Abstract
A new method is reported for the synthesis of the α,β-unsaturated nitrone moiety characteristic of the stephacidin/avrainvillamide family of bioactive prenylated indole alkaloids. Application to the synthesis of stephacidin analogs and a potential biological probe are showcased.
The stephacidins and related family members (Figure 1) are complex compounds of the alkaloid class broadly defined as containing a prenyl unit juxtaposed on an indole framework.1 The combination of structural intrigue and biological potential has stirred significant interest in both the synthesis2 and the study of fundamental biochemical mechanisms3 that are operative in the anti-cancer activity they exhibit. In 2005 we reported a synthesis of 1–3 by an approach that was notably inefficient in the final steps (Figure 1).4
Scheme 1.
Development of an improved protocol for indole oxidation. (a) NaBH3CN (40 equiv), AcOH, 20 °C, 24 h, >95%; (b) DMDO (1.1 equiv), MeOH/AcOEt (1:1), −40 °C, 1 h; (c) chloranil (2 equiv), THF, 70 °C, 40 min, 82% over two steps.7
Specifically, the conversion of stephacidin A (1) to avrainvillamide (2) was stymied by chemoselectivity issues that prevented clean and efficient oxidation. As shown in Figure 2, the general strategy of Somei5 was utilized to construct the characteristic α,β-unsaturated nitrone system. Thus, the indole (A) is first reduced to the indoline oxidation state (B) followed by a reoxidation that commences with N-oxidation (to N-hydroxyindoline C) and two sequential dehydrogenation events to deliver E via D in a single step. Whereas the Gribble reduction6 (A to B) proceeds in high yield, the Somei oxidation (B to E) in the stephacidin series is low yielding. In this letter an interim solution to this problem is put forth that utilizes dimethyldioxirane (DMDO) to chemoselectively access N-hydroxyindoles (isolable) followed by a separate dehydrogenation step. The overall efficiency and scalability of this protocol is superior to those previously employed, permitting the synthesis of stephacidin analogs and biological probes.
Scheme 2.
Synthesis of avrainvillamide probe 4. Reagents and Conditions: (a) proline 10 (1 equiv), HATU (1.1 equiv), DIPEA (3 equiv), DMF, 20°C, 12 h, 74%; (b) 10% Pd/C (20% w/w), H2, MeOH, 20°C, 18 h; (c) MeOH/DMF (1:1), 100 °C, 4 h, 85%; (d) KHMDS (1.1 equiv), MOMCl (1.2 equiv), THF, −78°C to 20°C, 2.5 h, 57%; (e) Mg0, MeOH, 50 °C, 4 h (85%); (f) BnBr (3 equiv), DBU (3 equiv), CH3CN/CH2Cl2 (1:1), 20 °C, 3 h, 59%; (g) LDA (2.2 equiv), Fe(acac)3 (2.2 equiv), −78 to 20 °C, 47%; (h) PPh3 (5 equiv), Br2 (5 equiv), CH2Cl2, 0 °C, 18 h, 43–70%; (i) MeMgBr (10.0 equiv), toluene, 20°C, 10 min; (j) 10% Pd/C (20% w/w), H2, MeOH, 20°C, 18 h; (k) BzCl (3 equiv), TEA (3 equiv), CH2Cl2, 20 °C, 12h; (l) Burgess reagent (10 equiv), benzene, 50 °C, 5 min, 74% over 4 steps; (m) 190 °C, 30 min, 59%; (n) K2CO3, MeOH/CH2Cl2 (1:1), 20 °C, 5 h, 81%; (o) 16 (10 equiv), TEA (3 equiv), acetone, 20 °C, 12 h 70%; (p) NaBH3CN (40 equiv), AcOH, 20 °C, 24 h, >95%; (q) DMDO (1.1 equiv), MeOH/AcOEt (1:1), −40 °C, 1h; (r) chloranil (2 equiv), THF, 70 °C, 40 min, 98% over two steps.
During our investigations into the synthesis of a biological probe, it became apparent that our method for generating the α,β-unsaturated nitrone via Gribble reduction and subsequent Somei oxidation was insufficient.
