Unified Total Synthesis of the Limonoid Alkaloids: Strategies for the De Novo Synthesis of Highly Substituted Pyridine Scaffolds

Abstract

Highly substituted pyridine scaffolds are found in many biologically active natural products and therapeutics. Accordingly, numerous complementary de novo approaches to obtain differentially substituted pyridines have been disclosed. This article delineates the evolution of the synthetic strategies designed to assemble the demanding tetrasubstituted pyridine core present in the limonoid alkaloids isolated from Xylocarpus granatum, including xylogranatopyridine B, granatumine A and related congeners. In addition, NMR calculations suggested structural misassignment of several limonoid alkaloids, and predicted their C3-epimers as the correct structures, which was further validated unequivocally through chemical synthesis. The materials produced in this study were evaluated for cytotoxicity, anti-oxidant effects, anti-inflammatory action, PTP1B and Nlrp3 inflammasome inhibition, which led to compelling anti-inflammatory activity and anti-oxidant effects being discovered.

Keywords: DFT; Limonoid alkaloid; NMR computation; granatumine A; pyridine synthesis; terpenoid; total synthesis; xylogranatopyridine B.

Scheme 1.

Select pyridine-containing limonoid alkaloids

Scheme 2.

Hypothetical biosynthetic pathways to xylogranatopyridine B and bislactone limonoid alkaloids

Scheme 3.

Three strategies to access xylogranatopyridine B

Scheme 4.. First-generation synthetic effort toward xylogranatopyridine B through Risch pyridine synthesis^a

^aReagents and conditions: (1) NaH (1.1 equiv), THF, 65 °C, 2 h, then HMDS (0.15 equiv), 15 m, then MeI (1.1 equiv), 0 °C, 3 h, 79%; (2) N,N-dimethylmethyleneiminium chloride (4–6, 1.1 equiv), MeCN, 80 °C, 1 h; (3) 3–3 (1.2 equiv), 3–2 (1.0 equiv), NH₄OAc (3.0 equiv), EtOH, 80 °C, 14 h, then O₂, 1 h, 16% over two steps; (4) LDA (1.1 equiv), 3-furaldehyde (1.2 equiv), THF, −78 °C, 0.5 h; (5) Ac₂O (3.5 equiv), DMAP (0.2 equiv), pyridine (5.0 equiv), CH₂Cl₂, 0 to 23 °C, 51% over two steps, 4:1 dr; (6) KOt-Bu (1.2 equiv), THF, −78 °C, 0.5 h; (7) Burgess reagent (1.5 equiv), PhMe, 80 °C, 1 h, 36% over two steps.

Scheme 5.. Second-generation synthetic effort toward xylogranatopyridine B through Stille coupling^a

^aReagents and conditions: (1) I₂ (1.3 equiv), pyridine (2.7 equiv), CH₂Cl₂, 0 to 23 °C, 3 h, 67%; (2) LDA (1.2 equiv), 3-furaldehyde (1.2 equiv), −78 °C, THF, 0.5 h; (3) Ac₂O (2.0 equiv), DMAP (0.1 equiv), pyridine (3.0 equiv), CH₂Cl₂, 0 to 23 °C, 0.5 h, 61% over two steps, 6:1 dr; (4) KOt-Bu (1.2 equiv), −78 °C, THF, 15 m; (5) SOCl₂ (2.0 equiv), pyridine (3.0 equiv), CH₂Cl₂, 0 to 23 °C, 0.5 h, 48% over two steps; (6) Pd(dppf)Cl₂ (10 mol %), Sn₂(n-Bu)₆ (1.2 equiv), 1,4-dioxane, 100 °C, 12 h, 52%.

