Synthesis and Characterization of Fatty Acid Conjugates of Niacin and

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Synthesis and Characterization of Fatty Acid Conjugates of Niacin and Salicylic Acid Chi B Vu, Jean E Bemis, Ericka Benson, Pradeep Bista, David Carney, Richard Fahrner, Diana Lee, Feng Liu, Pallavi Lonkar, Jill C Milne, Andrew J Nichols, Dominic Picarella, Adam Shoelson, Jesse Smith, Amal Ting, Allison Wensley, Maisy Yeager, Michael Zimmer, and Michael R Jirousek J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01961 • Publication Date (Web): 19 Jan 2016 Downloaded from http://pubs.acs.org on January 25, 2016

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Journal of Medicinal Chemistry

Synthesis and Characterization of Fatty Acid Conjugates of Niacin and Salicylic Acid Chi B. Vu,* Jean E. Bemis, Ericka Benson, Pradeep Bista, David Carney, Richard Fahrner, Diana Lee, Feng Liu, Pallavi Lonkar, Jill C. Milne, Andrew J. Nichols, Dominic Picarella, Adam Shoelson, Jesse Smith, Amal Ting, Allison Wensley, Maisy Yeager, Michael Zimmer and Michael R. Jirousek. Catabasis Pharmaceuticals, One Kendall Square, Suite B14202, Cambridge, MA 02139. KEYWORDS: niacin, omega-3 fatty acid, EPA, fatty acid niacin conjugate, SREBP, salicylic acid, DHA, fatty acid salicylate conjugate, NF-κB.

ABSTRACT: This report describes the synthesis and preliminary biological characterization of novel fatty acid niacin conjugates and fatty acid salicylate conjugates. These molecular entities were created by covalently linking two bioactive molecules – either niacin or salicylic acid to an omega-3 fatty acid. This methodology allows the simultaneous intracellular delivery of two bioactives in order to elicit a pharmacological response that could not be replicated by administering the bioactives individually or in combination. The fatty acid niacin conjugate 5 has been shown to be an inhibitor of the sterol regulatory element binding protein (SREBP), a key regulator of cholesterol metabolism proteins such as PCSK9, HMG-CoA reductase, ATP

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citrate lyase and NPC1L1. On the other hand, the fatty acid salicylate conjugate 11 has been shown to have a unique anti-inflammatory profile based on its ability to modulate the NF-κB pathway through the intracellular release of the two bioactives.

INTRODUCTION In recent years, the traditional drug discovery paradigm of optimizing a lead candidate toward a single therapeutic target has been challenged by the successful implementation of a number of cases involving polypharmacology. This approach, sometimes referred to as network or pathway pharmacology,1,2 involves the targeting of multiple therapeutic targets in order to achieve a desired pharmacological outcome. Imatinib, for instance, was successfully developed as a kinase inhibitor of BCR-ABL, c-KIT, and PDGFRs for the treatment of chronic myeloid leukemia.3 Since then, numerous other kinase inhibitors have been introduced against a vast array of therapeutic targets.4 With these kinase inhibitors, pathway pharmacology was used to rationally target a small subset of kinases known to be up-regulated in certain forms of cancer. Outside of the oncology field, pathway pharmacology has also been used in the design of the anti-psychotic agent ziprasidone and the bronchodilator GSK 961081. With ziprasidone, two small pharmacophores were merged into one single molecular entity in order to modulate multiple 5HT and dopamine receptors (5-HT2A, 5-HT2c, 5-HT6, 5-HT7, α1, D2 and D3 receptors).5 With GSK 961081, a non-hydrolysable linker was used to covalently join a muscarinic antagonist with a β2 receptor agonist in order to modulate two distinct GPCR targets.6 In this report, we describe a novel method of exploring pathway pharmacology involving the simultaneous intracellular delivery of two bioactives. With this methodology, the two bioactives are first joined covalently


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by the use of a plasma-stable linker. This process can provide a high level of extracellular selectivity since the two molecules have been rendered essentially inactive by the covalent linking process. However, once delivered inside cells, intracellular enzymes degrade the linker and thereby release the two bioactives, allowing them to exert their effect on the different therapeutic pathways or targets. Our conjugation methodology allows us to gain synergy by targeting multiple pathways in the same cells at the same time. Furthermore, it enables us to solve a difficult problem that arises with the simple co-administration of any two bioactive molecules, namely, the different PK and tissue distribution profile of the two bioactives. When the two bioactive molecules are covalently linked, however, they are delivered inside cells at the same time in equimolar concentrations. What we have observed is that the pharmacological effect that could be produced by the simultaneous delivery of the bioactives is unique and cannot not be replicated by administering the individual components or a combination of the individual components. We will demonstrate the utility of our methodology with two examples. The first example involves the use of fatty acid niacin conjugates in the area of lipid synthesis; and the second example involves the use of fatty acid salicylate conjugates in the area of NF-κB inhibition to counteract inflammation.

