Synthesis of the C₁–C₂₃ Fragment of the Archazolids and Evidence for V-ATPase but not COX Inhibitory Activity

Abstract

A convergent synthesis of a C₁–C₂₃ fragment of the archa­zolids has been completed based on a high-yielding Stille coupling to construct the substituted Z,Z,E-conjugated triene. After removal of the protecting groups, the resulting tetrol exhibited evidence for inhibition of the vacuolar-type ATPase (V-ATPase) but not cyclooxygenase (COX) inhibitory activity.

Key words

archazolid - natural product - Stille reaction - V-ATPase - cyclooxygenase - total synthesis.

The archazolid natural products[1] (A–F, Figure [1]) constitute a family of highly potent (subnanomolar IC₅₀) and selective vacuolar-type ATPase (V-ATPase) inhibitors that have shown promising activity against a number of particularly aggressive and lethal cancers including trastuzumab-resistant breast cancer,[2] glioblastoma multiforme (GBM),[3] and T-cell acute lymphoblastic leukemia (T-ALL).[4] More recent studies indicate that the archazolids also block iron metabolism and thereby mediate a therapeutic effect in breast cancers,[5] they modulate release of tumor-promoting cytokines,[6] and when combined with the p53 activator nutlin-3a, synergistically induce tumor cell death.[7]

Figure 1 Structure of the archazolid natural products

All members consist of a structurally similar 24-membered macrolactone and thiazole/carbamate side chain. Glycosylation at either the C₇- or C₁₅-hydroxyls (archazolids C and E, respectively) significantly reduces their V-ATPase inhibitory activity,[1d] indicating that these two groups form important interactions with the enzyme. Interestingly, these same hydroxyls are connected by a Z,Z,E-conjugated triene unique to the archazolids.

Figure 2 Reported synthetic approaches to the archazolid conjugated triene

Several synthetic strategies have been reported for the pharmacophorically relevant conjugated triene region of the archazolids (Figure [2]). Menche’s group ultimately utilized a three-step aldol condensation after attempted Horner–Wadsworth–Emmons reactions failed,[8] which then required a late-stage enantioselective CBS reduction to install the C₁₅ hydroxyl. Two palladium-catalyzed cross-couplings have also been described, one a successful, albeit low-yielding, Stille reaction[9] and the other a similar yet simpler ­Negishi coupling.[10] Recently, our group reported a synthesis of the archazolid triene by cross-metathesis (CM).[11] While promising, the convergence of this CM strategy is limited, for example due to the requirement of using a cis homodimer and issues associated with metathesis back-biting.[12c] Combined with nonideal aspects of the other synthetic approaches (e.g., low yields or requisite late-stage manipulations), we continued to explore alternative disconnections.

Herein we report the synthesis of a C₁–C₂₃ fragment of the archazolids based on a high-yielding and convergent Stille coupling for construction of the conjugated triene (Scheme [1]). The choice of coupling partner identity (i.e., which was the organostannane and halide) proved critical to the success of this reaction. After removal of the protecting groups, the V-ATPase and cyclooxygenase (COX) inhibitory activities of the resulting tetrol 1 were then assayed. The results suggest some level of V-ATPase inhibition by this compound but no significant interaction with COX.

Scheme 1 Novel Stille coupling-based synthesis of the archazolid triene containing compound 1

When considering alternative non-metathesis based archazolid syntheses, we nonetheless wanted to make use of the chemistry developed for these prior approaches.[12] To that end, aldehyde 2 was identified as a suitable starting point (Scheme [2]). This compound had been previously prepared en route to dihydroarchazolid B.[12c] We argue that saturation of the C₂–C₃ olefin (Figure [1]) would not be expected to negatively impact archazolid biological activity, but may simplify their synthesis and improve stability. In preparation for possible palladium-catalyzed cross-couplings, aldehyde 2 was first converted into vinyl iodide 3 in 85% as an 8:1 mixture of Z/E isomers by ¹H NMR analysis at the newly formed alkene.[13]

Scheme 2 Vinyl iodide synthesis for cross-coupling

Our ‘western hemisphere’ synthesis[12b] was adapted to produce an appropriate vinyl stannane coupling partner, accomplished through the use of phosphonate 4 which is available in two steps from known Weinreb amide 5 [14] (Scheme [3]). Horner–Emmons olefination[15] with aldehyde 6 [16] gave ketone 7 which was then reduced and methylated as previously described.[12b] Unfortunately all attempts to couple stannane 8 and iodide 3 failed. In each, unreacted iodide 3 was observed, suggesting difficulties in the oxidative addition step of the catalytic cycle, perhaps sterically hindered by the γ-methyl group.