Scheme 6.. Third-generation synthetic effort toward xylogranatopyridine B through Liebeskind pyridine synthesis^a

^aReagents and conditions: (1) LDA (1.1 equiv), 3-furaldehyde (1.2 equiv), −78 °C, THF, 0.5 h, 88%; (2) Ac₂O (2.0 equiv), pyridine (3.0 equiv), DMAP (0.1 equiv), 0 °C, CH₂Cl₂, 0.5 h, 94%; (3) LiTMP (2.5 equiv), −78 to 23 °C, THF, 1 h, then Burgess reagent (3.0 equiv), 60 °C, 2 h, 67%; (1’) LiCl (0.1 equiv), CuI (5 mol %), TMSCl (1.1 equiv), MeMgBr (1.2 equiv), −40 °C, THF, 10 m; (2’) AgNO₂ (1.2 equiv), TIPSCl (1.4 equiv), −40 °C, MeCN, 2 h, then −40 to −20 °C, 2 h, 63% over two steps; (3’) Ph₃P=CH₂ (3.0 equiv), PhMe, 23 to 60 °C, 1.5 h, 77%; (4a’) RCl (1.1 equiv), Et₃N (2.0 equiv), DMAP (0.1 equiv), CH₂Cl₂, 0 to 23 °C, 1 h, R = Pentafluorobenzoyl (PfBz), 83%, R = Piv, 89%; (4b’) For R = Bz, Bz₂O (1.1 equiv), Et₃N (2.0 equiv), DMAP (0.1 equiv), CH₂Cl₂, 0 to 23 °C, 1 h, 80%; For R = Ac, Ac₂O (2.0 equiv), Et₃N (3.0 equiv), DMAP (0.1 equiv), CH₂Cl₂, 0 to 23 °C, 1 h, 76%. ^bYield of the crude reaction mixture on 0.2 mmol scale, was determined by ¹H NMR using 1,3,5-trimethoxybenzene as an internal standard.

Scheme 7.. Optimization of fragment coupling partners for the Liebeskind pyridine synthesis

^aYield of the crude reaction mixture on 0.1 mmol scale, was determined by ¹H NMR using 1,3,5-trimethoxybenzene as an internal standard. ^b16 h. See also Scheme SI1.

Scheme 8.. Synthesis of xylogranatopyridine B utilizing a late-stage benzylic oxidation^a

^aReagents and conditions: (1) Cu(OAc)₂ (1.0 equiv), quinuclidine (2.0 equiv), 3–6 (1.0 equiv), 3–7 (1.5 equiv), 60 °C, DMF, 12 h, then O₂ (1 atm), 43%; (2) Cr(V) = Na[OCr(O₂COC(CH₃)C₂H₅)₂] (10.0 equiv), 15-crown-5 (0.5 equiv), 75 °C, MeCN, 14 h, 56% 8–1 + 27% 3–1; (2’) 10 mol % Mn(OAc)₃·2H₂O, tBuOOH (4.0 equiv), MeCN, 23 °C, 16 h, 34% 8–1 + 47% 3–1; (3) Zn(TMP)₂ (1.4 equiv), [Pd(allyl)Cl]₂ (5 mol %), diethyl allyl phosphate (1.2 equiv), 85 °C, 1 h, 67%; (4) TBSOTf (0.05 equiv), 8–4 (1.5 equiv), CH₂Cl₂, 0 to 23 °C, 0.5 h, 74%, 1:1 dr at C5; (5) TMAF (1.5 equiv), MeI (2.0 equiv), 4Å MS, DME, −40 to 23 °C, 1 h, 23% 1–1 (1:1 dr at C10) + 26% 8–6; (5’) Et₂Zn (2.0 equiv), CH₂I₂ (4.0 equiv), 0 to 23 °C, PhMe, 6 h, then TBAT (10.0 equiv) in THF, 12 h, 75%, >20:1 dr; (6’) [PtCl₂(C₂H₄)]₂ (10 mol %), CH₂Cl₂, 23 °C, 5 h, 69% 1–1 + 4% 8–8. ^bYield of the crude reaction mixture on 0.01 mmol scale, was determined by ¹H NMR using 1,3,5-trimethoxybenzene as an internal standard. Conversion of starting material in parenthesis. ^c1:1 dr. ^dReaction mixture was heated to 100 °C for 12 h. ^eReaction mixture was heated to 60 °C. ^fReaction was conducted at 23 °C. See also Scheme SI2–4.