RESULTS AND DISCUSSION Fatty acid niacin conjugates are molecular entities created by covalently linking niacin to an omega-3 fatty acid, namely (5Z,8Z,11Z,14Z,17Z)-eicosa-5,8,11,14,17-pentaenoic acid (EPA). Both niacin7-9 and EPA10 are well-known lipid lowering agents. EPA, in particular, has been shown to inhibit sterol regulatory element binding proteins (SREBPs) activity in a PPARα-


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independent manner.11,12 The SREBP pathway, in turn, is critical in modulating fatty acid synthesis and maintaining cholesterol homeostasis.13 SREBP-1 and SREBP-2 are the two different forms of SREBP proteins. SREBP-1 is known to regulate production of genes involved in fatty acid synthesis and oxidation such as FASN and SCD-1.14 SREBP-2, on the other hand, controls expression of genes involved in cholesterol homeostasis, which include HMG-CoA reductase, NPC1L1 and PCSK9.15 We hypothesized that if both niacin and EPA could be delivered together inside cells, a number of lipid synthesis pathways could be simultaneously affected to produce a unique biological response. In order to achieve simultaneous delivery to cells, the two drugs need to be covalently linked to form a single molecular conjugate. The resulting molecular conjugate also needs to be sufficiently stable in the plasma to remain intact during systemic circulation. Once delivered inside targeted cells, however, intracellular enzymes hydrolyze this molecular conjugate down to the individual components. As a fatty acid, when EPA is covalently linked to other moieties via an amide or ester bond, those bonds could be processed by intracellular enzymes such as fatty acid amide hydrolase (FAAH),16 Nacylethanolamine hydrolyzing acid amidase (NAAA),17 or monacylglycerol lipase (MAGL).18 Figure 1 lists the five possible fatty acid niacin conjugates (1-5), each with a different linker that enables the covalent linkage of niacin to EPA. Compounds 1-4 were prepared by the straightforward steps outlined in the Supporting Information Section. The diamide derivative 5 was prepared according to the general Scheme 1. Nicotinic acid was reacted with tert-butyl (2aminoethyl)carbamate in the presence of EDC/HOBT and Et3N to afford tert-butyl (2(nicotinamido)ethyl)carbamate. Upon treatment with HCl in dioxane, the HCl salt of N-(2aminoethyl)nicotinamide was obtained. This was then coupled with the fatty acid EPA using EDC/HOBT and Et3N to afford compound 5. This general Scheme 1 was also used to prepare a


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number of analogs that are pertinent to this report, namely the fatty acid niacin conjugates 7-10 and the fatty acid salicylate conjugates 11 and 12. Compounds 1-5 were first assessed for plasma stability. As shown in Figure 2, the diamide derivative 5 was the only analog among the 5 tested that showed complete plasma stability across all 4 species. Even at the two hour time point, essentially all of the parent compound was still present. We then investigated the cellular uptake of these fatty acid niacin conjugates using confocal microscopy. In order to facilitate the imaging process, we replaced the omega-3 fatty acid EPA in 5 with the fluorescent fatty acid cis-parinaric acid in order to form the corresponding fatty acid niacin conjugate 10.19 HuH-7 cells were treated with either cis-parinaric acid (25 µM) or the fluorescent fatty acid niacin conjugate 10 (25 µM) for 30 min, fixed with 4% formaldehyde and mounted with anti-fade DAPI. Figure 3A shows that the free fatty acid (i.e. cis-parinaric acid) does get into cells; but it is highly concentrated in the lysosomes, and is not distributed to the endoplasmic reticulum (ER). In sharp contrast, the fatty acid niacin conjugate 10 exhibits an ER-like (peri-nuclear) pattern, with less distribution to the lysosomes (Figure 3B). Endocytosis is believed to be the cellular uptake mechanism for these fatty acid niacin conjugates. A pre-incubation experiment with Dynasore, a known endocytosis inhibitor, was carried out. As shown in Figure 3C, treatment with 80 µM of Dynasore abolished the ER cellular localization of compound 10, indicating that these fatty acid niacin conjugates were taken up by cells by active endocytosis, likely via clathrin-coated vesicles.20 This uptake mechanism for the fatty acid niacin conjugates is distinct from what has been reported with free fatty acids. When unsaturated omega-3 fatty acids, such as EPA, are applied to cells, they are usually incorporated first into the phospholipids and then released intracellularly by the ratelimiting action of phospholipase A1 (PLA1) and phospholipase A2 (PLA2).21 Since the fatty


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acid niacin conjugates are taken up inside cells by active endocytosis, their intracellular distribution to the ER is different; and so is the biological response that they could elicit. Distribution of the fatty acid niacin conjugate to the ER also allows it to be localized in the same subcellular compartment as the enzyme FAAH.22 This would allow FAAH to hydrolyze the fatty acid niacin conjugate and release the individual components. The fatty acid niacin conjugate 5 was then evaluated in a number of assays to see if it could be hydrolyzed in targeted tissues down to the individual components. The rat liver lysate experiment was carried out initially to examine the hydrolysis of compound 5. This hydrolysis experiment was carried out by incubating compound 5 in rat liver lysate at 37 °C and monitored for the disappearance of the starting material. As shown in Figure 4, there was a time-dependent hydrolysis of compound 5 in this assay. Furthermore, this hydrolysis is FAAH-dependent since addition of the FAAH inhibitor PF-384523 almost completely abrogated the hydrolysis of compound 5. In humans, two forms of FAAH, FAAH1 and FAAH2, share 20% sequence homology and have different tissue distribution profiles.24 We expressed and purified recombinant FAAH1 enzyme to determine its role in hydrolysis of compound 5. As shown in Figure 5, recombinant FAAH1 hydrolyzed the amide bond between the fatty acid and the linker and released EPA in a time-dependent fashion. Again, the addition of the FAAH inhibitor PF-3845 also inhibited this particular hydrolysis. The hydrolysis of compound 5 and the formation of intracellular EPA were further confirmed in a cell-based assay using HepG2 cells. As shown in Figure 6, incubation of compound 5 in HepG2 cells resulted in a time-dependent hydrolysis. In this cell line, formation of intracellular EPA was monitored by using a deuterium-labeled EPA-d5, in order to distinguish the fatty acid that was being released from compound 5 over the endogenous EPA. Compound 9, the EPA-d5 version of compound 5, was prepared according to the same procedure outlined previously in