Scheme 3 Vinyl stannane synthesis and first attempts at Stille coupling

Gratifyingly, switching the sense of organometallic/halide in these reactions now led to success with the couplings. Specifically, iododestannylation[17] of 8 gave iodide 9, and iodide 3 was converted into stannane 10 by lithium–halogen exchange and trapping with Bu₃SnCl (Scheme [4]).[18] These two compounds underwent very efficient coupling using Fürstner’s conditions,[19] providing 11 in 82% yield.

Scheme 4 Reagents and conditions: (a) I₂, CH₂Cl₂, –10 °C, 70%; (b) t-BuLi then Bu₃SnCl, 90%; (c) 10 (1.0 equiv), Pd(PPh₃)₄ (5 mol%), CuTC (2.0 equiv), [Ph₂PO₂][NBu₄] (3.0 equiv), THF, 15 h, 82%.

We were intrigued about the possibility of a compound of type 11 inhibiting V-ATPase function. Others have commented on the utility of natural product derived fragments for drug discovery,[20] particularly those that maintain essential pharmacophoric features. Previously we had tested both ‘western’ (12) and ‘eastern’ (13) hemispheres of the archazolids using an Arabidopsis V-ATPase assay and found that neither displayed measurable inhibitory activity (Figure [3]).[12b] However, these compounds lacked the important linked C₇- and C₁₅-hydroxyls present in tetrol 1.

Figure 3 Synthetic archazolid fragments; only compound 1 bears the linked C₇- and C₁₅-hydroxyls known to be important for V-ATPase inhibitory activity

As indicated in Figure [4], compound 1 [21] displayed dose-dependent growth inhibition of etiolated Arabidopsis.[22] A key component in the etiolated habit is stem elongation driven by V-ATPase-mediated cell expansion,[23] such that monitoring seedling stem length provides a measurement for V-ATPase activity. Previously, we demonstrated that stem elongation in Arabidopsis seedlings is inhibited by known V-ATPase inhibitors concanamycin A[24] and bafilomycin,[23] with concanamycin A exhibiting four times the potency of bafilomycin.[12b] The IC₅₀ value for compound 1 in this assay was approximately two orders of magnitude greater than that of concanamycin A and thus also the archazolids.[25] Nonetheless, this modest activity is significant given the major structural differences and overall simplification of 1 relative to the natural product.

Figure 4 Select Arabidopsis V-ATPase assay results[22]

Based on a recent report by Reker et al.,[26] we may also suspect other biochemical targets for compound 1. In their study, archazolid A (ArcA) was dissected into four hypothetical fragments (e.g., ArcA-1, Figure [5]) from which potential targets were predicted. This computational exercise identified primarily proteins associated with arachidonic acid (e.g., cyclooxygenase, COX). However when tested, the COX-2 inhibitory activity of ArcA was weak (24 ± 6% inhibition at 10 μM). Reker et al. suggest this disconnect between predicted hypothetical fragment activities and the actual natural product could be due to the COX active site being buried, allowing for binding of smaller fragments (e.g., ArcA-1 and arachidonic acid) but not ArcA.[26]

Figure 5 Structures of synthetic archazolid fragment 1, a hypothetical archazolid fragment ArcA-1 used by Reker,[26] archazolid A (ArcA), and arachidonic acid

We saw compound 1 as an opportunity to add additional experimental data to this theoretical work, representing a fragment similar to the hypothetical fragment ArcA-1. Interestingly, compound 1 did not show a dose response of greater than 5% inhibition of COX-1 or COX-2 at concentrations between 1 and 200 μM.[27] It is possible that compound 1 (C₂₃) is similarly too large compared to ArcA-1 (C₁₇) and arachidonic acid (C₂₀) to bind COX. Alternatively, the inactivity of 1 could suggest that a carboxylic acid terminus is critical, which is known to be important for other COX inhibitors.[28] However, this would not explain the measureable activity of ArcA which also lacks a carboxylic acid. Other factors such as binding entropies[29] might also therefore need to be considered (with ArcA being more conformationally restricted than 1) to understand the differential COX-inhibitory activity of these compounds.

