Optimized routes to 7-carboxymethyl-pterin a useful building block for medicinal chemists Zachary Bennett, Kassidy Grumbles, Dr. Jeffery Pruet | Valparaiso University

Abstract Methods Path B Results Future Work Pterins are a class of nitrogen-heterocycle with wide Two general routes to 7-CMP were envisioned (Figure 3): O Na2SO3 O To continue this project, a third route (Path C) will be O H2O application in the field of medicinal chemistry. The NH2 O H N investigated. Similar to Path B, this route relies on a NH2 HN 30 min HN importance of pterin derivatives stems from discovery of Path A HN Path B H N N NH O H 95% regiospecific Minisci reaction, this time via H2N N NH2 2 2 H2N N N numerous biologically relevant pterins, such as folate and H2SO4 decarboxylative generation of the acyl radical. O O bioterin. Due to this biological relevance, several pterin-based N N O O HN HN Scheme 5: Synthesis of unsubstituted pterin N AgNO3 N HN O O inhibitors have been developed for various biological targets. Na2S2O8 HN H2N N N H2N N N MeO O K H2SO4 O 6-methyl regioisomer also possible The sulfate salt of the pyrimidine was condensed with glyoxal, H N N N In this regard pterins can be viewed as a privileged scaffold, as 2 H2N N N [Acyl radical insertion] removing the issue of regiosiomers (Scheme 5). O the discovery of new pterin analogs gives rise to a vast array [Oxidation] Figure 4: New route under investigation (Path C)