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Scheme 1 using the appropriate deuterium-labeled fatty acid. The requisite EPA-d5 was prepared by total synthesis using the reaction sequence outlined by Sandri and Viala.25 As shown in Figure 7, there was a time-dependent rate of formation of EPA-d5 during the hydrolysis of compound 9 in human HepG2 cell lines. The assays described above represent some initial steps aimed at understanding the hydrolysis of compound 5. The actual hydrolysis of compound 5 in an in vivo setting is most likely much more complex due to the large arrays of amidases and hydrolases that are available across various tissues and species. Thus, it is more appropriate to evaluate the oral bioavailability, tissue distribution and hydrolysis of compound 5 in an actual animal model. When niacin is administered to rodents, dogs, monkeys or humans, one of the major plasma metabolites is nicotinuric acid.26 In a typical PK experiment where niacin was administered as a single oral dose of 30 mg/kg to Sprague Dawley rats (plasma collection at t = 0.25, 0.5, 1, 2, 4 and 6 hr) the Cmax and AUClast of niacin were 7467 ± 2246 ng/mL and 6980 ± 3173 hr*ng/mL respectively. In the same PK experiment, the Cmax and AUClast of nicotinuric acid were 369 ± 80 ng/mL and 466 ± 359 hr*ng/mL respectively. Both niacin and its nicotinuric acid metabolite were cleared fairly rapidly and neither one was detected in the plasma after 2 hours. When the fatty acid niacin conjugate 5 was dosed at 100 mg/kg po (i.e. the same molar equivalent as 30 mg/kg of niacin) a low level of the parent compound was detected in the plasma (for compound 5, the Cmax and AUClast were 212 ± 74 ng/mL and 172 ± 67 hr*ng/mL respectively). Interestingly, even though niacin was not detected in the plasma, the molar equivalent dose of compound 5 produced much higher plasma levels of nicotinuric acid (Cmax and AUClast were 1131 ± 306 ng/mL and 3484 ± 1013 hr*ng/mL respectively). Furthermore, this type of fatty acid niacin conjugate allowed the delivery of nicotinuric acid over a much longer period; at the 6 hour time point, a significant


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level of nicotinuric acid could still be detected in the plasma (>200 ng/mL). Compound 5 was then evaluated in a PK/tissue distribution study in order to further verify that the fatty acid niacin conjugate could be hydrolyzed down to the individual components in targeted tissues. Plasma and tissue samples were processed and analyzed by LC/MS/MS for the presence of 1) the parent compound, 2) the niacin linker metabolite 13, 3) the linker-EPA metabolite 17, 4) niacin and 5) nicotinuric acid 16 (Figure 8). As shown in Table 1, low concentration of the parent compound 5 was detected in the plasma and liver. A significant quantity of the parent compound was, however, present in the intestine (6,747 ng/g). The desired hydrolysis of compound 5 did indeed take place and the key nicotinuric acid metabolite was detected not only in the plasma, but also in both tissues (nicotinuric acid liver concentration = 1356 ng/g; nicotinuric acid intestine concentration = 1387 ng/g). The hydrolysis of the amide bond between EPA and the linker portion also occurred in targeted tissues and the niacin linker metabolite was detected not only in plasma but also in both tissues (liver concentration of niacin linker metabolite = 10,910 ng/g; intestine concentration of niacin linker metabolite = 23,899 ng/g). Lower concentration of the linker EPA metabolite was detected in the plasma and the two isolated tissues. Even though niacin itself was not detected in the plasma and liver tissue, it was detected in intestinal tissues (concentration = 279 ng/g). This tissue distribution study showed that compound 5 was extensively hydrolyzed in the intestine and a lower level of the intact parent compound was delivered to the liver. Changing the nature of the linker group could also change the rate of hydrolysis of the corresponding fatty acid niacin conjugate and its tissue distribution considerably. In compounds 7 and 8, the ethylenediamine linker has been replaced with (S)pyrrolidin-3-amine and N1-methylethane-1,2-diamine respectively. This substitution should render the amide bond between the diamine linker and the fatty acid more resistant to hydrolysis


