Structure–Activity Studies on Bis-Sulfonamide SHIP1 Activators

The SH2-containing inositol polyphosphate 5-phosphatase 1 (SHIP1) enzyme opposes the activity of PI3K and therefore is of interest in the treatment of inflammatory disorders. Recent results also indicate that SHIP1 promotes phagolysosomal degradation of lipids by microglia, suggesting that the enzyme may be a target for the treatment of Alzheimer’s disease. Therefore, small molecules that increase SHIP1 activity may have benefits in these areas. Recently we discovered a bis-sulfonamide that increases the enzymatic activity of SHIP1. A series of similar SHIP1 activators have been synthesized and evaluated to determine structure–activity relationships and improve in vivo stability. Some new analogs have now been found with improved potency. In addition, both the thiophene and the thiomorpholine in the parent structure can be replaced by groups without a low valent sulfur atom, which provides a way to access activators that are less prone to oxidative degradation.


Introduction
Inositol phospholipids are critical participants in the PI3K/AKT pathway, a key cell signaling axis in eukaryotic cells.The phosphorylation pattern on the inositol acts as a recognition element for intracellular protein kinases, which, after binding, initiate phosphorylation cascades that contribute to signaling between the cell membrane and the nucleus.Inositol phosphorylation is tightly controlled by inositol kinases and phosphatases, as this signaling influences cell survival [1][2][3][4][5] and inflammatory processes [6][7][8].The major enzymes involved in metabolizing inositol phospholipids include PI3K, PTEN, SHIP1/2, and INPP4A/B (Figure 1).Modulation of these enzymes can be clinically useful.For example, the activity of phosphatidylinositol-3-kinases (PI3K) has been shown to have profound effects on cellular physiology [9,10].Once activated, PI3K rapidly synthesizes PI(3,4,5)P 3 from phosphatidylinositol-4,5-bisphosphate (PI(4,5)P 2 ) (Figure 1).PI(3,4,5)P 3 then recruits and activates the protein phosphatase AKT, which phosphorylates other protein kinases, transmitting signals from the cell membrane to the nucleus.Interest in PI3K has led to a number of small molecules that are clinically useful for the treatment of cancer [11].The successes with PI3K inhibitors have led to the investigation of other enzymes that metabolize inositol phospholipids, and we have been active in evaluating small molecules that modulate the activity of SHIP [10].In contrast to PTEN (phosphatase and tensin homolog), which directly reverses the PI3K reaction to generate PI(4,5)P 2 from PI(3,4,5)P 3 , SHIP generates PI(3,4)P 2 , which can be further dephosphorylated by INPP4.Notably, the PH domain of AKT recognizes both PI(3,4,5)P 3 and PI(3,4)P 2 , however, and both phosphoinositol species are needed for the full activation of AKT [12].The activity of AKT may be influenced by modulation of either PI3K or SHIP, and therefore SHIP modulation may also provide a means to treat a number of these disease states [10]. of AKT may be influenced by modulation of either PI3K or SHIP, and therefore SHIP modulation may also provide a means to treat a number of these disease states [10].Recently we have been investigating SHIP1 modulators and their therapeutic use in Alzheimer's disease (AD) [13,14].Several genome-wide association studies have identified single nucleotide polymorphisms in the INPP5D (SHIP1) gene linked to AD risk [15][16][17][18].Both SHIP1 and SHIP2 are expressed in microglia [19], and thus both SHIP paralogs could potentially limit microglial homeostatic functions that control amyloidosis and remove dead or dying neurons [19].SHIP1 expression is increased in the brains of late-onset AD patients and also in 5XFAD mice, which are a common AD model system [20][21][22].
While these results would seem to implicate SHIP1 inhibition as a potential treatment option for AD, other studies suggest a more complex situation [23].Two groups have published analyses of SHIP1 mutations in 5XFAD mice but have arrived at differing conclusions.One study, which used germline SHIP1 haploinsufficient 5XFAD mice, found that plaque burden was reduced, and behavioral studies in these mice demonstrated a preserved cognitive function [24], supporting the use of SHIP inhibitors.A second study used a conditional knockout of SHIP1 restricted to microglia and macrophages, finding that SHIP1 is required to limit Aβ plaque burden [25], which would implicate the use of SHIP1 activators as a treatment option.One hypothesis for these differing results is that up-or downregulation of SHIP1 enzymatic activity could be beneficial depending on the disease stage.For example, in early stages, patients may benefit from enhanced microglial homeostatic function that is induced by activation, while in late stages, antagonism of SHIP1 may instead be beneficial to reduce damage from dysfunctional microglia.The role of SHIP1 in AD is therefore still under investigation, with potential benefits of up-or downregulation of the enzyme still being evaluated.
New small molecule SHIP1 activators are therefore needed to explore their effects in AD model systems to define the role of SHIP1 in AD.SHIP is known to be activated by the binding of its product, PI (3,4)P2, to an allosteric site on the enzyme near the C2 domain [26].While some SHIP1 activators (often referred to as enzyme agonists [26][27][28][29]) have Recently we have been investigating SHIP1 modulators and their therapeutic use in Alzheimer's disease (AD) [13,14].Several genome-wide association studies have identified single nucleotide polymorphisms in the INPP5D (SHIP1) gene linked to AD risk [15][16][17][18].Both SHIP1 and SHIP2 are expressed in microglia [19], and thus both SHIP paralogs could potentially limit microglial homeostatic functions that control amyloidosis and remove dead or dying neurons [19].SHIP1 expression is increased in the brains of late-onset AD patients and also in 5XFAD mice, which are a common AD model system [20][21][22].