Reasoning that the indoline nitrogen should be susceptible to hydroxylation with a highly electrophilc oxidant, indoline 6 was treated with 1.1 equivalents of DMDO at −40 °C to furnish the N-hydroxyindole 7 in nearly quantitative yield. The intermediate hydroxy indoline presumably oxidized spontaneously to furnish 7. Mild dehydrogenation using p-chloranil cleanly led to the desired nitrone 8 in 82% overall isolated yield (Scheme 1).7
This two-step procedure is simple to perform and proved critical for the preparation of several avrainvillamide analogs. The synthesis of one such analog, a biological probe specifically designed for Cravatt’s ABPP method,8 is delineated in Scheme 2.
Thus, we began with HATU mediated amide bond formation between acid 9 and amine 10 (Scheme 2). In contrast to our route to 1, 2, and 3 in which the benzopyran double bond poses chemoselectivity challenges to standard hydrogenolysis of the Cbz group, 11 can be treated with H2/Pd/C followed by heating to effect deprotection and diketopiperazine formation. Treatment of the intermediate diketopiperazine with base followed by MOMCl protected the amide nitrogen and set the stage for oxidative coupling (57% over two steps). Unfortunately, tosyl protected derivative was not amenable to oxidative coupling presumably due to the acidic methyl protons of the tolyl group. Exchanging of the tosyl group for a benzyl (12) alleviated this problem. Removal of the tosyl group with Mg0 in MeOH required slightly elevated temperatures (50 °C) but proceeded in good yield (85%). Benzyl protection was effected using benzyl bromide and DBU to give 12 in 59% yield. With 12 in hand the key oxidative coupling was studied and after several attempts the use of LDA and Fe(acac)3 followed by MeOH quench at low temperature was found to produce the expected cyclic compound in 47% yield. As observed in our other substrates, MOM removal was capricious under all conditions examined; PPh3•Br2 gave the best results (43–70%). With the MOM group removed, Grignard addition followed by dehydration with Burgess reagent gave the corresponding olefin 13 in 97% yield over two steps. Several reaction conditions were examined for the cyclization of 13 but 14 was never observed due to the incompatibility of 13 with Brönsted acids. The benzyl group was removed to reveal the free phenol in hopes that it would provide a subtle modulation in reactivity and allow for successful ring closure. This was also unsuccessful. It was postulated that the electron donating effects of the oxygen substituent on the indole mired the cyclization. Our previous studies have shown this cyclization to be effective on unsubstituted indole substrates.4a In an attempt to approximate the electronics of an unsubstituted indole, the benzoyl ester was formed from the free phenol and olefin 15 efficiently cyclized when heated at 190 °C. The benzoyl moiety was easily cleaved by treatment with K2CO3 and the free phenol derivative was treated with acid chloride 16 in the presence of TEA. The resulting ester was then reduced under Gribble conditions to yield indoline 17. Using previously reported oxidation conditions (SeO2 or Na2WO4•2H2O) the ester side chain was readily cleaved. Alternatively, using DMDO as an oxygen source at −40 °C produced N-hydroxyindoline in good yield and direct treatment with chloranil installed the desired α,β unsaturated nitrone (4) in 98% yield over two steps.
Initial testing in the Cravatt lab demonstrated that 4 showed high levels of protein labeling in breast cell (T47D) lysates at concentrations ranging from 1–200 μM upon visualization by in-gel fluorescence following click chemistry with rhodamine-azide.
Subsequent competition studies were performed on avrainvillamide mimic 8, in which cell lysates were incubated with 100 μM of 8 prior to the addition of 1–10 μM of 4. These studies demonstrated the disappearance of a doublet around the 50 kD range upon pre-treatment with 8, suggesting a possible selective protein target for avrainvillamide (2) in the 50 kD molecular weight region.
In summary an efficient two-step protocol for the chemoselective preparation of avrainvillamide and stephacidin analogs containing the hallmark α,β unsaturated nitrone has been developed. Application of this method to the improved synthesis of analog 8 and bioprobe 4 will enable further studies of the biochemical functions of this alkaloid family.
Figure 1.
Selected stephacidin family members (1–3) and target “probe” 4.
Figure 2.
The general method for conversion of indoles to α,β unsaturated nitrones.
Acknowledgments
We thank Erantie Weerapana of the Cravatt lab for biological tests. We are grateful to Universidad De Oviedo for predoctoral fellowship (M.E.S.) and Università degli Studi di Siena for postdoctoral fellowship (E.P.). Financial support for this work was provided by The Scripps Research Institute and the NIH (NIGMS).
Footnotes
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References and notes
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