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by FAAH. Figure 9 shows the hydrolysis of compounds 5, 7 and 8 using the recombinant FAAH-1. Compound 7 was more resistant to FAAH-1 hydrolysis, when compared to compound 5. For compound 8, there was essentially no hydrolysis of the fatty acid amide bond under the assay conditions. Since the fatty acid conjugates 7 and 8 are more resistant to hydrolysis, more of the intact parent compound should be delivered to the liver. PK studies using doublecannulated rats were carried out to compare the amount of parent compound that could potentially be delivered to the liver. For these PK studies, Sprague Dawley rats that have been surgically implanted with indwelling jugular vein cannula (JVC) and portal vein cannula (PVC) were used; and serial blood collection was carried out at both the portal and jugular vein (t = 10, 20, 40 min and 1, 2, 4 and 6 hrs post dose). Table 2 summarizes selected PK parameters for compounds 5, 7 and 8 in this double cannulated rat PK study.27 Consistent with the tissue distribution study, we saw that compound 5 was extensively hydrolyzed in the intestine, as indicated by the higher portal concentration of the niacin-linker metabolite 13 (Cmax = 2,061 ± 433 ng/mL) and a lower portal concentration of the parent compound (Cmax = 418 ± 122 ng/mL). On the other hand, with the more hydrolysis-resistant compound 7 and 8, we observed less metabolism of the parent compound in the intestine; and as a result, a higher portal concentration of the parent compound was observed along with a lower portal concentration of the niacin linker metabolite. Compared to compound 5, a higher level of the parent compound was also observed in the peripheral circulation for the more hydrolysis-resistant compounds 7 and 8 (peripheral Cmax = 564 ± 226 and 400 ± 100 ng/mL respectively). Compound 8 was then evaluated in a rat tissue distribution study where animals (n = 6) were dosed with 100 mg/kg q.d. for 3 days. Four hours after the last dose, plasma and tissues were collected and analyzed for the parent compound 8 along with the metabolites 15, 16, and 19.28 Even though compound 8 was


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dosed at a lower dose of 100 mg/kg, there was a significantly greater concentration of the corresponding parent compound (> 60 fold increase) in the liver, when compared with compound 5 (Figure 10). This result demonstrated the dramatic shift in the tissue distribution of the intact parent compound upon decreasing the rate of hydrolysis of the amide bond between the fatty acid EPA and the linker. Since the components are only released intracellularly with the fatty acid niacin conjugate 5, our approach could potentially allow one to avoid the unpleasant flushing side effect of niacin. It is widely known that niacin is capable of binding to and activating GPR109A, a Gi-coupled GPCR. This process, in turn, promotes the release of prostaglandin D2 (PGD2) from skin cells. This eicosanoid is a vasodilator that is responsible for the cutaneous flushing. Our analyses demonstrated that the fatty acid niacin conjugate 5 did not interact with the GPR109A receptor. Interaction of conjugate 5 with the GPR109A receptor, was tested using an adenylate cyclase inhibition assay in human epidermal A431 cell line that expressed GPR109A.29 One of the activities of niacin binding to GPR109A is the inhibition of adenylate cyclase. Adenylate cyclase converts ATP to cAMP and this activity is stimulated by forskolin. As shown in Figure 11, niacin inhibited forskolin-mediated cAMP production in a dose-dependent manner with an EC50 of 0.73 µM. In this assay, niacin was able to inhibit approximately 50% of the cAMP signal produced by forskolin. In sharp contrast, compound 5 did not functionally activate GPR109A. No significant inhibition of cAMP production was observed when these epidermal cells were incubated with compound 5 over the same dose range. In essence, the molecules that are being covalently linked have been rendered inactive in the extracellular space. Covalently linking niacin to a fatty acid moiety using the plasma stable linker described above thus represents a potential new way of delivering the niacin component without inducing the flushing side effects.


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After having confirmed that the fatty acid niacin conjugate 5 could undergo the appropriate hydrolysis to afford the desired components in targeted tissues, we then focused our attention on the biological effect that it could induce. The SREBP proteins have been known to be essential for cholesterol biosynthesis as well as for the uptake and biosynthesis of various fatty acids. HMG-CoA reductase and PCSK9 are two of the target genes that are known to be regulated by the mature form of the SREBP-2. Both EPA and niacin, at very high concentration, could affect SREBP-2 activity.11,12 At the lower concentration of 50 μM, however, both EPA and niacin, showed little activity on SREBP-2 maturation when they were incubated in human HepG2 cells. In sharp contrast, when the fatty acid niacin conjugate 5 was incubated in human HepG2 cells, there was a decrease in the production of mature SREBP-2 protein. The effect was dose dependent and the decrease in SREBP-2 protein could be observed at the 25 and 50 μM concentration (Figure 12). Statins have previously been shown to increase plasma level of PCSK9 through its effect on LDL levels and SREBP-2.30-32 In this cell assay, the effect of administering atorvastatin on SREBP-2 was also quite pronounced. As shown in Figure 12, when human HepG2 cells were incubated with 10 μM of atorvastatin, there was a significant increase in the production of mature SREBP-2 protein. However, treatment of human HepG2 cells with a combination of 10 μM of atorvastatin with 25 μM of the fatty acid niacin conjugate 5 resulted in a significant decrease in the production of mature SREBP-2 protein. Since PCSK9 is an immediate downstream gene of SREBP-2, a decrease in the production of the SREBP-2 protein would naturally lead to a decrease in the expression of PCSK9. A dose-dependent decrease in the expression of PCSK9 mRNA levels in HepG2 cells was observed when cells were treated with compound 5 is (Figure 13). This effect on the reduction of SREBP-2 maturation and reduced expression of PCSK9 mRNA should translate to a corresponding effect