While these results would seem to implicate SHIP1 inhibition as a potential treatment option for AD, other studies suggest a more complex situation [23].Two groups have published analyses of SHIP1 mutations in 5XFAD mice but have arrived at differing conclusions.One study, which used germline SHIP1 haploinsufficient 5XFAD mice, found that plaque burden was reduced, and behavioral studies in these mice demonstrated a preserved cognitive function [24], supporting the use of SHIP inhibitors.A second study used a conditional knockout of SHIP1 restricted to microglia and macrophages, finding that SHIP1 is required to limit Aβ plaque burden [25], which would implicate the use of SHIP1 activators as a treatment option.One hypothesis for these differing results is that up-or downregulation of SHIP1 enzymatic activity could be beneficial depending on the disease stage.For example, in early stages, patients may benefit from enhanced microglial homeostatic function that is induced by activation, while in late stages, antagonism of SHIP1 may instead be beneficial to reduce damage from dysfunctional microglia.The role of SHIP1 in AD is therefore still under investigation, with potential benefits of up-or downregulation of the enzyme still being evaluated.
New small molecule SHIP1 activators are therefore needed to explore their effects in AD model systems to define the role of SHIP1 in AD.SHIP is known to be activated by the binding of its product, PI(3,4)P 2 , to an allosteric site on the enzyme near the C2 domain [26].While some SHIP1 activators (often referred to as enzyme agonists [26][27][28][29]) have demonstrated activity as anti-inflammatories, most suffer from poor bioavailability or low efficacy [13,[30][31][32].Recently we discovered a bis-sulfonamide (K306, 1, Figure 2) that has significant activity as a SHIP1 activator [14].K306 enhanced phagolysosomal degradation of synaptosomes and dead neurons in microglial models, which occurred in a SHIP1-dependent manner, revealing a new function for SHIP1 in the brain that could be exploited to enhance microglial function.The K306 structure is related to a series of sulfonamide-based endothelial lipase inhibitors [33].Sulfonamides have been used previously as phosphate mimics in the development of phosphatase inhibitors, however [34,35], so one of the sulfonamides in the K306 structure may mimic a phosphate on PI(3,4)P 2 , the endogenous ligand for allosteric activation of SHIP1 [36].Interestingly, K306 was shown to bind to a new (as yet undefined) allosteric site on the protein, as other activators lost activity when the C2 domain of the protein was deleted [36], yet K306 maintained agonism with this C2-less SHIP1 construct [13,14].
Molecules 2023, 28, x FOR PEER REVIEW 3 of 24 demonstrated activity as anti-inflammatories, most suffer from poor bioavailability or low efficacy [13,[30][31][32].Recently we discovered a bis-sulfonamide (K306, 1, Figure 2) that has significant activity as a SHIP1 activator [14].K306 enhanced phagolysosomal degradation of synaptosomes and dead neurons in microglial models, which occurred in a SHIP1-dependent manner, revealing a new function for SHIP1 in the brain that could be exploited to enhance microglial function.The K306 structure is related to a series of sulfonamidebased endothelial lipase inhibitors [33].Sulfonamides have been used previously as phosphate mimics in the development of phosphatase inhibitors, however [34,35], so one of the sulfonamides in the K306 structure may mimic a phosphate on PI (3,4)P2, the endogenous ligand for allosteric activation of SHIP1 [36].Interestingly, K306 was shown to bind to a new (as yet undefined) allosteric site on the protein, as other activators lost activity when the C2 domain of the protein was deleted [36], yet K306 maintained agonism with this C2-less SHIP1 construct [13,14].Initial in vivo evaluation of K306 in a murine model demonstrated a short half-life, requiring a multiple-dosing regimen to achieve anti-inflammatory responses [14].This may be due to rapid oxidation of the sulfides in K306 by cytochrome P450 (CYP) enzymes, which are prevalent in mice [37,38].Evaluation of the K306 structure by SwissADME [39] showed a high likelihood of oxidation, particularly of the sulfide in the thiomorpholine (Figure 2).A lower possibility of oxidation on the thiophene was noted, as the aromaticity of the system and steric effects slow these reactions.The thiophene is still a concern, however, as this heterocycle is known to undergo oxidation by CYP enzymes and can lead to toxic metabolites [40,41].
Given that compound 1 provides a platform for a new class of SHIP1 activators with a potentially new binding mode and interesting biological effects in AD models, synthetic studies were initiated with two primary objectives: (1) determine key structure-activity relationships and (2) find molecules less prone to oxidative degradation.In considering the K306 structure, the molecule may be readily divided into four distinct fragments: the sulfonamide fragment A, the aromatic linker fragment B, the amino acid fragment C, and a second sulfonamide fragment D (Figure 2).Studies to modify each of these fragments were undertaken.Once the most active constituent of each fragment was determined, hybrid molecules that could have even greater potency may be explored by combining the best subunits.

