A Streamlined Synthetic Approach to SPMs Nicholas Wourms, Alisa Litvintsev, Jodi C. Westcott, and David Fichtner Cayman Chemical, 1180 E. Ellsworth Rd., Ann Arbor, MI 48108

HH HH HH 2-deoxy-D-ribose Background CHCH CHCH · Inexpensive and readily available from multiple vendors, which permits scalability HH LXLAXA HH HHLXBLXB 4 4 4 4 · Provides preferred syn diol required for SPM synthesis Specialized pro-resolving lipid mediators (SPMs) are a class of compounds that are · Enantiomerically pure, avoiding any enrichments or difficult purifications biosynthesized from polyunsaturated fatty acids (PUFAs) and exhibit pro-resolving 1 · Allows for bilateral carbon-carbon coupling capabilities. Although the subset lipoxins were initially discovered nearly 35 years ago, HH HH HH · Allows for selective protections of the hydroxyl groups the role of SPMs in the progression and resolution of inflammation is still being HH · Payne rearrangement can be utilized to form internal and external epoxides explored and new SPMs are being discovered to this day. CHCH · Lactol can be forced open to the aldehyde to improve reactivity MaresinMaresin 1 1 CHCH MaresinMaresin 2 2 · For resolvin D4 and resolvin E3, D-ribose is used in lieu of 2-deoxy-D-ribose SPMs are key in numerous anti-inflammatory processes that occur within the body including for the diol source4 host defense, pain, organ protection, and tissue remodeling. SPMs are all derived from free

PUFAs released at the onset of inflammation including arachidonic acid and the ω-3 HH HH fatty acids: eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and docosapentaenoic CHCH CHCH Glycidol 2 acid (DPA). This results in several structural similarities in the SPMs that can be exploited HH HH HH MethylMethyl Terminal Terminal Source Source CarboxylicCarboxylic Acid Acid Source Source to create a scalable and streamlined synthetic approach to multiple derivatives. In fact, · Commercially available in both enantiomerically enriched forms from HH MethylMethyl succinyl/glutaryl succinyl/glutaryl chloride chloride HH HH the research community has been able to synthesize over 20 SPMs from just a few HH CHCH 1-butyne1-butyne multiple vendors ResolvinResolvin D1 D1 ResolvinResolvin D2 D2 commercially available starting materials. By manipulating these building blocks, a major · Fairly inexpensive, permitting scalability Cl Cl portion of any of the SPMs and their derivatives can be synthesized. Five of these integral ResolvinResolvin E1 E1 x x CMeCMe · Desired stereochemistry set early in synthesis HH CHCH HH · Allows for bilateral carbon-carbon coupling pieces and the qualities that make them useful in synthesis are outlined in Figure 1. HH HH HH · Protection of primary alcohol allows for opening the epoxide with a nucleophilic attack