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on the secretion of PCSK9. When human HepG2 cells are treated with compound 5, the resulting cell culture supernatants can be analyzed for secreted PCSK9 levels using a humanspecific PCSK9 ELISA kit. The results from this type of assay are shown in Figure 14A and they clearly demonstrated the unprecedented synergistic effect of compound 5 on secreted PCSK9 levels in human HepG2 cells. In this assay, EPA did not produce any effect on secreted PCSK9 levels at the highest tested concentration of 50 μM (when cells were incubated with EPA at doses that were greater than 50 µM, cellular toxicity was observed). Niacin, likewise, did not produce any noticeable effect at doses as high as 10 mM. The combination of EPA and niacin also did not produce any effect on secreted PCSK9 levels at the highest tested concentration of 50 µM. In sharp contrast with the individual components or the combination of the individual components, the fatty acid niacin conjugate 5 showed a synergistic inhibition on secreted PCSK9 and had an IC50 value of 17 µM. In addition to its effects on PCSK9, high concentrations of niacin have been reported to inhibit the secretion of Apolipoprotein B (ApoB). ApoB is the major apolipoprotein found in VLDL and low density lipoprotein (LDL) particles secreted from the liver into circulation. An assay was established to study the effects of Compound 5 on ApoB secretion from the human hepatocyte line, HepG2. HepG2 cells were incubated with Compound 5, EPA, niacin or EPA plus niacin. After 18 hours, the amount of ApoB present in the cell culture supernatant was measured using a commercially available ELISA kit. As shown in Figure 14B, compound 5 significantly inhibited ApoB secretion in a dose dependent manner at a significantly lower concentrations than its component parts. (Figure 14B, IC50 of 27 µM). These data demonstrate a synergistic activity of compound 5 as compared to the combination of EPA and niacin in this assay. The measure of cellular toxicity by Alamar Blue was not significantly different across all treatment groups


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Compound 5 was evaluated orally in the ApoE*3-Leiden mice33 for its ability to lower PCSK9 and other lipid parameters. These transgenic mice were generated using a genomic construct isolated from human ApoE*3 Leiden, a genetic mutation that is characterized by familial dysbetalipoproteinemia. ApoE*3 Leiden mice, when fed on a high cholesterol Western-type diet, have been shown to have a dramatic increase in total plasma cholesterol and triglyceride levels. Compound 5 was dosed over a 16-week period in order to assess plasma PCSK9, total cholesterol, and triglyceride levels, liver LDL-receptor protein level, lipoprotein profile as well as other atherosclerotic endpoints. At the conclusion of the study, ApoE*3-Leiden mice that have been treated with compound 5 showed a significant reduction in PCSK9 levels, LDL particles including VLDL and LDL cholesterol, as well as plasma triglycerides. The entire data set for this ApoE*3-Leiden study is presented in a separate communication, along with a more detailed discussion on the mechanism of action of compound 5.34 In addition to niacin, we have also explored the possibility of co-delivering salicylic acid and an omega-3 fatty acid to cells using an analogous linker system. The fatty acid salicylate conjugate 11 was prepared by covalently linking salicylic acid to a different omega-3 fatty acid, namely (4Z,7Z,10Z,13Z,16Z,19Z)-docosa-4,7,10,13,16,19-hexaenoic acid (DHA). The same ethylenediamine linker used in the preparation of the fatty acid niacin conjugate 5 was employed in this particular example. By itself, salicylic acid, has been reported to be an inhibitor of NFκB-mediated gene expression by suppressing IKK kinase activity.35-37 On the other hand, the omega-3 fatty acid DHA has been shown to inhibit NF-κB through multiple mechanisms. DHA has been shown to inhibit IκBα phosphorylation by binding to GPR120 and repartitioning TAB1 away from the TLR4 receptor that mediates the LPS signal.38 DHA has also been shown to induce cytoplasmic retention of pro-inflammatory NF-κB proteins through p105 overexpression,


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thereby favoring the nuclear translocation of the p50-p50 transcriptional repressor homodimer.39 Additionally, DHA can be converted to the cyclopentanone ring containing A4 and J4neuroprostanes that are analogous in structure to PGJ2.40,41 PGJ2 has been shown to directly bind cysteine residues in the DNA binding domains of both the p50 and p65 subunits of NFkB, preventing them from binding to DNA.42,43 The simultaneous intracellular delivery of salicylic acid and DHA would therefore allow the modulation of the NF-κB axis via multiple pathways. We have evaluated the fatty acid salicylate conjugate 11 in a number of cellular assays and have observed a pattern of cellular uptake and hydrolysis that is similar to that observed previously with the fatty acid niacin conjugate 5. In order to assess the cellular uptake of fatty acid salicylate conjugates by confocal microscopy, we have prepared the fatty acid conjugate 12, wherein the DHA portion has been replaced with the fluorescent cis-parinaric acid. Figure 3D shows that the fatty acid salicylate conjugates are taken up into cells and are co-localized in the ER compartment, similar to what was previously observed with the fatty acid niacin conjugate 5. Distribution of the fatty acid salicylate conjugate 11 to the ER also facilitated its hydrolysis by FAAH. We have studied the hydrolysis of 11 in a rat liver lysate assay and have observed that its hydrolysis was FAAH-dependent. As shown in Figure 15, the addition of the FAAH inhibitor PF-3845 almost completely abrogated the hydrolysis of 11. The hydrolysis of the fatty acid salicylate 11 has also been studied in vivo in the appropriate PK and tissue distribution studies. When salicylic acid is dosed orally to rodents, dogs, monkeys and human, salicyluric acid 21 is detected as a major metabolite (Figure 16).44 This observation is consistent with the fatty acid salicylate conjugate 11 being hydrolyzed intracellularly in targeted tissues down to the individual components (i.e. salicylic acid), resulting in salicyluric acid as one of the major metabolites. In a PK experiment, compound 11 was dosed orally to Sprague