Results and Discussion
Initially, the sulfonamide of fragment A was varied to determine the necessity for the thiomorpholine and to verify that oxidation of the thiomorpholine sulfide was leading to inactivation.The synthesis of these analogs closely followed the reported synthesis of K306 [33], beginning with the formation of a sulfonamide between the known sulfonyl chloride 2 [42] and different amines (    Initial in vivo evaluation of K306 in a murine model demonstrated a short half-life, requiring a multiple-dosing regimen to achieve anti-inflammatory responses [14].This may be due to rapid oxidation of the sulfides in K306 by cytochrome P450 (CYP) enzymes, which are prevalent in mice [37,38].Evaluation of the K306 structure by SwissADME [39] showed a high likelihood of oxidation, particularly of the sulfide in the thiomorpholine (Figure 2).A lower possibility of oxidation on the thiophene was noted, as the aromaticity of the system and steric effects slow these reactions.The thiophene is still a concern, however, as this heterocycle is known to undergo oxidation by CYP enzymes and can lead to toxic metabolites [40,41].
Given that compound 1 provides a platform for a new class of SHIP1 activators with a potentially new binding mode and interesting biological effects in AD models, synthetic studies were initiated with two primary objectives: (1) determine key structure-activity relationships and (2) find molecules less prone to oxidative degradation.In considering the K306 structure, the molecule may be readily divided into four distinct fragments: the sulfonamide fragment A, the aromatic linker fragment B, the amino acid fragment C, and a second sulfonamide fragment D (Figure 2).Studies to modify each of these fragments were undertaken.Once the most active constituent of each fragment was determined, hybrid molecules that could have even greater potency may be explored by combining the best subunits.

Results and Discussion
Initially, the sulfonamide of fragment A was varied to determine the necessity for the thiomorpholine and to verify that oxidation of the thiomorpholine sulfide was leading to inactivation.The synthesis of these analogs closely followed the reported synthesis of K306 [33], beginning with the formation of a sulfonamide between the known sulfonyl chloride 2 [42] and different amines (Table 1).Reduction of the nitro group with iron powder in acetic acid then gave the corresponding aromatic amines 3-11.Amide formation with Boc-glycine mediated by HATU provided the carbamates 12-20.Removal of the Boc-protecting groups with TFA (which also produces amine 21) followed by sulfonamide formation with 4-fluorobenzenesulfonyl chloride gave the bis-sulfonamides 22-29.with Boc-glycine mediated by HATU provided the carbamates 12-20.Removal of the Bocprotecting groups with TFA (which also produces amine 21) followed by sulfonamide formation with 4-fluorobenzenesulfonyl chloride gave the bis-sulfonamides 22-29.

Entry
Aminothiophene Amide Bis-Sulfonamide Analog with Boc-glycine mediated by HATU provided the carbamates 12-20.Removal of the Bo protecting groups with TFA (which also produces amine 21) followed by sulfonami formation with 4-fluorobenzenesulfonyl chloride gave the bis-sulfonamides 22-29.with Boc-glycine mediated by HATU provided the carbamates 12-20.Removal of the Boc protecting groups with TFA (which also produces amine 21) followed by sulfonamid formation with 4-fluorobenzenesulfonyl chloride gave the bis-sulfonamides 22-29.

Entry
Aminothiophene Amide Bis-Sulfonamide Analog with Boc-glycine mediated by HATU provided the carbamates 12-20.Removal of the Bocprotecting groups with TFA (which also produces amine 21) followed by sulfonamide formation with 4-fluorobenzenesulfonyl chloride gave the bis-sulfonamides 22-29.with Boc-glycine mediated by HATU provided the carbamates 12-20.Removal of the Bo protecting groups with TFA (which also produces amine 21) followed by sulfonami formation with 4-fluorobenzenesulfonyl chloride gave the bis-sulfonamides 22-29.with Boc-glycine mediated by HATU provided the carbamates 12-20.Removal of the Bo protecting groups with TFA (which also produces amine 21) followed by sulfonamid formation with 4-fluorobenzenesulfonyl chloride gave the bis-sulfonamides 22-29.with Boc-glycine mediated by HATU provided the carbamates 12-20.Removal of the Bocprotecting groups with TFA (which also produces amine 21) followed by sulfonamide formation with 4-fluorobenzenesulfonyl chloride gave the bis-sulfonamides 22-29.with Boc-glycine mediated by HATU provided the carbamates 12-20.Removal of the Bo protecting groups with TFA (which also produces amine 21) followed by sulfonami formation with 4-fluorobenzenesulfonyl chloride gave the bis-sulfonamides 22-29.with Boc-glycine mediated by HATU provided the carbamates 12-20.Removal of the Bo protecting groups with TFA (which also produces amine 21) followed by sulfonamid formation with 4-fluorobenzenesulfonyl chloride gave the bis-sulfonamides 22-29.with Boc-glycine mediated by HATU provided the carbamates 12-20.Removal of the Bocprotecting groups with TFA (which also produces amine 21) followed by sulfonamide formation with 4-fluorobenzenesulfonyl chloride gave the bis-sulfonamides 22-29.