O of potential drug candidates. 7-carboxymethyl-pterin (7-CMP) O FeSO , H O After determining which pathway is the most efficient, the N O 4 2 2 O N [Fischer-Esterification] HN H SO has previously been shown as a useful scaffold for the rapid HN N O O 2 4 10-15 min. N 7-carboxymethyl pterin will be used as a building block in H2N N N CO2Me HN HN generation of structurally diverse pterin amides. We are H2N N N CO2H OMe O the construction of several new potential inhibitors of H2N N N 33-51% H N N N currently exploring multiple routes towards 7-CMP to assess 7-carboxymethyl Pterin (7-CMP) 3 equiv. 2 fungal methionine synthase (MetSyn). Due to significant O the most efficient method of generating this useful scaffold. Figure 3: Two routes explored in the synthesis of 7-CMP Scheme 6: Regiospecific radical acylation, providing 7-CMP structural differences between the fungal and mammalian We intend to use 7-CMP as a building block in our search for Path A relies on the oxidation of 7-methyl pterin, which Due to the resonance of a pterin, nucleophilic attack by the MetSyn, selectively targeting this fungal enzyme will allow selective inhibitors of fungal methionine synthase. typically forms with the 6-methyl isomers, while path B uses a acyl radical occurs at C7 (Scheme 6). This allows for a access to anti-fungal drugs without risk of disrupting the Background regiospecific Minisci-type radical insertion to provide the relatively quick and easy single step to producing the pterin patients biochemistry. Using the DBU-promoted amidation (Fig. 3), several new inhibitor candidates can be rapidly Pterins such as folate and biopterin play key roles in cell desired product. As paths A and B both require the triamino- ester regiospecifically.7 The product precipitates upon the made from 7CMP. One such substituted 7-pterin amide is division, degradation of nucleic acids and amino acids, pyrimidine, this common intermediate was synthesized as completion of the reaction, due to its decreased basicity. One shown in Figure 5, whereby the folate and methionine neurotransmitter biosynthesis, and nitric oxide production.1 shown in Scheme 1. complication is the use of excess sulfuric acid diminishes this binding domains are simultaneously bound by the inhibitor O O O selective precipitation, further complicated by the Due to this biological relevance, several pterin-based HNO2 Na2(S2O4) AcOH NO NH OH, H O NH candidate. This is a promising result in the development of inhibitors have been developed for targets such as ricin, HN HN 4 2 HN 2 incompatibility of the reaction to a basic work-up due to iron a strong competitive inhibitor. H N N NH 89% Reflux leishmaniases, nitric oxide synthase, and dihydrofolate 2 2 H2N N NH2 H2N N NH2 hydroxide co-precipitation. 89% reductase.2-4 Likely the most widely known medically relevant Optimized conditions for this Minsici-type reaction were Scheme 1: Synthesis of common triaminopyrimidine intermediate pterin is the anti-folate drug methotrexate. assessed by varying the concentration of iron (Table 1). One attractive O CO2H Preliminary results suggest improved yields may be accessible NH2 NH2 N biological target is Path A Results N H N N with varying iron. N N CO2H fungal methionine H2N N N NH2 Path A relies on the condensation of the pyrimidine with H2N N N 7-CMP Triamterene Methotrexate synthase (MetSyn). a) b) Pterin used Equiv. of * treats hypertension chemotherapy agent pyruvaldehyde. Under acidic conditions, this reaction Entry recovered Yield This enzyme is (mmol) FeSO4 used OH (mmol) O O predominantly gives the undesired 6-methyl isomer. However Figure 5: Side-by-side comparison of the MetSyn active site bound to a) N N crucial for all HN HN O 1 22 1.5 8.9 41% H in basic condition this is reversed as the more nucleophilic folate and methionine, and b) a pterin-based inhibitor prepared previously. S S N H2N N N organisms, and H2N N N N CO2H H nitrogen exists as the free-base and readily reacts with the 2 22 1.0 11.3 51% anti-Shigella agent anti-Ricin agent O inhibition of the more electrophilic aldehyde (Scheme 2). 3 22 0.5 7.2 33% fungal form would References Figure 1: Therapeutic pteridines O O O * NaHCO Definitive trends in yields may need additional data provide an anti- NH2 O O 3 N N 1) Shane, B. Folate in Health and Disease; 2nd ed.; CRC Press, 2010 HN H2O HN HN fungal agent.5 Due to significant differences between the 30min Reflux Table 1: The effect of varying iron quantities on reaction yield. 2) Pruet, J. M.; Jasheway, K. R.; Manzano, L. A.; Bai, Y.; Anslyn, E. V.; H2N N NH2 H 90% H2N N N H2N N N fungal and mammalian forms of MetSyn, inhibitors can be Robertus, J. D. Eur J Med Chem 2011, 46, 3608. engineered to specifically bind and deactivate the fungal 95 : 5 3) Tulloch, L. B.; Martini, V. P.; Iulek, J.; Huggan, J. K.; Lee, J. H.; Scheme 2: Synthesis of 7-methyl pterin, and its isomer Comparison of Paths enzyme, effectively killing the infection without harming the Gibson, C. L.; Smith, T. K.; Suckling, C. J.; Hunter, W. N. J Med Chem 2010, 53, 221. patient. As MetSyn is a folate-dependent enzyme, we have Due to the mixture of isomers, the dominant 7-methyl isomer Both paths have their individual merits on pitfalls. While Path was first separated prior to oxidation overnight to the A benefits from slightly improved yield and milder conditions, 4) Matter, H.; Kotsonis, P.; Klingler, O.; Strobel, H.; Frohlich, L. G.; Frey, aimed to generate a library of pterin-based inhibitors to serve A.; Pfleiderer, W.; Schmidt, H. H. J Med Chem 2002, 45, 2923. as folate mimics in our designed inhibitors. For our inhibitor corresponding 7-carboxypterin (Scheme 3). it suffers from extended reaction times and mixtures of regiosiomers. Path B clearly benefits from fast access to the 5) Ubhi, D.; Kago, G.; Monzingo, A. F.; Robertus J. D. J. Mol. Biol. 2014, scaffold, we chose 7-carboxymethyl-pterin (7CMP). This O O 426, 1839. 1) KMnO4 , NaOH product, without regioisomeric concern. contains the desired pterin-core, and benefits from a recently N N HN 24 hr, Reflux HN 6) Pruet, J. M., Saito, R., Manzano, L.A., Jasheway, K.R., Wiget, P.A., 6 developed DBU-promoted amidation reaction (Figure 2). OH Kamat, I., Anslyn, E.V., Robertus, J.D. ACS Medicinal Chemistry H N N N 2) HCl H N N N Total 2 76% 2 Overall Letters 2012, 3, 588. O N O # of steps reaction Pros Cons N yield 7) Pruet, J. M.; Robertus, J. D.; Anslyn, E. V. Tettrahedron Lett. 2010, HN HN O time OMe N Scheme 3: Permanganate oxidation of 7-methyl pterin to 7-carboxypterin H2N N N N 51(18), 2539 O OMe H2N N N N O • Must separate N 7-carboxypterin subsequently underwent Fischer-esterification • Benign Path A 3 52% 48.5 hrs regioisomers in methanol to provide the desired 7-CMP (Scheme 4). conditions Acknowledgements N • overnight reactions MeOH N O O O N We thank Valparaiso University and the faculty members in the N N MeOH, H SO (cat) N N N HN 2 4 chemistry department for their support in this research. H2N N N 24 hr, Reflux HN • Fast • Hazardous reaction O O OH N R NH O Path B 2 48% ~45 min • Regiospecific conditions Specifically we would like to thank Dr. Pruet for guidance and HN 2 H2N N N H N N N H 76% 2 N R O • Rust by-products assistance in this project. H2N N N O O Figure 2: A facile reaction for generating new pterin-amides from 7-CMP Scheme 4: Fischer-esterification to form 7-carboxymethyl pterin Table 2: Comparison of routes thus far

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