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Dawley rats at 300 mg/kg and plasma was collected at t = 0, 0.5, 1, 2, 4, 8 and 24 hr for analysis of the parent compound the potential metabolites shown in Figure 16. Compound 11 was orally bioavailable and Cmax for the parent compound was 477 ± 193 ng/mL, along with an AUClast of 1945 ± 1027 Hr*ng/mL. Even though the linker-DHA metabolite 22 could not be detected in the plasma, the salicylate-linker metabolite 20 was detected in the plasma (Cmax = 484 ± 80 ng/mL; AUClast = 2611 ± 501 Hr*ng/mL). In this oral rat PK study, we also observed the presence of salicylic acid (Cmax = 75 ± 125 ng/mL; AUClast = 229 ± 132 Hr*ng/mL) which indicated that the amide bond between salicylate and the ethylenediamine linker is degradable in cells and targeted tissues. Concurrent with the release of salicylic acid, we also observed the presence of the salicyluric acid metabolite 21 (Cmax = 563 ± 76.8 ng/mL; AUClast = 2937 ± 493 Hr*ng/mL). Compound 11 was then evaluated in a rat tissue accumulation study over a 21 day period. In this study, male Sprague Dawley rats, in groups of 4, were dosed orally with 300 mg/kg of compound 11 once a day for 14 days. Animals were sacrificed on days 1, 3, 7 and 14; plasma and tissues were collected 4 hr after dosing in order to determine the levels of the parent compound 11, salicylic acid, and salicyluric acid 21. The tissues examined included brain, epididymal fat, liver and muscle. Another group of animals was dosed orally with 300 mg/kg of compound 11 for 14 days and then subjected to a 7-day washout cycle; after which plasma and tissues were collected on day 21. As shown in Figures 17A, steady state levels of the parent compound were achieved in the various tissues and plasma after 7 days of dosing with the fatty acid salicylate conjugate 11. Higher concentration of the parent compound was detected in the tissues than in the plasma. Among the tissues that were examined, epididymal fat contained the highest tissue concentration of the parent compound 11 (concentration = 771.96 ± 176.92 ng/g). This was followed by the muscle and liver, wherein the tissue concentration of the parent


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compound 11 was 425 ± 88.87 ng/g and 330.74 ± 118.82 ng/g respectively. There was no significant level of the parent compound 11 measured in the brain tissue at any of the time points tested. The corresponding plasma concentration of the parent compound 11 at this 7-day time point was 115.51 ± 28.45 ng/mL. After 14 days of dosing, animals were subjected to a 7-day washout cycle. Our data indicate that the parent compound 11 was completely cleared out of plasma and tissues within 7 days (Figure 17A). Complete hydrolysis of the fatty acid salicylate conjugate 11 occurred and salicylic acid was detectable both in the plasma and tissues. Figure 17B shows the plasma and tissue concentration of salicylic acid over the course of the 21 day study. Steady state levels of salicylic acid were achieved after 7 days of dosing with 11 and a plasma concentration of 154.20 ± 65.43 ng/mL was observed. During this period, steady state levels were also achieved in the tissues, and a higher concentration of salicylic acid was observed in the liver (235.91 ± 95.14 ng/g) and muscle (265.73 ± 73.0 ng/g). Low levels of salicylic acid were detected both in the epididymal fat (65.03 ± 65.02 ng/g) and in the brain (53.32 ± 26.95 ng/g). Salicyluric acid is a phase 2 metabolite formed through the intracellular conjugation of salicylic acid and glycine. The levels of the metabolite salicyluric acid achieved a relatively constant state after 7 days of dosing across the tissues sampled (Figure 17C). As with salicylic acid, the highest concentrations were observed in plasma (764.41 ± 21.98 ng/mL) and liver tissues (506.48 ± 82.44 ng/g), with lower salicyluric acid measured in fat (203.71 ± 80.19 ng/g), muscle (165.21 ± 14.0 ng/g) and brain tissues (114.43 ± 8.87 ng/g). As observed previously with the parent compound 11, both salicylic acid and salicyluric acid were completely cleared from the plasma and tissues after a 7-day washout cycle.


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Having confirmed that the individual components could be released intracellularly, we then turned our attention to the pharmacological response that could be produced by the fatty acid salicylate conjugate 11. In an NF-κB reporter assay, compound 11 showed significantly more potent and synergistic inhibition of NF-κB than salicylic acid or DHA, its two major components. As shown in Figure 18, salicylic acid only showed inhibition of NF-κB at high concentration of 3.2 mM, which is consistent with the previously reported values.45 Likewise, DHA could only modestly inhibit NF-κB at a concentration of 78.7 µM. In sharp contrast, compound 11 was able to inhibit NF-κB with an IC50 value of 25.4 µM. This synergistic inhibition of NF-κB could not be replicated with the individual components or a combination of the individual components, even at much higher concentration. Inhibition of NF-κB could have many potential therapeutic applications, particularly in the area involving the enhancement of muscle regeneration in injured and diseased skeletal muscle.46,47 Serum levels of many NF-kB dependent pro-inflammatory chemokines including MMP-9,48 and cytokines including TNF-α,49 have been reported to be elevated relative to controls in boys with Duchenne muscular dystrophy, underscoring the potential utility of NF-κB modulation in this disease. We have since evaluated compound 11 in a number of preclinical models of Duchenne muscular dystrophy. These included the mdx mouse model and the golden retriever model. Detailed discussion of the results from those studies can be found in an upcoming communication.50 In summary, we have described a novel method of exploring pathway pharmacology through the use of fatty acid niacin and fatty acid salicylate conjugates. In our methodology, conjugates comprised of two covalently-linked bioactives are activated by specific intracellular enzymes in order to release the two bioactives inside cells and thereby allow them to simultaneously modulate multiple therapeutic pathways. Compared to the simple co-administration of the two