Entry
Aminothiophene Amide Bis-Sulfonamide Analog   While most of the analogs with variations in fragment A were easy to handle, t sulfone analog 23 proved difficult.During the final sulfonamide formation to access 2 an insoluble solid was formed and filtered from the reaction mixture.This solid was po tulated to be the sulfone 23 but was insoluble in all solvents (DMSO, acetone, methan chloroform, and water), so verification of the structure by NMR was not feasible.A com bustion analysis did match the formula for the desired product, but given the insolubili of this substance, it was not evaluated further.
The structure of the aryl group in fragment B of the parent structure was also varie The synthesis of these systems began with the requisite nitro-substituted sulfonyl chlori (30, 34, and 38, Scheme 1), which were then used in a sulfonamide formation with thi morpholine.Attempts to directly couple a glycine N-sulfonamide to the aromatic ami were unsuccessful, so completion of the molecules was accomplished in a stepwise fas While most of the analogs with variations in fragment A were easy to handle, th sulfone analog 23 proved difficult.During the final sulfonamide formation to access 2 an insoluble solid was formed and filtered from the reaction mixture.This solid was po tulated to be the sulfone 23 but was insoluble in all solvents (DMSO, acetone, methano chloroform, and water), so verification of the structure by NMR was not feasible.A com bustion analysis did match the formula for the desired product, but given the insolubilit of this substance, it was not evaluated further.
The structure of the aryl group in fragment B of the parent structure was also varied The synthesis of these systems began with the requisite nitro-substituted sulfonyl chlorid (30, 34, and 38, Scheme 1), which were then used in a sulfonamide formation with thio morpholine.Attempts to directly couple a glycine N-sulfonamide to the aromatic amin were unsuccessful, so completion of the molecules was accomplished in a stepwise fash While most of the analogs with variations in fragment A were easy to handle, the sulfone analog 23 proved difficult.During the final sulfonamide formation to access 23, an insoluble solid was formed and filtered from the reaction mixture.This solid was postulated to be the sulfone 23 but was insoluble in all solvents (DMSO, acetone, methanol, chloroform, and water), so verification of the structure by NMR was not feasible.A combustion analysis did match the formula for the desired product, but given the insolubility of this substance, it was not evaluated further.
The structure of the aryl group in fragment B of the parent structure was also varied.The synthesis of these systems began with the requisite nitro-substituted sulfonyl chloride (30, 34, and 38, Scheme 1), which were then used in a sulfonamide formation with thiomorpholine.Attempts to directly couple a glycine N-sulfonamide to the aromatic amine were unsuccessful, so completion of the molecules was accomplished in a stepwise fash- While most of the analogs with variations in fragment A were easy to handle, the sulfone analog 23 proved difficult.During the final sulfonamide formation to access 23, an insoluble solid was formed and filtered from the reaction mixture.This solid was postulated to be the sulfone 23 but was insoluble in all solvents (DMSO, acetone, methanol, chloroform, and water), so verification of the structure by NMR was not feasible.A combustion analysis did match the formula for the desired product, but given the insolubility of this substance, it was not evaluated further.
The structure of the aryl group in fragment B of the parent structure was also varied.The synthesis of these systems began with the requisite nitro-substituted sulfonyl chloride (30, 34, and 38, Scheme 1), which were then used in a sulfonamide formation with thiomorpholine.Attempts to directly couple a glycine N-sulfonamide to the aromatic amine were unsuccessful, so completion of the molecules was accomplished in a stepwise fashion.After addition of thiomorpholine to the sulfonyl chlorides, the nitro groups were reduced, and an amide coupling was performed with Boc-glycine.Deprotection of the Boc group and sulfonamide formation with 4-fluorobenzenesulfonyl chloride then gave analogs 33, 37, and 41.
Molecules 2023, 28, x FOR PEER REVIEW 5 (30,34, and 38, Scheme 1), which were then used in a sulfonamide formation with morpholine.Attempts to directly couple a glycine N-sulfonamide to the aromatic am were unsuccessful, so completion of the molecules was accomplished in a stepwise f ion.After addition of thiomorpholine to the sulfonyl chlorides, the nitro groups wer duced, and an amide coupling was performed with Boc-glycine.Deprotection of the group and sulfonamide formation with 4-fluorobenzenesulfonyl chloride then gave logs 33, 37, and 41.
Scheme 1. Synthesis of analogs varying fragment B of K306.
The amino acid fragment of the activator (fragment C) was then modified.This accomplished by using different amino acids as coupling partners following the gen pathway shown in Table 2 below.The amine 3 was coupled with a Boc-protected am acid utilizing HATU as the coupling agent.Removal of the Boc-protecting group was followed by sulfonamide formation with 4-fluorobenzenesulfonyl chloride.This led t synthesis of compounds 48-53.The amino acid fragment of the activator (fragment C) was then modified.This was accomplished by using different amino acids as coupling partners following the general pathway shown in Table 2 below.The amine 3 was coupled with a Boc-protected amino acid utilizing HATU as the coupling agent.Removal of the Boc-protecting group was then followed by sulfonamide formation with 4-fluorobenzenesulfonyl chloride.This led to the synthesis of compounds 48-53.

