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Synthesis of Conformationally Liberated Yohimbine Analogues and Evaluation of Cytotoxic Activity

A modular synthetic approach to strategically unique structural analogues of the alkaloid yohimbine is reported. The overall synthetic strategy couples the transition-metal-catalyzed decarboxylative allylation of 2,2-diphenylglycinate imino esters with a scandium triflate-mediated highly endo-selective intramolecular Diels–Alder (IMDA) cycloaddition to generate a small collection of de-rigidified yohimbine analogues lacking the ethylene linkage between the indole and decahydroisoquinoline units. One compound generated in this study contains an unprecedented pentacyclic urea core and appears to demonstrate increased cytotoxicity against the gastric cancer cell line SGC-7901 in comparison to a pancreatic cancer cell line (PATU-8988) and a normal human gastric mucosal cell line (GES-1).


INTRODUCTION
Yohimbinoids are a large family of polycyclic indole alkaloids that have long attracted synthetic and medicinal chemists alike due to the combination of dense functionality and diverse bioactivity. 1 The eponymous member of the family, yohimbine (1), is a potent pan-subtype α2-adrenergic receptor (α2-AR) antagonist that is a component used in traditional medicines and is a frequent chemical probe of α2-AR activity for various physiological processes. 2 There is a growing body of evidence demonstrating the potent cytotoxic activity of various yohimbinoids 3 and synthetic derivatives of the yohimbine framework. 4 For example, Ma and coworkers determined that yohimbine inhibited the proliferation of two different pancreatic cancer cell lines. 3b Recent innovative studies by Luesch and Huigens demonstrated that yohimbine can be used as a starting point to generate libraries of complex and selective small molecule modulators of other G-protein-coupled receptors (GPCRs) implicated in the proliferation of various cancers. 4 These reports epitomize the unwavering interest in generating novel yohimbine analogues with useful biological activities.
Several racemic 5 and asymmetric 6 total syntheses of 1 have been reported. Despite the tactical diversity displayed in the previously reported syntheses of 1 and structural derivatives, all these approaches are unified in their use of tryptamine (e.g., 2a) or the corresponding bromide 2b, resulting in a dearth of yohimbine structural derivatives that lack the rigidifying twocarbon linker in the C ring joining the indole and aliphatic amine (bold in Scheme 1A). This ethylene linker enforces coplanarity between the indole and piperidine D-ring units in 1, a factor that is predicted to account for the alkaloid's lack of selectivity in antagonistic activity against the various α2-AR subtypes. 2 To the best of our knowledge, only the aforementioned efforts from Luesch and Huigens have generated and evaluated yohimbine analogues, in which the C-ring ethylene linker has been severed. Specifically, they cleaved the C−N bond linking the C and D rings of (+)-1 with cyanogen bromide and converted the resulting N-cyano primary bromide into a low micromolar inhibitor of CCR8, a GPCR implicated in the cell migration of malignant melanomas. 4 As part of our broader campaign focused on the functionalization of semistabilized 2-azaallyl anions, 7−9 we recognized that the transition-metal-catalyzed decarboxylative allylation (DcA) strategy, advanced by our group 7 and others, 10 could allow for the rapid and modular access to novel "conformationally liberated" analogues of the yohimbine scaffold lacking the rigidifying C-ring ethylene linker (Scheme 1B). Specifically, subjection of the imine formed by the condensation of indole aldehydes 6 with 2,2-diphenylglycinate ester 7 to our Pd-7 or Ni-catalyzed 8 DcA conditions, followed by reductive amination with known O-Boc-protected glutaconaldehyde (5), 6a would quickly afford trienes 4. An endoselective intramolecular Diels−Alder (IMDA) cycloaddition would then complete the formation of the parent conformationally liberated yohimbine scaffold 3, which could be derivatized further by various functional group transformations. We were confident in the plausibility of this approach given that both Jacobsen (Scheme 2A) 6b and Hiemstra (Scheme 2B) 6a employed a similar endo-selective IMDA process in their syntheses of (+)-1, albeit with more conformationally restricted precursors in which the C-ring was already established.