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bioactives, our conjugation methodology offers a number of advantages such as extracellular selectivity and mechanistic synergy by the simultaneous delivery of the two bioactives to the various intracellular targets. Both fatty acid conjugates of niacin and salicylate were able to produce a pharmacological effect that could not be replicated by administering the individual components or a combination of the individual components. For the fatty acid niacin conjugate 5, a distinctive effect on SREBP inhibition was observed, along with a down-regulation of cholesterol and other lipid metabolism genes, including PCSK9. For the fatty acid salicylate conjugate 11, the intracellular release of a salicylic acid and a DHA molecule produced an unprecedented and synergistic inhibition of NF-κB. The fatty acid niacin conjugate 5 has since been advanced into human clinical trials in order to evaluate its effect on the various lipid parameters. The fatty acid salicylate conjugate 11 has also been advanced into the clinic and is currently being evaluated in patients with Duchenne muscular dystrophy. The results from those clinical trials will be disclosed in due course.

EXPERIMENTAL SECTION General information: All chemical reagents and solvents were commercially available and used as received. Fatty acids and acid chlorides were purchased from Nu-Chek Prep. Reactions were generally run under argon or nitrogen. 1H NMR (400 MHz) and 13C NMR (100 MHz) spectra were recorded on a Varian 400MHz Unity Inova system in CDCl3, unless otherwise noted. Chemical shifts are expressed in parts per million (ppm, δ); coupling constants are in hertz (Hz). Splitting patterns describe multiplicities s (singlet), d (double), t (triplet), q (quartet), m (multiplet), br (broad). High resolution mass spectroscopy data was analyzed by direct flow


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injection, utilizing electrospray ionization (ESI) on a Waters Qtof API US instrument in the positive mode. Analytical and other mass spectra were collected on an Agilent Technologies 1200 series system with an Agilent Technologies 6120 Quadruple LC-MS detector in positive mode. A SiliCycle C18 XDB,3 x 100 mm column was used with a gradient of H2O and acetonitrile each with 0.1% formic acid; UV detection at 254 nm and 210 nm. Normal phase flash chromatography was accomplished on Teledyne Isco systems using pre-packed silica gel columns. Sample purity was determined by LC-MS; all compounds were of >95% purity, as determined by at least two different HPLC methods. For the hydrolysis experiments and PK/tissue distribution study, the samples were analyzed on the Agilent 6410 Triple Quad LC/MS mass spectrometer. Separation was achieved using a Gemini 3 µM C6 phenyl column with a gradient using water containing 0.1% formic acid (solvent A) and methanol containing 0.1% formic acid (solvent B). General procedure used to prepare N-(2-((5Z,8Z,11Z,14Z,17Z)-icosa-5,8,11,14,17pentaenamido)ethyl)nicotinamide (5) and other fatty acid conjugates (6-12): In a typical run, a mixture containing tert-butyl (2-aminoethyl)carbamate (1 g, 6.24 mmol), niacin (730 mg, 5.93 mmol), EDC (1.45 g, 7.50 mmol), HOBT (1.25 g, 7.50 mmol) and Et3N (1.8 mL, 12.50 mmol) in 20 mL CH2Cl2 was stirred at rt for 18 h. The resulting reaction mixture was washed with halfsaturated NH4Cl, brine, dried (Na2SO4) and concentrated under reduced pressure. Purification by silica gel chromatography (gradient elution, CH2Cl2 to 10% MeOH in CH2Cl2) afforded tertbutyl (2-(nicotinamido)ethyl)carbamate (1.4 g, 85%). This material was taken up in 5 mL of THF along with 8 mL of 4 N HCl in dioxane (32 mmol). The resulting reaction mixture was stirred at rt for 8 h and then concentrated under reduced pressure to afford the HCl salt of N-(2aminoethyl)nicotinamide in essentially quantitative yield. This material was then taken up in 20