Entry Amino Acid
Additionally, analogs of K306 with different groups in fragment D were also synthesized starting from amine 21 (Table 3).Modification of this region of the molecule was accomplished by adding different electrophiles to the amine 21.A number of different groups on the sulfonamide were synthesized, leading to 64-71.These additions were not restricted to sulfonyl chlorides as coupling partners, with acid chlorides and chloroformates also being investigated, leading to the synthesis of 72 and 73.acid utilizing HATU as the coupling agent.Removal of the Boc-protecting group was then followed by sulfonamide formation with 4-fluorobenzenesulfonyl chloride.This led to the synthesis of compounds 48-53.Additionally, analogs of K306 with different groups in fragment D were also synthesized starting from amine 21 (Table 3).Modification of this region of the molecule was accomplished by adding different electrophiles to the amine 21.A number of different groups on the sulfonamide were synthesized, leading to 64-71.These additions were not restricted to sulfonyl chlorides as coupling partners, with acid chlorides and chloroformates also being investigated, leading to the synthesis of 72 and 73.acid utilizing HATU as the coupling agent.Removal of the Boc-protecting group was then followed by sulfonamide formation with 4-fluorobenzenesulfonyl chloride.This led to the synthesis of compounds 48-53.Additionally, analogs of K306 with different groups in fragment D were also synthesized starting from amine 21 (Table 3).Modification of this region of the molecule was accomplished by adding different electrophiles to the amine 21.A number of different groups on the sulfonamide were synthesized, leading to 64-71.These additions were not restricted to sulfonyl chlorides as coupling partners, with acid chlorides and chloroformates also being investigated, leading to the synthesis of 72 and 73.Additionally, analogs of K306 with different groups in fragment D were also synthesized starting from amine 21 (Table 3).Modification of this region of the molecule was accomplished by adding different electrophiles to the amine 21.A number of different groups on the sulfonamide were synthesized, leading to 64-71.These additions were not restricted to sulfonyl chlorides as coupling partners, with acid chlorides and chloroformates also being investigated, leading to the synthesis of 72 and 73.acid utilizing HATU as the coupling agent.Removal of the Boc-protecting group was then followed by sulfonamide formation with 4-fluorobenzenesulfonyl chloride.This led to the synthesis of compounds 48-53.Additionally, analogs of K306 with different groups in fragment D were also synthesized starting from amine 21 (Table 3).Modification of this region of the molecule was accomplished by adding different electrophiles to the amine 21.A number of different groups on the sulfonamide were synthesized, leading to 64-71.These additions were not restricted to sulfonyl chlorides as coupling partners, with acid chlorides and chloroformates also being investigated, leading to the synthesis of 72 and 73.Additionally, analogs of K306 with different groups in fragment D were also synthesized starting from amine 21 (Table 3).Modification of this region of the molecule was accomplished by adding different electrophiles to the amine 21.A number of different groups on the sulfonamide were synthesized, leading to 64-71.These additions were not restricted to sulfonyl chlorides as coupling partners, with acid chlorides and chloroformates also being investigated, leading to the synthesis of 72 and 73.Additionally, analogs of K306 with different groups in fragment D were also synthesized starting from amine 21 (Table 3).Modification of this region of the molecule was accomplished by adding different electrophiles to the amine 21.A number of different groups on the sulfonamide were synthesized, leading to 64-71.These additions were not restricted to sulfonyl chlorides as coupling partners, with acid chlorides and chloroformates also being investigated, leading to the synthesis of 72 and 73.Additionally, analogs of K306 with different groups in fragment D were also synthesized starting from amine 21 (Table 3).Modification of this region of the molecule was accomplished by adding different electrophiles to the amine 21.A number of different groups on the sulfonamide were synthesized, leading to 64-71.These additions were not restricted to sulfonyl chlorides as coupling partners, with acid chlorides and chloroformates also being investigated, leading to the synthesis of 72 and 73.Additionally, analogs of K306 with different groups in fragment D were also synthesized starting from amine 21 (Table 3).Modification of this region of the molecule was accomplished by adding different electrophiles to the amine 21.A number of different groups on the sulfonamide were synthesized, leading to 64-71.These additions were not restricted to sulfonyl chlorides as coupling partners, with acid chlorides and chloroformates also being investigated, leading to the synthesis of 72 and 73.Additionally, analogs of K306 with different groups in fragment D were also synthesized starting from amine 21 (Table 3).Modification of this region of the molecule was accomplished by adding different electrophiles to the amine 21.A number of different groups on the sulfonamide were synthesized, leading to 64-71.These additions were not restricted to sulfonyl chlorides as coupling partners, with acid chlorides and chloroformates also being investigated, leading to the synthesis of 72 and 73.Additionally, analogs of K306 with different groups in fragment D were also synthesized starting from amine 21 (Table 3).Modification of this region of the molecule was accomplished by adding different electrophiles to the amine 21.A number of different groups on the sulfonamide were synthesized, leading to 64-71.These additions were not restricted to sulfonyl chlorides as coupling partners, with acid chlorides and chloroformates also being investigated, leading to the synthesis of 72 and 73.Additionally, analogs of K306 with different groups in fragment D were also synthesized starting from amine 21 (Table 3).Modification of this region of the molecule was accomplished by adding different electrophiles to the amine 21.A number of different groups on the sulfonamide were synthesized, leading to 64-71.These additions were not restricted to sulfonyl chlorides as coupling partners, with acid chlorides and chloroformates also being investigated, leading to the synthesis of 72 and 73.Additionally, analogs of K306 with different groups in fragment D were also synthesized starting from amine 21 (Table 3).Modification of this region of the molecule was accomplished by adding different electrophiles to the amine 21.A number of different groups on the sulfonamide were synthesized, leading to 64-71.These additions were not restricted to sulfonyl chlorides as coupling partners, with acid chlorides and chloroformates also being investigated, leading to the synthesis of 72 and 73.Additionally, analogs of K306 with different groups in fragment D were also synthesized starting from amine 21 (Table 3).Modification of this region of the molecule was accomplished by adding different electrophiles to the amine 21.A number of different groups on the sulfonamide were synthesized, leading to 64-71.These additions were not restricted to sulfonyl chlorides as coupling partners, with acid chlorides and chloroformates also being investigated, leading to the synthesis of 72 and 73.

