scaffold accommodates polar functional groups at C2 and C3 and maintains SHIP inhibition, however polar groups are not tolerated at C17. A basic amine is required at C2 or C3 in order to exhibit SHIP inhibition. A combination of an amine at C3 and a nonpolar alkyl chain provides inhibitors with selectivity for SHIP1 over SHIP2. Enzyme kinetic studies were then undertaken with inhibitor 46, and these studies support the inhibitor binding in the active site of the SHIP phosphatase domain. In silico docking of the aminosteroids in an x-ray structure of the SHIP1 phosphatase domain predict that the steroid rests in the active site of SHIP, with the ammonium group inhabiting the metal binding site. This would place the alkyl chain at C17 near the P4IM loop, which may explain the SHIP1 selectivity of C17-alkyl aminosteroids, as this region of the enzyme is quite different between SHIP1 and SHIP2. While the C17-alkyl chain imparts selective inhibition of SHIP1, it also leads to inhibitors with lower water solubility. The removal of the C17 alkyl chain led to pan-SHIP1/2 inhibitors with much improved water solubility, and some of these inhibitors (like 46). While the loss of paralog selectivity may at first seem disconcerting, in some cases this may be beneficial as it proactively prevents paralog compensation, where diseased cells utilize SHIP2 when SHIP1 is selectively inhibited. Diamine 46 showed promising results in cell based and murine models of AD, and is currently being studied in other model systems. This work provides a basis for the synthesis of future analogs for both selective SHIP1 inhibitors and pan-SHIP1/2 inhibitors, which may be useful in studying the role of SHIP in AD and other disorders.