In summary, we have developed an efficient synthesis of the archazolid macrolactone (C₁–C₂₃) framework. The resulting fragment 1 displayed evidence for inhibition of the V-ATPase, in line with the importance of properly linked C₇- and C₁₅-hydroxyls for archazolid/V-ATPase binding. Compound 1 was also assayed for COX inhibition based on previously reported predicted activities for a structurally similar hypothetical fragment. The results showed no significant COX-inhibitory activity for 1 (<5% inhibition at concentrations up to 200 μM) suggesting certain structural requirements for COX binding (i.e., carboxylic acid or macrocycle). Current efforts are aimed at advancing our understanding of archazolid structure–activity-relationships,[30] by utilizing 1 as a starting point to further probe the archazolid/V-ATPase and archazolid/COX interactions.

No conflict of interest has been declared by the author(s).

Acknowledgment

Financial support from the National Institutes of Health (R15GM101580) is gratefully acknowledged.

• References and Notes

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• 18b The Z,Z-stereochemistry of the resulting stannane was confirmed by detailed NMR analysis. See Supporting Information.
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• 21 Compound 11 To a solution of 9 (36 mg, 0.058 mmol, 1.0 equiv) and 10 (45 mg, 0.058 mmol, 1.0 equiv) in degassed THF (1.2 mL) was added [Ph₂PO₂][NBu₄] (75 mg, 0.17 mmol, 3.0 equiv), CuTC (28 mg, 0.07 mmol, 2.5 equiv), and Pd(PPh₃)₄ (7 mg, 0.006 mmol, 0.1 equiv), and the mixture was stirred for 15 h. The reaction was quenched with aq NaHCO₃ (15 mL) and extracted with MTBE (2 × 15 mL). The combined organic extracts were dried over MgSO₄, filtered, and concentrated in vacuo. Purification by flash chromatography on silica (20:1 to 10:1 hexanes–EtOAc) gave 11 (46 mg, 82%) as an oil. [α]_D ²⁰ –5.2 (c 0.5, CH₂Cl₂). IR (ATR): 3062, 2983, 1736, 1614, 1415, 1274, 1267, 1129, 1078, 930 cm^–1. ¹H NMR (500 MHz, C₆D₆): δ = 6.80 (d, J = 16.0 Hz, 1 H), 6.44 (ddd, J = 15.3, 10.7, 0.9 Hz, 1 H), 6.17 (d, J = 10.4 Hz, 1 H), 6.02 (s, 1 H), 5.90 (dd, J = 16.0, 7.2 Hz, 1 H), 5.58 (dd, J = 15.4, 8.0 Hz, 1 H), 5.34 (dd, J = 8.4, 0.8 Hz, 1 H), 5.29 (ddd, J = 9.8, 2.0, 1.0 Hz, 1 H), 5.07 (d, J = 7.4 Hz, 1 H), 4.29 (dd, J = 9.0, 6.0 Hz, 1 H), 3.63 (d, J = 10.0 Hz, 1 H), 3.58–3.53 (m, 2 H), 3.47 (dd, J = 9.7, 6.2 Hz, 1 H), 3.38 (dd, J = 9.7, 6.8 Hz, 1 H), 3.18 (s, 3 H), 2.71 (m, 1 H), 2.42 (m, 1 H), 1.96 (m, 2 H), 1.91 (s, 3 H), 1.86 (m, 1 H), 1.89 (d, J = 1.0 Hz, 3 H), 1.71 (d, J = 1.0 Hz, 3 H), 1.59 (s, 3 H), 1.50 (m, 2 H), 1.39 (m, 2 H), 1.08 (s, 6 H), 1.03 (s, 6 H), 1.02 (s, 3 H), 1.01 (s, 6 H), 1.00 (s, 3 H), 0.99 (d, J = 7.0 Hz, 3 H), 0.98 (s, 3 H), 0.96 (d, J = 7.0 Hz, 3 H), 0.95 (s, 6 H), 0.94 (d, J = 7.0 Hz, 3 H), 0.93 (s, 3 H), 0.19 (s, 3 H), 0.17 (s, 3 H), 0.13 (s, 6 H), 0.08 (s, 6 H), 0.05 (s, 6 H). ¹³C NMR (125 MHz, C₆D₆): δ = 137.8, 136.0, 134.9, 134.7, 133.4, 133.3, 133.0, 132.5, 130.9, 130.3, 126.3, 125.4, 73.8, 72.9, 68.5, 63.4, 63.4, 56.0, 43.7, 41.5, 40.6, 40.1, 33.1, 