31%
Evaluation of the effects of these analogs on SHIP1 utilizing the malachite green assay for phosphatase activity showed some interesting trends in reactivity (Figure 3).The malachite green assay is based on the quantification of the green complex formed between malachite green dye, molybdate, and free orthophosphate under acidic conditions [43].The rapid color formation from this reaction can be conveniently measured and quantified at 620 nm on a plate reader.As the malachite reaction requires free phosphate, the quantity of free phosphate generated from a substrate over a short time (in this case 20 min) can be measured and directly related to phosphatase activity.For in vitro assays with SHIP, PI(3,4,5)P 3 -diC8 [44] is used as the substrate, as the endogenous PI(3,4,5)P 3 aggregates without a lipid membrane, and is not recognized by SHIP.The malachite results showed that analogs that were truncated and missing fragment D sulfonamide or the amino acid and sulfonamide fragments (3 and 21) were less effective SHIP1 activators than K306.Sulfoxide 22, where the thiomorpholine sulfide has been oxidized to a sulfoxide, also showed no SHIP1 activation, which is consistent with oxidation of the sulfur being a problem that needs to be addressed in this class of SHIP1 activators.Interestingly sulfoxide 22 showed inhibition of SHIP1.As the endogenous ligand of allosteric activation of SHIP1 is the product PI(3,4)P 2 , and this is similar in structure to PI(3,4,5)P 3 , the active site and the allosteric binding site may share recognition elements that allow small structural changes to switch the role of the molecule from activator to inhibitor.Replacement of the thiomorpholine with a morpholine or a pyrrolidine (24 and 25) also resulted in a loss of activity.The use of a more lipophilic piperidine did provide an active analog (26) that was more active than K306, however.Acyclic systems in fragment A were also explored with analogs 27-29.A rough trend that favored the less polar derivatives was noted with these analogs, with the butylamine analog 29 being the most active.This may indicate that this fragment of the inhibitor is binding in a nonpolar region of the protein.
thiomorpholine with a morpholine or a pyrrolidine (24 and 25) also resulted in a loss of activity.The use of a more lipophilic piperidine did provide an active analog (26) that was more active than K306, however.Acyclic systems in fragment A were also explored with analogs 27-29.A rough trend that favored the less polar derivatives was noted with these analogs, with the butylamine analog 29 being the most active.This may indicate that this fragment of the inhibitor is binding in a nonpolar region of the protein.The analogs with different aromatic spacer groups (fragment B, compounds 33, 37, and 41) were then evaluated.Replacing the 2-methylthiophene with a 2-chlorothiophene gave analog 33, which was nearly equipotent as the parent K306 structure.The 2-chlorothiophene that was chosen as chlorine may slow oxidation of the thiophene sulfur due to steric and electronic effects.Attempts were also made to replace the thiophene ring with a more stable benzene ring.This led to the synthesis of benzenesulfonamides 37 and 41.Some activity was maintained with a phenyl ring when the sulfonamide and the amide were in a 1,4-orientation (sulfonamide 41); however, the 1,3-orientation (compound 40) was significantly less active.
Evaluation of analogs with changes in amino acid fragment (fragment C) was then undertaken (compounds 48-53, Figure 3).Analogs employing both enantiomers of alanine and phenylalanine were first investigated, with the results appearing to indicate that the presence of an (S) stereocenter led to compounds with better activator activity than the (R) enantiomers.Analogs bearing a phenylglycine and a proline in fragment C showed significantly improved activator activity compared to the parent compound.
Modification of the second sulfonamide fragment (fragment D) was then investigated.Deletion of the fluorine (as in 64) or incorporation of a less polar group on the benzenesulfonamide (as in 65 and 66) led to a loss of activator activity.Switching to the more polar 4-nitro-substituted benzenesulfonamide 67 gave an increase in the activity, so the 3-nitro and 2-nitro sulfonamides were also synthesized and evaluated.While the 3-nitrobenzenesulfonamide analog 68 was disappointing, the 2-nitrobenzenesulfonamide 69 was quite active.While the activity was promising, nitro groups can be problematic in the development of bioactive small molecules, as nitroreductases often will reduce the nitro group to an electrophilic nitroso derivative, which can have mutagenic effects and exhibit hepatotoxicity [45].While the nitro group in 69 was relatively hindered, this was still a concern, so some other bioisosteres for a nitro group were then evaluated beginning with the trifluoromethyl group [46] in 70, which had also showed good activator activity.An ester has also been used as a nitro bioisostere [47], but incorporation of the ester next to the sulfonamide led to the spontaneous cyclization of the sulfonamide nitrogen to form the benzoic sulfimide 71.Sulfimide 71 displayed promising SHIP1 activator activity in the malachite green assay, nearly as potent as 69, demonstrating that a polar group in this position is beneficial for bioactivity.Other functional groups in lieu of the sulfonamide in fragment D including the benzamide 72 and the carbamate 73 have little effect on SHIP activity.