Scheme 9.

Four strategies to access (+)-granatumine A

Scheme 10.. First-generation synthetic effort toward (+)-granatumine A through Liebeskind pyridine synthesisa ^a

^aReagents and conditions: (1) 5 mol % (S,S)-(+)-N,N′-Bis(3,5-di-tert-butylsalicylidene)-1,2-cyclohexanediaminomanganese(III) chloride, 4-phenylpyridine-N-oxide (5 mol %), aq. NaOCl (1 equiv), CH₂Cl₂, 0 to 23 °C, 43%, 89:11 er, 10:1 dr; (2) 1 mol % [Rh(COD)(OH)]₂, PhMe₂SiH (1.3 equiv), THF, 23 to 60 °C, 2 h, 89%; (3) O₃, acetone, −78 °C 0.5 h, then Jones reagent (2.0 equiv), 0 to 23 °C, 2 h, 51%; (4) N,N-dimethylmethyleneiminium chloride (1.1 equiv), MeCN, 70 °C, 1 h. ^bYield of the crude reaction mixture on 0.01 mmol scale, was determined by ¹H NMR using 1,3,5-trimethoxybenzene as an internal standard. Conversion of starting material in parenthesis.

Scheme 11.. Second-generation synthetic effort toward (+)-granatumine A through alkylation^a

^aReagents and conditions: (1) LDA (1.5 equiv), ZnCl₂ (2.0 equiv), −40 °C, THF, 0.5 h, then allyl acetate (1.2 equiv), 1 mol % [Pd(allyl)Cl]₂, 60 °C, 5 h, 76%; (2) LDA (1.2 equiv), 3-furaldehyde (1.2 equiv), −78 °C, THF, 0.5 h, 82%; (3) 10 mol % (+)-tetramisole (11–5), Ac₂O (0.5 equiv), PhMe, 0 °C, 10 h, 41% 11–6 (91:9 er) + 46% (–)-11–4; (4) LiHMDS (4.0 equiv), −78 to 23 °C, 4 h, then Burgess reagent (4.0 equiv), 60 °C, 3 h, 80%; (5) SeO₂ (2.5 equiv), Na₂HPO₄ (5.0 equiv), 1,4-dioxane, 100 °C, 14 h, 31% 11–7 + 28% 9–7 + 27% 11–1; (6) PBr₃ (1.2 equiv), CH₂Cl₂, 0 to 23 °C, 43%; (6”) Dess−Martin periodinane (1.5 equiv), CH2Cl2, 23 °C, 1 h, 52% over two steps; (7) NaSO₂Ph (1.5 equiv), DMF, 45 °C, 1 h, 94%; (1’) HO–NH₂·HCl (2.0 equiv), NaOAc (1.5 equiv), EtOH, 23 °C, 0.5 h; (2’) TBSCl (1.5 equiv), imidazole (3.5 equiv), DMF/CH₂Cl₂ (1:2), 0 to 23 °C, 14 h, 69% over two steps; (3’) NBS (1.2 equiv), AIBN (0.1 equiv), CCl₄, 70 °C; (4’) NaSO₂Ph (2.0 equiv), DMF, 45 °C, 18 h, 2:1 dr; (5’) TBSCl (1.5 equiv), imidazole (3.5 equiv), DMF/CH₂Cl₂ (1:2), 0 to 23 °C, 14 h, 48% over three steps. See also Scheme SI5.