RESULTS AND DISCUSSION
2.1. Synthesis. Our initial studies began with what was expected to be a routine imine condensation between aldehyde 6a and amino ester 7 (Table 1). Surprisingly, this transformation proved to be particularly challenging. The Bocprotecting group was readily hydrolyzed at elevated temperatures, and the electrophilic acrylate ester was prone to several side reactions. All standard methods for imine condensation were not acceptable means to generate the desired imine 8a. For example, heating to reflux in toluene with a Dean−Stark trap equipped with the reaction vessel led to the near-complete decomposition of the starting materials (Table 1, entry 1). Switching the solvent to dichloromethane and adding anhydrous magnesium sulfate as a desiccant to the refluxing reaction mixture simply resulted in the deprotection of the Boc carbamate in aldehyde 6a with a negligible imine condensation (entry 2). Similarly, heating the reagents in 1,2-dichloroethane with a buffered mixture of MgSO 4 and triethylamine resulted in almost quantitative transfer of the N-Boc group from indole 6a to amine 7 (entry 3). The best results following the traditional protocol were obtained using a laborious two-step/ two-reaction vessel process involving a combination of TiCl 4 and Et 3 N (entry 4), 11 but even with this modification, the isolated yield for imine 8a was moderate (46%). During these studies, we observed that the amine and aldehyde were both relatively low-melting compounds and were often obtained as honeylike oils. Since the condensation is a reversible reaction, we hypothesized that the higher concentration of the starting materials achieved in a solvent-free melt might be helpful. Accordingly, aldehyde 6a and amine 7 were combined and heated to 70°C under otherwise standard atmospheric conditions to afford the desired imine in 58% yield after 2 h (entry 5). The only other compounds observed in the reaction mixture were the starting materials; no products from decomposition or Boc-group migration were detected. This result encouraged us to develop the concept further by introducing low pressure using a standard laboratory vacuum pump to remove the water generated during the condensation. It should be noted that for the imine condensation between these substrates, water is the lowest boiling species present during the reaction. We were gratified to discover that simply heating a 1:1 mixture of 6a and 7 without any additional solvent or dehydrating agent at 70°C for 1 h in an open vial  ACS Omega http://pubs.acs.org/journal/acsodf Article and an additional 2 h under reduced pressure afforded imine 8a in a very high yield and purity (94%, entry 7). Directly heating a mixture of 6a and 7 under vacuum without premixing at atmospheric pressure afforded imine 8a in a reduced yield (81%, entry 6). This high-yielding vacuum-assisted solvent-free imine condensation strategy has proven to be quite general for the synthesis of various 2,2-diphenylglycinate imino esters, 7e,f,8 including those shown in Figure 1. It should be noted that the condensation does fail, however, to directly construct highly congested imines such as N,O-di-Boc-protected 8c ( Figure 1). Imine 8c could be generated in 64% isolated yield, however, by reacting alcohol 8d with Boc 2 O, DMAP, and Et 3 N in THF (see the Experimental Section). With a collection of imines 8 and 9 in hand, the proposed transition-metal-catalyzed DcA reaction was next investigated beginning with non-stereoselective reaction conditions (Table  2A). Previous studies indicated that the bidentate ligand 1,1′bis(diphenylphosphino)ferrocene (dppf) was appropriate for the construction of racemic homoallylic imines via a Pdcatalyzed decarboxylative generation and allylation of 2-azaallyl anions from imino esters. 