General Experimental Information
All anhydrous reactions were run under a positive pressure of argon.Dichloromethane (DCM) was dried by passage through an alumina column.1,2-Dichloroethane (DCE), diisopropylethylamine (DIPEA), triethylamine (TEA), and dimethylacetamide (DMA) were freshly distilled from calcium hydride before use.Tetrahydrofuran (THF) was freshly distilled from Na/benzophenone still before use.Ethyl acetate (EA) and hexanes were purchased from commercial sources and used as received.Silica gel column chromatography was performed using 60 Å silica gel (230-400 mesh).Copies of 1 H NMR and 13 C NMR spectra are available online in the supplementary materials.

General Procedure for the Synthesis of Sulfonamides 3-11
The amine (2 equiv) and 5-methyl-4-nitrothiophene-2-sulfonyl chloride (3) (1 equiv) were dissolved in 1,4-dioxane (1.0 M) and heated to 60 • C. The mixture was stirred for 1 h at 60 • C, after which the reaction was cooled to rt and 20 mL of water was added.The mixture was extracted with DCM (3 × 20 mL), and the organics were dried over MgSO 4 , filtered, and concentrated.The crude mixture was purified via silica gel chromatography, yielding the nitrothiophene intermediate.This nitrothiophene was dissolved in acetic acid (0.3 M) and iron power (5 equiv) was added.The mixture was heated to 60 • C and stirred for 1.5 h followed by removal of acetic acid in vacuo.The residue was taken up in EA and the iron powder was filtered away.The solution was washed with saturated NaHCO 3 until a pH of 8 was reached.The organic layer was then dried over Na 2 SO 4 , filtered, and concentrated.The residue was purified via silica gel chromatography, yielding the desired amine.

General Procedure for the Synthesis of Sulfonamides 22-29
The Boc-protected amine was dissolved in TFA (0.5 M) and stirred at rt for 0.5 h.The solvent was then removed in vacuo to provide the amine TFA salt.This amine salt was dissolved in dry DCM (0.7 M) and dry TEA was added (2.2 equiv).The desired sulfonyl chloride or other electrophile (1.1 equiv) was then added and the mixture was stirred at rt for 24 h under argon.The reaction was then taken up in EA and washed with H 2 O and 5% HCl.The organic layer was then separated, dried over Na 2 SO 4 , filtered, and concentrated.The residue was then purified by trituration from DCM or by silica gel chromatography, yielding the pure product.

Conclusions
The K306 bis-sulfonamide structure was modified to investigate structure-activity relationships.The thiomorpholine subunit is shown to be a potential liability, as its oxidation leads to inactive or insoluble analogs.Replacement of the thiomorpholine with a piperidine or a butyl sulfonamide provides structures that also act as SHIP1 activators without this problem.Substitutions in the aromatic fragment of K306 (fragment B) show that there is some tolerance for change in this area, with a 2-chlorothiophene and a 1,4disubstituted benzene ring able to take the place of the thiophene.Substitution of the glycine for a proline or phenylglycine in the amino acid fragment of the molecule (fragment C) appears to provide more potent activators.The 4-fluorobenzenesulfonamide in fragment D can also be modified, with the incorporation of electron-withdrawing groups on this benzensulfonamide being beneficial to the activator activity.The results of this study are now being applied to the development of new SHIP activators with improved potency and bioavailability.In the future, we hope to use these molecules to define the allosteric binding site where the binding of small molecules to SHIP1 leads to increased enzyme activity.In addition, we plan to evaluate the anti-inflammatory properties of these molecules in model systems of a number of disease states, including Alzheimer's disease.

Figure 2 .
Figure 2. Analysis of K306 structure and potential CYP oxidation sites.

Figure 2 .
Figure 2. Analysis of K306 structure and potential CYP oxidation sites.

9 a
Amine 21 is the product of the TFA deprotection and was isolated before formation of the sulfo amide.b The sulfone 23 was insoluble (see text).

9 a
Molecules 2023, 28, x FOR PEER REVIEW 5 of Amine 21 is the product of the TFA deprotection and was isolated before formation of the sulfon amide.b The sulfone 23 was insoluble (see text).

25 a
Molecules 2023, 28, x FOR PEER REVIEW 5 of Amine 21 is the product of the TFA deprotection and was isolated before formation of the sulfonamide.b The sulfone 23 was insoluble (see text).

a
Amine 21 is the product of the TFA deprotection and was isolated before formation of the sulfonamide.b The sulfone 23 was insoluble (see text).

