Azanorbornadienes as Thiol-Reactive Cleavable Linkers

Abstract

Azanorbornadienes (ZNDs), prepared from pyrroles, undergo Michael reaction with thiols followed by retro-Diels–Alder (rDA) cleavage to release the starting pyrrole and a thiomaleate. Somewhat less reactive in this regard than furan-derived oxanorbornadienes, ZNDs have an additional point of variability at the pyrrole nitrogen center. Sulfonylated ZNDs were far more stable toward rDA cleavage than acylated analogues, tert-Butoxycarbonyl (Boc) examples were much less reactive with thiols, rendering the rDA step slower than the initial conjugate addition.

Graphical Abstract

Pyrrole and its derivatives play an important role in biology and drug development,¹ being present in at least 30 clinically approved small-molecule medications against a wide variety of indications (excluding the much larger number of pharmaceutical agents containing variations on the indole moiety). We describe here studies of pyrroles as analogues to furans in Diels-Alder and retro-Diels-Alder chemistry that enables the use of oxanorbornadiene (ONDs) as thiol-responsive cleavable linkers (Figure 1).^2–6 In this sequence, Diels-Alder cycloaddition with electron-deficient alkynes are used to construct compact OND moieties (1) that retain significant Michael acceptor ability. Conjugate addition of thiols is particularly facile, resulting in adducts that decompose by retro-Diels-Alder fragmentation at rates (k₂) that vary widely depending on the nature of the substituents.

Figure 1.

Thiol-triggered retro-Diels-Alder cleavage reactions of oxanorbornadienes (ONDs, top) and azanorbornadienes (ZNDs, bottom).

N-Acylpyrroles have long been known to participate in Diels-Alder reactions with electron-deficient dienophiles,^7–11 but no information on thiol-triggered retro-Diels-Alder (rDA) reactivity appeared in the literature until a recent report from Moreno-Vargas, Bernardes, and colleagues on the chemistry of N-Boc pyrrole adducts of tosylacetylene (2, R¹-R⁴ = H, R⁵ = Boc, R⁵ = SO₂p-tolyl, R⁶ = H).¹² An important advantage to this system is the avoidance of potential furan-associated metabolic toxicity, since acylpyrroles are less likely to undergo analogous P450 processing.¹³ We describe here our initial exploration of the performance of azanorbornadienes (ZNDs) derived from dimethylacetylene dicarboxylate (DMAD) as a prototypical electron-deficient alkyne, focusing mostly on the rates of pyrrole release by rDA reaction.

Consistent with prior reports, the assembly of ZNDs from pyrroles required higher temperatures than Diels-Alder reactions of furans. ZNDs 2a-f were prepared from a selection of substituted pyrroles (3a-f) with hydroxyl substituents from which cargo molecules could be attached (Figure 2). Four different electron-withdrawing substituents were used on the pyrrole nitrogen, including acyl (Ac), t-butoxycarbonyl (Boc), methanesulfonyl (Ms) and p-tolylsulfonyl (Ts). Yields (14-65%) were lower than typically observed for Diels-Alder reactions of furans at similar molar ratios of reactants. While we did not investigate this thoroughly, the yields seemed to correspond to the establishment of less-favorable equilibria between forward and retro-Diels-Alder reactions, as pyrrole was always observed in the reaction mixtures even in the presence of excess alkyne.

Figure 2.

Preparation of ZND electrophiles from pyrroles; yields refer to isolated, purified products.

All of the ZNDs were found to be reactive with thiols under base-catalyzed conditions, initially tested in CDCl₃. The example (2b) shown in Figure 3 is unique in its relatively fast nature. All of the other ZND electrophiles required 20 minutes or longer to completely react with 3 equivalents of β-mercaptoethanol under identical conditions [2c = 20 min; 2d = 2 h; 2a, 2e, 2f = discussed below].

Figure 3.