31.4, 30.3, 28.5, 26.7, 26.6, 26.5 25.4, 24.8, 24.8, 24.0, 21.0, 19.1, 18.9, 18.8, 17.4, 17.2, 17.1, 16.2, 14.3, 11.2, 9.8, 8.7, –3.0, –3.6, –4.2, –4.3, –4.4, –4.7, –4.8, –4.9. HRMS (ESI⁺): m/z calcd for C₅₅H₁₀₈O₅Si₄Na⁺ [M + Na]⁺: 983.7172; found 983.7179. Compound 1 To a solution of 11 (25 mg, 0.026 mmol) in THF (1.2 mL) at 0 °C was added pyridine (0.3 mL) and HF·py (60% HF, 0.2 mL), and the mixture was allowed to slowly warm to r.t. for 42 h. The reaction was cooled to 0 °C, diluted with EtOAc (15 mL), and quenched with aq NaHCO₃ (15 mL). The layers were separated, and the aqueous phase was re-extracted with EtOAc (2 × 15 mL). The combined organic extracts were dried over MgSO₄, filtered, and concentrated in vacuo. Purification by flash chromatography on silica (1:1 to 1:2 to 0:1 hexanes– EtOAc) gave 1 (7 mg, 58%) as an oil. [α] _D ²⁰ –6.0 (c 0.5, CH₂Cl₂). IR (ATR): 3350, 3955, 2929, 2872, 1716, 1688, 1525, 1471, 1418, 1369, 1244, 1126, 1084, 1008, 963, 919, 828, 730 cm^–1. ¹H NMR (500 MHz, CD₃OD): δ = 6.52 (dd, J = 16.0, 0.7 Hz, 1 H, H13), 6.29 (ddd, J = 15.0, 10.8, 0.7 Hz, 1 H, H₂0), 5.90 (d, J = 11.0 Hz, 1 H, H19), 5.74 (s, 1 H, H11), 5.70 (dd, J = 15.8, 5.9 Hz, 1 H, H14), 5.57 (dd, J = 15.2, 7.6 Hz, 1 H, H21), 5.09 (d, J = 9.0 Hz, 1 H, H9), 5.07 (d, J = 8.0, 1.0 Hz, 1 H, H6), 4.46 (dd, J = 6.0, 3.5 Hz, 1 H, H15), 4.05 (dd, J = 9.0, 6.1 Hz, 1 H, H7), 3.48 (t, J = 6.0 Hz, 2 H, H1a/b), 3.38 (d, J = 8.4 Hz, 1 H, H17), 3.37 (dd, J = 10.6, 5.5 Hz, 1 H, H23a), 3.33 (dd, J = 10.6, 6.5 Hz, 1 H, H23b), 3.09 (s, 3 H, OMe), 2.36–2.28 (m, 2 H, H8/22), 1.97 (t, J = 7.3 Hz, 2 H, H4a/b), 1.81 (d, J = 1.2 Hz, 3 H, Me10), 1.70 (s, 3 H, Me12), 1.64 (m, 1 H, H16) 1.58 (d, J = 1.0 Hz, 3 H, Me5), 1.56 (d, J = 1.0 Hz, 3 H, Me18), 1.48–1.40 (m, 4 H, H2/3), 0.96 (d, J = 7.0 Hz, 3 H, Me22), 0.81 (d, J = 7.0 Hz, 3 H, Me8), 0.59 (d, J = 7.0 Hz, 3 H, Me16). ¹³C NMR (125 MHz, CD₃OD): δ = 139.3, 138.7, 134.7, 134.4, 133.6, 132.7, 131.5, 130.5, 129.9, 127.3, 126.9, 90.4, 73.1, 73.0, 71.7, 68.2, 63.0, 56.3, 42.8, 41.3, 41.2, 40.7, 33.3, 25.3, 25.0, 20.5, 17.2, 17.0, 16.3, 11.1, 10.7. HRMS (ESI⁺): m/z calcd for C₃₁H₅₂O₅Na⁺ [M + Na]⁺: 527.3712; found 527.3729.
• 22 Assays were conducted side-by-side for compound 1 and concanamycin at concentrations of 0.125, 0.25, 0.5, 1.0, 2.0, 10.0, and 20.0 μM. See Supporting Information for details.
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Due to short supply, archazolids themselves were not assayed. However, reported V-ATPase inhibitory activity of concanamycin A and archazolid A are similar. The IC₅₀ values for concanamycin A and archazolid A of purified M. sexta holoenzyme were reported as 0.8 nmol/mg V-ATPase and 0.5 nmol/mg V-ATPase, respectively. Archazolid:
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Concanamycin:
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• 27 A COX activity assay kit was purchased from Cayman Chemical (https://www.caymanchem.com/product/760151, accessed Oct. 5, 2016). See Supporting Information for details.
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