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mL of CH2Cl2 along with (5Z,8Z,11Z,14Z,17Z)-eicosa-5,8,11,14,17-pentaenoic acid (>95% purity, 1.5 g, 5.04 mmol), EDC (1.22 g, 6.36 mmol), HOBT (1.1 g, 6.36 mmol) and Et3N (4.4 mL, 31.8 mmol). The resulting reaction mixture was stirred at rt for 16 h. It was then washed with half-saturated NH4Cl, brine, dried (Na2SO4) and concentrated under reduced pressure. The resulting residue was purified by silica gel chromatography (gradient elution, CH2Cl2 to 6% MeOH in CH2Cl2) to afford 1.68 g of N-(2-((5Z,8Z,11Z,14Z,17Z)-icosa-5,8,11,14,17pentaenamido)ethyl)nicotinamide (compound 5, 60% overall yield, >95% purity by LC/MS). 1H NMR (400 MHz, CDCl3) δ 9.06 (d, J = 2.3 Hz, 1H), 8.72 (dd, J = 4.9, 1.7 Hz, 1H), 8.11 (dt, J = 8.0, 2.0 Hz, 1H), 7.64 (s, 1H), 7.38 (ddd, J = 7.9, 4.9, 0.9 Hz, 1H), 6.10 (d, J = 6.3 Hz, 1H), 5.46 – 5.25 (m, 9H), 3.65 – 3.49 (m, 4H), 2.90 – 2.73 (m, 8H), 2.22 (dd, J = 8.4, 6.9 Hz, 2H), 2.14 – 1.98 (m, 4H), 1.70 (p, J = 7.4 Hz, 2H), 0.97 (t, J = 7.5 Hz, 3H); 13C NMR (100 MHz, DMSO-d) δ 20.0, 25.1, 26.2, 34.9, 38.1, 38.8, 39.1, 39.3, 39.5, 39.7, 39.9, 123.3, 128.9, 127.6, 127.7, 127.8, 127.9, 127.9, 128.0, 128.1, 129.4, 129.9, 131.5, 134.8, 148.4, 151.7, 164.9, 172.3; HRMS (ES+) Calcd for C28H40N3O2 (M + H+) m/z 450.3121, found 450.3121. The same general experimental procedure was employed for the synthesis of fatty acid salicylate conjugate 11, substituting niacin for salicylic acid. 1H NMR (400 MHz, CDCl3) δ 12.44 (br s, 1 H), 8.01 (m, 1H), 7.52 (dd, J = 1.3, 7.6 Hz, 1H), 7.36 (m, 1H), 6.95 (m, 1H), 6.84 (m, 1H), 6.36 (m, 1H), 5.29-5.37 (m, 12H), 3.49-3.53 (m, 4H), 2.72-2.85 (m, 4H), 2.77-2.85 (m, 10H), 2.062.42 (m, 3H), 0.97 (t, J = 7.5 Hz, 3H).

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C NMR (100 MHz, DMSO-d) δ 14.3, 20.6, 23.4, 25.5,

25.6, 25.7, 36.3, 39.4, 41.6, 76.8, 77.1, 77.4, 114.2, 118.3, 118.9, 126.2, 127.0, 127.7, 127.9, 128.1, 128.6, 129.7, 132.1, 134.2, 161.5, 170.8, 175.0; High resolution MS (ES+) Calcd for C31H42N2O3 (M + H+) m/z 491.3274, found 491.3278.


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Fluorescent imaging using HuH-7 cells: Human hepatocellular carcinoma cells (HuH-7) were obtained from Japan Cell Research Bank (JCRB). Two million cells were plated overnight in 10% FBS DMEM on a 4-well slide chamber (Nunc Lab-Tek II Chamber Slide System, Thermoscientific). Cells were infected with CellLight ER-RFP, BacMam 2.0 (Molecular Probes, C10591) or CellLight Late Endosomes-RFP, BacMam 2.0 (Molecular Probes, C10589) for 48 hours. Cell medium was replaced with DMEM plus compound 10 (prepared in 10% FBS), or compound 12 for 30 minutes and fixed with 4% paraformaldehyde for 2 minutes at 37 °C and washed two times with phosphate buffered saline for 5 minutes per wash. Slides were mounted with ProLong Gold antifade reagent with DAPI (Molecular Probes). Images were taken with a Zeiss 510 LSM confocal microscope with a 63X lens. Three lasers at 408 nm, 488 nm and 561 nm were used for DAPI, fatty acid conjugates 10 or 12, and ER/lysosome/endosomes-tracker respectively. For the endocytosis imaging, HuH-7 cells were infected with CellLight ER-RFP, BacMam 2.0 (Molecular Probes) for 48 hours. Cells were pretreated with 80 µM dynasore and then treated with compound 10 for 30 minutes, fixed with 4% formaldehyde for 2 minutes at 37 o

C and mounted with anti-fade DAPI (Molecular Probes). Images were taken on a Zeiss 510

LSM confocal microscope with 63X lens. Plasma stability studies: The in vitro stability of compounds 1-5 was studied in human, mouse, beagle and rat plasma. The plasma from the different species was diluted to 50% with 0.05 M PBS (pH 7.4) at 37 °C. Test compounds were dissolved in DMSO to a final concentration of 10mM and then diluted to 1mM in MeOH. The reactions were initiated by the addition of the test compounds to 500 µL of preheated plasma in a 96-well plate to yield a final concentration of 5 µM. The assays were performed at 37 °C and conducted in triplicate. Samples (50 µL) were taken at 0, 30, 60 and 120 min and 200 µL acetonitrile containing an appropriate internal


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standard (200 ng/mL) was added. The samples were subjected to vortex mixing for 1 min and then centrifugation at 4°C for 30 min at 4,000 rpm. The clear supernatants were then analyzed by LC-MS-MS under electrospray ionization in the positive multiple reactions monitoring (MRM) mode. Hydrolysis experiment using rat liver lysate: The rat liver lysate hydrolysis experiment was performed in Eppendorf tubes, by adding the necessary amount of water for the assay first. A 10x buffer solution with final concentrations of HEPES and EDTA of 50 mM and 1 mM respectively was prepared from 500 mM HEPES and 10 mM EDTA in water. Rat liver lysate was removed from -80oC storage, thawed and centrifuged at 4000g for 5 min. A volume of the supernatant was transferred to the Eppendorf tube yielding a final concentration in the assay of 3 mg/mL rat liver lysate. The tube was gently inverted to mix the components. The reactions are then place in a 37 oC incubator for 20 min allowing the reaction mixture to reach 37 oC before the reaction was initiated. To initiate the hydrolysis reaction, the samples were removed from the incubator and the compounds of the invention were added, as a DMSO solution, prepared in a manner to allow for

Synthesis and Characterization of Fatty Acid Conjugates of Niacin and

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