Journal of Medicinal Chemistry Recently, we have also pioneered a synthesis of novel cADPR derivatives that are highly stable both chemically and enzymati- cally. The 8-bromo N1-cyclic inosine 5′-diphosphoribose (8-Br- N1-cIDPR) thus obtained proved to be a novel agonist of Ca2+ release in intact cells.28,29 The opposed bioactivities of the classical antagonist 8-Br-cADPR and 8-Br-cIDPR thus show that the smallest of structural changes (i.e., NH2 → CO at C-6, as with changes at C-8 and C-3′) can transform a cADPR antagonist into an agonist. Furthermore, the enhanced stability of 8-Br-cIDPR allows direct chemical modification at the 8-position, including to the parent N1-cIDPR, (2, Figure 1), which is roughly equipotent to cADPR in permeabilized T cells.30 This stable analogue was recently cocrystallized with the wild-type ectoenzyme CD38, a multifunctional enzyme responsible for the formation and metabolism of cADPR, providing insight into substrate binding and the catalytic process.31 The chemoenzymatic approach relies on the selectivity of Aplysia cyclase that, while promiscuous, may not always recog- nize the required NAD+ analogue as substrate. Moreover, while NAD+ analogues generally cyclize at N1, some are hydrolyzed to the corresponding linear ADPR analogue. Another exception, ethenonicotinamide adenine 5′-dinucleotide, in which the N1 posi- tion is substituted, cyclizes through the N7 nitrogen by ADPRC.32 NHD+, a close analogue of NAD+ but bearing a 6-keto group on the purine ring rather than an amino group, cyclizes at N7, giving the fluorescent, biologically inactive N7-cIDPR, as indeed does the Figure 2. N7 cyclization of NHD+ by Aplysia cyclase. guanosine congener NGD+ (Figure 2).33 The reasons for this are not fully understood. 6-Thio NHD+ (where a 6-mercaptopurine + replaces the hypoxanthine ring of NHD ) has yet to be explored as a potential substrate for ADP-ribosyl cyclase. Since the only modifications at position 6 in the cyclic nucleotide explored to date (NH2 and CO) result in major changes in biological activity, we anticipated that the 6-thio (CS) derivative in particular would be of significant interest. The synthesis of C-6 modified cADPR analogues has never been straightforward. NAD+ analogues with substituents at C-6 such as SMe, NH(CH2)6NH2 or H all fail to cyclize when incu- bated with ADPRC.7 The corresponding hydrolyzed product is formed instead. The simple N-methylated analogue, 6-N-methyl Article + nicotinamide adenosine 5′-dinucleotide (6-NMe-NAD ) has, however, not yet been investigated. N1-cIDPR was prepared via both 8-Br-NHD+ and 8-Br-N1-cIDPR to bypass the problem encountered with N7 cyclization.30 To the best of our knowledge, even a total synthesis was never attempted for such a class of compound, presumably because of the difficulty of incorporating an intact “northern” ribose into the molecule. In order to further investigate the requirements for N1 versus N7 cyclization inter alia, we report here syntheses of both 6-NMe-NAD+ and 6-thio NHD+ and an investigation of their cyclization behavior. ■ RESULTS AND DISCUSSION Synthesis of 6-N-Methyl NAD+. 6-NMe adenosine 7 was earlier synthesized from 1-N-methyladenosine via a Dimroth rearrangement that involves an opening and recyclization of the adenine ring.34 We synthesized 7 by a different synthetic route that could provide wider flexibility for other modifications longer term. 6-Chloropurine 4 was first activated with TMS-triflate, then condensed with tetraacetyl ribose 5 to produce the 6-chloro protected nucleoside 6 in its β configuration (Scheme 1). Treat- ment with methylamine hydrochloride followed by acetate re- moval with methanolic ammonia produced 6-NMe adenosine 7 in good yield. Phosphorylation was achieved by using the esta- blished POCl3/TEP method but, in contrast to other nucleosides, both the 5′-monophosphate 8a and 3′,5′-bisphosphate 8b were formed as a mixture. Subsequent treatment with triphenyl- phosphine, morpholine, and dipyridyl disulfide produced two morpholidates. The desired monomorpholidate 9a was then easily isolated by ion-exchange chromatography and condensed with β-NMN+ to form the target 6-NMe-NAD+ 10. Incubation of 6-NMe-NAD+ 10 with Aplysia cyclase gene- rated only the hydrolyzed product 6-NMe-ADPR 11, confirmed by 1H NMR spectroscopy and a molecular ion of 572 in the electrospray mass spectrum. The latter is in accord with the structure of a linear nucleotide rather than the cyclic 6-NMe- cADPR, as it differs by 17 mass units for the additional hy- droxyl group. Moreover, the presence of two NMR doublets at 5.24 and 5.14 ppm represents a typical anomeric proton pattern for the terminal ribose hydroxyl in the α and β configurations. This result is perhaps not too surprising based upon another report of the hydrolysis of C-6 substituted NAD+ analogues, but these had more radical changes.7 It was initially assumed that cyclization of 6-NMe-NAD+ is most likely blocked by the steric hindrance brought about by the free rotation of the methyl group. However, an early review7 reported that the compound with an H at C-6 rather than an amino group is apparently also unable to cyclize, indicating that the cyclization catalyzed by Aplysia cyclase might (not surprisingly) involve the participation of the C-6 amino group. It may be possible that the steric hindrance of the methyl group disturbs the electrophilic attack at the N1 position and there- fore blocks the cyclization. It seems likely that the steric bulk of the NMe might interfere with the approach to the known covalent E179 enzyme−ribose intermediate for cyclization.35 For hydrolysis to occur requires the formation of the 6-NMe- NAD+−enzyme covalent complex, which allows water to attack the ribosyl C-1″ rather than N1. Another explanation could be interference with the actual binding of 6-NMe-NAD+ to Aplysia cyclase by reducing the H-bonding sites in the active site. Synthesis of 6-Thio NHD+. 6-Thio NHD+ was first re- ported by Atkinson et al.36 and synthesized by coupling 6-thio IMP (14) with nicotinamide mononucleotide with dicyclohex- ylcarbodiimide, a method developed by Todd et al.37 The low 1479 dx.doi.org/10.1021/jm201127y | J. Med. Chem. 2012, 55, 1478−1489