Scheme 1 .
Scheme 1. Synthesis of analogs varying fragment B of K306.
as the coupling agent.Removal of the Boc-protecting group was then followed by sulfonamide formation with 4-fluorobenzenesulfonyl chloride.This led to the synthesis of compounds 48-53.
as the coupling agent.Removal of the Boc-protecting group was then followed by sulfonamide formation with 4-fluorobenzenesulfonyl chloride.This led to the synthesis of compounds 48-53.
formation with 4-fluorobenzenesulfonyl chloride.This led to the synthesis of compounds 48-53.
formation with 4-fluorobenzenesulfonyl chloride.This led to the synthesis of compounds 48-53.

Figure 3 .
Figure 3. Evaluation of SHIP1 Activation in the Malachite Green Assay.Percentage of phosphatase activity for SHIP1 and SHIP2 enzyme as determined in the malachite green assay as compared to a vehicle control (veh, DMSO) used as 100% activity.The assay was performed at 250 µM concentration for the small molecule activator with 100 µM PI(3,4,5)P 3 -diC8 as the substrate.Results are the mean of at least three trials.Values >100% are due to activation, while values <100% show inhibition of the enzyme.

Table
). Reduction of the nitro group with iron powder in acetic acid then gave the corresponding aromatic amines 3-11.Amide formation

Table 1 .
Synthesis of K306 Analogs Varying Fragment A.

Table 1 .
Synthesis of K306 Analogs Varying Fragment A.

Table 1 .
Synthesis of K306 Analogs Varying Fragment A.

Table 1 .
Synthesis of K306 Analogs Varying Fragment A.

Table 1 .
Synthesis of K306 Analogs Varying Fragment A.

Table 1 .
Synthesis of K306 Analogs Varying Fragment A.

Table 1 .
Synthesis of K306 Analogs Varying Fragment A.

Table 1 .
Synthesis of K306 Analogs Varying Fragment A.

Table 1 .
Synthesis of K306 Analogs Varying Fragment A.

Table 1 .
Synthesis of K306 Analogs Varying Fragment A.

Table 1 .
Synthesis of K306 Analogs Varying Fragment A.

Table 1 .
Synthesis of K306 Analogs Varying Fragment A.

Table 1 .
Synthesis of K306 Analogs Varying Fragment A.

Table 1 .
Synthesis of K306 Analogs Varying Fragment A.

Table 1 .
Synthesis of K306 Analogs Varying Fragment A.

Table 1 .
Synthesis of K306 Analogs Varying Fragment A.

Table 1 .
Synthesis of K306 Analogs Varying Fragment A.

Table 1 .
Synthesis of K306 Analogs Varying Fragment A.

Table 1 .
Synthesis of K306 Analogs Varying Fragment A.

Table 1 .
Synthesis of K306 Analogs Varying Fragment A.

Table 2 .
Synthesis of K306 Analogs Varying Fragment C.

Table 2 .
Synthesis of K306 Analogs Varying Fragment C.

Table 2 .
Synthesis of K306 Analogs Varying Fragment C.
a Isolated yield over 3 steps.

Table 3 .
Synthesis of K306 Analogs Varying Fragment D.

Table 2 .
Synthesis of K306 Analogs Varying Fragment C.
a Isolated yield over 3 steps.

Table 3 .
Synthesis of K306 Analogs Varying Fragment D.

Table 2 .
Synthesis of K306 Analogs Varying Fragment C.
a Isolated yield over 3 steps.

Table 3 .
Synthesis of K306 Analogs Varying Fragment D.

Table 2 .
Synthesis of K306 Analogs Varying Fragment C.
a Isolated yield over 3 steps.

Table 3 .
Synthesis of K306 Analogs Varying Fragment D.

Table 2 .
Synthesis of K306 Analogs Varying Fragment C.
a Isolated yield over 3 steps.

Table 3 .
Synthesis of K306 Analogs Varying Fragment D.

Table 2 .
Synthesis of K306 Analogs Varying Fragment C.
a Isolated yield over 3 steps.

Table 3 .
Synthesis of K306 Analogs Varying Fragment D.

Table 2 .
Synthesis of K306 Analogs Varying Fragment C.
a Isolated yield over 3 steps.

Table 3 .
Synthesis of K306 Analogs Varying Fragment D.

Table 2 .
Synthesis of K306 Analogs Varying Fragment C.
a Isolated yield over 3 steps.

Table 3 .
Synthesis of K306 Analogs Varying Fragment D.

Table 2 .
Synthesis of K306 Analogs Varying Fragment C.
a Isolated yield over 3 steps.

Table 3 .
Synthesis of K306 Analogs Varying Fragment D.

Table 2 .
Synthesis of K306 Analogs Varying Fragment C.
a Isolated yield over 3 steps.

Table 3 .
Synthesis of K306 Analogs Varying Fragment D.

Table 2 .
Synthesis of K306 Analogs Varying Fragment C.
a Isolated yield over 3 steps.

Table 3 .
Synthesis of K306 Analogs Varying Fragment D.

Table 3 .
Synthesis of K306 Analogs Varying Fragment D.

Table 3 .
Synthesis of K306 Analogs Varying Fragment D.

Table 3 .
Synthesis of K306 Analogs Varying Fragment D.