CHCH CHCH HydroxylHydroxyl Source Source · Substitution of the glycidol enantiomer provides the aspirin-triggered epimer by Resolvin D1 (RvD1) is one of the many SPMs derived from DHA. The synthesis of HH HH the same synthetic path as the parent SPM RvD1 advantageously utilizes three of the building blocks outlined in Figure 1. Both 2-deoxy-D-Ribose2-deoxy-D-Ribose D-RiboseD-Ribose HH 2-deoxy-D-ribose and enantiomerically enriched glycidol afford fixed stereochemistry ResolvinResolvin E2 E2 HH HH ResolvinResolvin D3 D3 ResolvinResolvin D4 D4 of the hydroxyl groups.1 Because of this, RvD1 can be synthesized in both higher yields HH HH 1-Butyne and in larger quantities than if an enantiomerically impure reagent was used. Incorporating HH CHCH HH HH CHCH HH HH 1-butyne not only completes the ω-chain but offers the flexibility for easy radiolabeling. CHCH · 1-butyne and 1-butyne-d are commercially available GlycidolGlycidol HH 5 Additional detail on the three synthons and their specific role pertaining to RvD1 are HH HH HH · Useful in completing ω-chain via organolithium addition to the THP-glycidyl outlined in Figure 2. · 1-butyne-d is an efficient way to radiolabel most of the D-class resolvins ResolvinResolvin E3 E3 HH 5 · 1-butyne-d5 is isotopically enriched, affording a d0 value of less than 1% ResolvinResolvin D5 D5 ResolvinResolvin D6 D6 Utilizing this Strategy for the Synthesis of RvD1 Methyl succinyl chloride/Methyl glutaryl chloride HCHC Current literature details the synthesis of RvD1 using 2-deoxy-D-ribose and HH · Commercially available, inexpensive HH HH2 2 2 2 1,3 H H (R)-glycidol. Using this chiral pool approach, RvD1 can be synthesized with the H H HCHC HCHC S S · Useful in the synthesis of 4/5-carbon hydroxy acid systems in many SPMs hydroxyl groups enantiomerically set at the beginning of the synthesis. S S · Ketoalkyne can be formed through Friedel-Crafts acylation HH HH · Midland reduction of the ketone affords desired stereochemistry of hydroxyl group CHCH 2-deoxy-D-Ribose is a key component in synthesizing RvD1. In some of the synthesis CHCH ® HH CHCH · (S)-Alpine-Borane is commercially available for use in Midland reduction papers for RvD1, the acetonide-protected lactol of 2-deoxy-D-ribose is utilized to MCTR1MCTR1 MCTR2MCTR2 3 build the compound. Unfortunately, the acetonide-protected lactol normally affords HH HH2 2 low-yielding reactions. This problem can be circumvented by forcing the lactol to its HCHC S S ProtectinProtectin D1 D1 Common Commercially Available Synthons open form, the aldehyde. This improves the reactivity significantly and allows the reactions HH to be run at lower temperatures, which provides better cis/trans selectivity. The lactol · [3-(Ethoxycarbonyl)propyl]triphenylphosphonium bromide CHCH is reacted with acid and propyl dithiol to force the ring open, forming the dithiolane. · Propyltriphenylphosphonium bromide MCTR3MCTR3 The primary alcohol can then be trityl protected selectively, while the syn diols can be · (Formylmethyl)triphenylphosphonium chloride TBDMS protected. Protecting the primary alcohol with the trityl group allows for selective · Methyl (triphenylphosphoranylidene)acetate deprotection that won’t affect the silyl ether protecting groups. This allows for addition References on either side of the synthon without affecting the other, offering not only flexibility 1. Serhan, C.N., Hamberg, M., and Samuelsson, B. Biochem. Biophys. Res. Commun. 118(3), 943-949 (1984). 2. Serhan, C.N. and Petasis, N.A. Chem. Rev. 111(10), 5922-5943 (2011). 3. Rodriguez, A.R. and Spur, B.W. Tetrahedron Lett. 53(51), 6990–6994 (2012). but high yields as well. HH H H 4. Nagarapu, L., Karnakanti, S., and Bantu, R., Tetrahedron 68(29), 5829-5832 (2012). HH H2H2 S S CHCH S S CHCH The monohydroxyl group in RvD1 is also pre-set at the beginning of the synthesis by HH HH H H 2 2 CHCH using (R)-glycidol. Glycidol is commercially available in both enantiomerically enriched CHCH HCHC forms, giving the flexibility to make both RvD1 and aspirin-triggered RvD1. The glycidol PCTR1PCTR1 PCTR2PCTR2 is THP protected before opening the epoxide by organolithium addition of 1-butyne.1 The addition and subsequent reduction of the 1-butyne completes the ω-chain. HH H H Commercially available 1-butyne-d5 can be utilized for radiolabeling RvD1. The 2 2 S S CHCH Rodriguez and Spur paper detailing the synthesis of RvD1 offers an alternative method HCHC for radiolabeling through Boland reduction of the 13(Z) olefin late in the synthesis.3 However, even with fresh deuterated solvents, this way of radiolabeling can be difficult Figure 1 PCTR3PCTR3 to monitor and is susceptible to exchange.

BrPh3PBrPh3P CEtCEt The innate properties of these three synthons, coupled with their low cost and wide HH TBSTBS TBSTBS 3 steps3 steps TBSTBS TBSTBSS S TBSTBS TBSTBS 5 steps5 steps HH availability, make a scalable and streamlined synthesis for RvD1 feasible. The strategies CEtCEt TrTr TrTr CEtCEt HH S S TBSTBS CHCH employed for the synthesis of RvD1 are also applicable to the synthesis of other resolvins. HH TrTr TBSTBS 4 steps HH Manipulating these same building blocks and the others outlined in Figure 1 allow for 4 steps synthesis of existing and future SPMs. I I HH THPTHP 7 steps7 steps HH ResolvinResolvin D1 D1 TBSTBS Figure 2