Scheme 12.. Third-generation synthetic effort toward (+)-granatumine A through Mukaiyama aldol^a

^aReagents and conditions: (1) TESOTf (1.2 equiv), Et₃N (2.0 equiv), CH₂Cl₂, 0 to 23 °C, 2h, 88%; (2) 9–7 (1.0 equiv), BF₃·OEt₂ (0.1 equiv), CH₂Cl₂, −78 to 23 °C; (3) Burgess Reagent (1.5 equiv), PhMe, 80 °C, 1.5 h, 67% over two steps; (4) HO–NH₂·HCl (1.5 equiv), NaOAc (2.0 equiv), EtOH, 80 °C, 14 h, 92%. ^bUB3LYP/6–311++G(d,p)//UB3LYP/6–31+G(d,p), ωB97x-D/6–311++G(d,p)//ωB97x-D/6–31+G(d,p), and M06–2X/6–311++G(d,p)//M06–2X/6–31+G(d,p) levels of theory were used in the computational evaluation. See also Table SI4–5.

Scheme 13.. Synthetic approaches to the 1,3-diketone fragment^a

^aReagents and conditions: (1’) KHMDS (1.1 equiv), MeOTf (1.1 equiv), THF, −78 to 23 °C, 1.5 h; (2’) Mn(OAc)₃·2H₂O (20 mol %), t-BuOOH (5.0 equiv), EtOAc, 65 °C, 14 h, 54% over two steps; (3’) AcOH/H₂SO₄ (1:1), 100 °C, 14 h, 82%; (1”) Pd(TFA)₂ (5 mol %), DMSO (10 mol %), O₂ (1 atm), AcOH, 80 °C, 12 h, 92%; (2”) Urea·H₂O₂ (3.0 equiv), DBN (3.0 equiv), H₂O (9.0 equiv), THF, 0 to 23 °C, 5 h, 67%, 1:1 dr; (3”) Pd(OAc)₂ (5 mol %), XPhos (5 mol %), PhMe, 120 °C, 15 h, 90%. ^b1,4-Dioxane was used as solvent unless otherwise specified. ^cYield of the crude reaction mixture, using 0.2 mmol 13–6, was determined by ¹H NMR using dibromomethane as an internal standard. Conversion of 13–6 in parenthesis. ^dToluene was used as solvent.

Scheme 14.. Fourth-Generation synthetic effort toward (+)-granatumine A, (+)-xylogranatin F, and (+)-granatoine employing a pyran-to-pyridine conversion^a

^aReagents and conditions: (1’) ethylenediammonium diacetate (0.7 equiv), 9–7 (1.5 equiv), (CH₂Cl)₂, 65 °C, 4 h, 47%; (2”) LiBH₄ (4.5 equiv), CeCl₃·7H₂O (4.5 equiv), CF₃CH₂OH/THF (1:1), 0 to 23 °C, 3 h, 61%; (3”) HO–NH₂·HCl (5.0 equiv), LiOAc·H₂O (6.0 equiv), MeOH, 80 °C, 12 h, 34%; (1”) ethylenediammonium diacetate (0.9 equiv), 9–7 (1.5 equiv), (CH₂Cl)₂, 65 °C, 4 h, 25%. See also Scheme SI6–8.

Scheme 15.. Computational investigation of the regioselective pyran formation and experimental evidence^a

^aCalculations were performed using ωB97x-D/6–311+G(2d,p)//ωB97x-D/6–31+G(d,p). Free energies (in kcal/mol). See also Scheme SI10 and Table SI6.

Scheme 16.. Structure revision of four bislactone limonoid alkaloids and structural corroboration of (+)-granatumine A guided by GIAO NMR computation^a

^aGeometry optimization calculations were performed using B3LYP/6–31+G(d,p) level of theory with the SMD solvation method (chloroform), while NMR single point calculations were conducted using mPW1PW91/6–311+G(2d,p) for all the above calculations. See also Table SI7–10.