Example of addition-fragmentation sequence followed by ¹H NMR (CDCl₃, 25°C), showing representative spectra. Starting concentrations: 2b = 0.056 M, β-mercaptoethanol = 0.17 M, triethylamine = 0.17 M

The half- lives of retro-Diels–Alder fragmentation were determined by ¹H NMR, starting promptly after the addition of excess β-mercaptoethanol and triethylamine, as shown in Figure 3. Concentration-vs-time values fit the first-order kinetic behavior anticipated for retro-Diels–Alder fragmentation; half-lives for these processes are summarized in Figure 4. The thiol adduct of an amide ZND (2b) was found to have a rate of rDA cleavage on the order of 6 hours at room temperature. Otherwise structurally similar adducts of sulfonamide ZNDs (2c, 2d), in contrast, were far more stable, requiring approximately 70 days for half of the material to undergo fragmentation. Previously-reported azanorbornadienes derived from acylpyrroles and a terminal alkynylsulfone showed thiol-triggered rDA rates between these, but much closer to the acyl derivative incorporating DMAD.¹²

Figure 4.

(black) Half-lives of rDA fragmentation of β-mercaptoethanol adducts of the indicated ZND molecules at 25 °C (unless otherwise indicated) in the indicated solvent. Results from ref 12 (entries S1 – S3, at 37 °C) are included for comparison, for which R⁵ = SO₂p-tol, R⁶ = H. (blue) Second-order rate constants for appearance of pyrrole products for cases in which β-mercaptoethanol addition is rate-determining.

This remarkable difference between N-acyl and N-sulfonyl pyrrole derivatives is likely to reflect fundamental distinctions between the bonding in these groups. Unlike the sp² hybridization of nitrogen in N-acyl moieties, sulfonyl nitrogen atoms are usually pyramidalized.^14–15 N-sulfonyl nitrogen centers are therefore more electron-rich, with inductive effects more important than resonance.¹⁵ We therefore suggest that the rDA process is accelerated for N-acyl cases because of the greater contribution of the sp²-N center to the developing aromaticity of the pyrrole product. Solvent effects also seem to be different from acyl vs. sulfonamide substituent as well. Thus, the rDA reaction of the β-mercaptoethanol adduct of mesyl ZND 2c was approximately three times faster in methanol-d₄ than in chloroform-d. In contrast, the rDA reaction of acetyl ZND 2b was slightly slower in the protic solvent (Figure 4).

Thiol addition rates appear to be much more sensitive to the substituents of ZND electrophiles than ONDs. It was found that N-Boc-ZNDs (2a, 2e and 2f) underwent relatively slow thiol addition, presumably because of steric hindrance, such that we were unable to observe the thiol adduct as an intermediate by NMR. Instead reactions of these electrophiles with β-mercaptoethanol proceeded directly to their corresponding pyrroles (3a, 3e and 3f) by virtue of rDA cleavage after rate-determining conjugate addition. Second-order rate constants in the range of 10⁻⁴ – 10⁻⁵ M⁻¹s⁻¹ were observed, with the slowest Michael acceptor being the most sterically burdened 2f, having substituents at both bridgehead positions.⁻ As previously determined for ONDs, and reported also for ZND S2,¹² we also assume that 2f undergoes faster rDA cleavage.

Competition experiments between ONDs and ZNDs are shown in Figure 5. Equal amounts of 2-substituted (6 and 2b) or 2,5-disubstituted (8 and 2f) electrophiles were treated in CDCl₃ with 0. 5 equivalents of β-mercaptoethanol and 1 equiv. of triethylamine. The OND reagents were found to be more reactive with thiol than the ZNDs, by a factor of approximately 69/31 in the first case and by >20-fold (only furan and no pyrrole observed) in the second. For the doubly bridgehead substituted electrophiles, approximately 10% of the reaction was observed to proceed via ZND attack in CD₃OD to give a small amount of pyrrole 3f (data not shown).

Figure 5.

Competition between OND and ZND electrophiles bearing one (top) or two (bottom) bridgehead substituents for thiol nucleophile in CDCl₃ at 25°C, monitored by ¹H NMR.

This initial survey of azanorbornadiene electrophiles has revealed generally slower reactions with thiols compared to oxanorbornadiene analogues, but dramatic differences in the retro-Diels-Alder behavior of the thiol adducts of N-acyl and N-sulfonyl variants. While the rates of each step were different for ZNDs compared to ONDs, the new nitrogen analogues provided similarly clean transformations to conjugate adduct and rDA fragments. We therefore find pyrrole analogues to be worthy of further development as thiol-responsive cleavable linkages.

ABBREVIATIONS

OND

oxanorbornadiene

ZND

azanorbornadiene

DMAD

dimethylacetylene dicarboxylate