Scheme 17.. Completion of the synthesis of (+)-granatumine A, the revised structures of (+)-xylogranatin G and (+)-xylogranatin F^a

^aReagents and conditions: (1’) SOCl₂ (7.0 equiv), CH₂Cl₂, 40 °C, 12 h, then NaOMe (10.0 equiv), MeOH, 70 °C, 5 h, 82%, 1:1 dr; (1”) SOCl₂ (7.0 equiv), CH₂Cl₂, 40 °C, 12 h, then Zn(OAc)₂ (10.0 equiv), AcOH, 100 °C, 12 h, 74%, 1:1 dr; (2”) K₂CO₃ (7.0 equiv), MeOH, 60 °C, 5 h, 87%. See also Scheme SI9.

Scheme 18.. Preliminary results of PTP1B inhibitory activities of selected bislactone limonoid alkaloids and analogs^a

^aXF = xylogranatin F (16–1), XG = xylogranatin G (16–5), C3-epi-GA = C3-epi-granatumine A (16–8), C3-deoxy-XF = C3-deoxy-xylogranatin F (9–1)

Scheme 19.. ROS inhibitory activity of granatumine A^a

^aA) Fluorescence images of HepG2 cells stained with DCFHDA for 30 min respectively. Before staining, cells were intact (control), pretreated with 2 mM H₂O₂ for 2 h (H₂O₂) or pretreated first with 50 μM granatumine A (1–2) for 24 h and then with H₂O₂ (2 mM) for 2 h. Scale bar: 25 μM. n = 3 (3 wells or slides/group, mean ±SD). B) The statistically quantified data on the cellular fluorescence intensity which were first pretreated with 0–50 μM granatumine A (1–2) for 24 h and then with H₂O₂ (2 mM) for 2 h. The H₂O₂ group is when the concentration of granatumine A (1–2) is 0 μM. The data were the mean ± SD and were normalized to the H₂O₂ group. n = 3 (3 wells or slides/group). NS: not significant, *P< 0.05, **P< 0.01, ***P<0.001. ****P<0.0001. Hoechst 33342: 2′-(4-Ethoxyphenyl)-6-(4-methylpiperazin-1-yl)-1H,3′H-2,5′-bi-1,3-benzimidazole. DCFHDA, 2′,7′-Dichlorofluorescein diacetate. See also Figure SI80–83.

Scheme 20.. Effects on reducing IL-1β production by several limonoid alkaloid natural products and synthetic intermediates^a

^a(A) Evaluation of a panel of compounds on Nlrp3 inflammasome activation. Production of IL-1β by bone marrow derived macrophages (BMDMs) treated with 1 μg/μL LPS and 1 μM of corresponding compound (4-hr.) and primed with 5 mM ATP (1-hr.) as measured by ELISA. Control samples (untreated, LPS, and LPS/ATP) and compound-treated samples all contain DMSO (1:1000). Data is expressed as the mean +/− SEM and is representative of one independent experiment with n = 4 biological replicates. Statistical significance was determined by one-way analysis of variance (ANOVA) with Dunnett’s post-hoc test for multiple comparisons and α = 0.05. P-values are marked as follows: ****p < 0.0001. (B) Immunoblot analysis of caspase-1 inhibition and cytotoxicity of C3-keto-xylogranatopyran (14–1). Caspase-1 p20 levels and LDH release from wildtype BMDMs stimulated with LPS (1 ug/mL) and ATP (5 mM) and treated with 62.5, 125, 250, 500, or 1000 nM C3-keto-xylogranatopyran (14–1) as measured by immunoblot and LDH cytotoxicity assay. Data are expressed as the mean ± SEM of one independent experiment with n = 4–6 biological replicates. Caspase-1 p20 protein levels were determined by quantifying band intensity of each sample and are represented as total protein relative to LPS + ATP treated condition. Statistical significance was determined by one-way analysis of variance (ANOVA) with Dunnett’s post-hoc test for multiple comparisons and α = 0.05. P-values are marked as follows: **p<0.01, ****p < 0.0001. LPS, lipopolysaccharides. LDH, lactate dehydrogenase. See also Figure SI84.