7a−c,10 Subjecting acrylate 8a to these standard reaction conditions in acetonitrile afforded the corresponding homoallylic imine (±)-10a in 50% average yield (Table 2A, entry 1). The remainder of the starting material was converted into a mixture of products, in which either the 2-azaallyl anion intermediate was protonated or the diphenylmethine carbon of the delocalized anion attacked the π-allylPd(II) electrophile. Similarly, the 3-chloroindolyl imine 8b was converted to DcA product (±)-10b in 59% average yield (entry 2), whereas 3-alkyl-substituted indole 8c afforded imine (±)-10c in a higher yield (75%, entry 3). Remarkably, even imine 8d was a viable substrate for the racemic DcA reaction conditions, affording the corresponding homoallylic imine 10d in an admittedly low 30% average isolated yield (entry 4). This represents, to the best of our knowledge, the first example of a free indole N−H successfully participating in a Pd-catalyzed DcA reaction involving a relatively basic 2-azaallyl anion intermediate. In comparison to acrylates 8, the unsubstituted allyl esters 9 proved to be superior substrates for the racemic Pd-catalyzed DcA reaction conditions. For example, nearly quantitative conversion of 3-chloroindolyl imine 9b into homoallylic imine (±)-11b was observed (95%,   (refs 7a and 8), the major/first-to-elute enantiomer is presumed to be the (S) configuration. entry 6), marking a significant improvement over the transformation of the corresponding acrylate 8b (entry 2). We next explored the asymmetric transition-metal-catalyzed DcA using the chiral bisphosphine ligand (S,S)-f-binaphane (12), 12 which was identified in previous studies to impart significant enantioselectivity to related DcA transformations involving allyl 2,2-diphenylglycinate imines. 7b,c,8 Unfortunately, the Pd-catalyzed asymmetric DcA of acrylate ester 8a in acetonitrile (Table 2B, entry 1) or dimethylsulfoxide (entry 2) generated the desired homoallylic imine in modest yields (48− 64%) and negligible enantiomeric ratios (er). Switching to a Ni(0) catalyst did not improve the er for the DcA of acrylate 8a (entry 4). The yields (42−86%) and er values (up to 68:32 in favor of the S enantiomer) improved somewhat when allyl ester 9a was subjected to the Pd-catalyzed procedures (entries 4 and 5). The highest yield (93%), with an improved er value (70:30), was observed when allyl ester 9a was subjected to our Ni-catalyzed asymmetric DcA reaction conditions using chiral ligand 12 at 60°C (entry 6). 8 Lowering the reaction temperature of the Ni-catalyzed process to 45°C slightly increased the er to 75:25 but at a significant expense to the yield (entry 7).
Chlorination of the 3-position of the indole moiety (9b) dramatically reduced the isolated yields and observed er values for both the Pd-and Ni-catalyzed DcA transformations (entries 8−10).
Preliminary efforts indicate that homoallylic imine 11a can be converted to the desired acrylate 10a via olefin cross metathesis (see the Supporting Information). The relatively modest er values obtained in these studies, however, highlight the need for more extensive chiral ligand screening for the Pdand Ni-catalyzed asymmetric DcA of challenging substrates such as imino esters 8 and 9. Nevertheless, these efforts provided sufficient quantities of the desired δ-imino acrylates 10a−c for exploring the remainder of our proposed synthetic Scheme 3. Synthesis of the Conformationally Liberated Yohimbinoid Framework strategy toward novel racemic conformationally liberated analogues of yohimbine.
Toward this end, we first investigated the critical IMDA cycloaddition starting with racemic imine 10a (Scheme 3). Selective hydrolysis of the benzophenone imine over the Boc group was achieved with a combination of aqueous HCl in ethyl acetate and careful monitoring of the reaction progress by TLC. Two-step reductive amination between the resulting amine 13a and O-Boc-glutaconaldehyde (5) 6a afforded the IMDA precursor 4a in 56% isolated yield. Heating a solution of 4a in toluene to 70°C for 24 h led to the near-complete conversion of the triene into a 2:1 mixture of diastereomers in which the endo-chair IMDA cycloadduct endo-14a predominated. The minor diastereomer (exo-14a) was presumed to arise via an exo-chair transition state (Scheme 3, inset). In their synthesis of yohimbine from tryptamine, Hiemstra and coworkers observed a similar endo/exo ratio (3:1 for the free indole; Scheme 2B) for the thermal IMDA cycloadditions of a more conformationally restricted triene. 6a Subjecting triene 4a to a combination of scandium triflate (4 equiv) in acetonitrile at ambient temperatures, however, catalyzed the IMDA reaction so as to afford the endo-chair cycloadduct 15a as a single diastereomer with concomitant cleavage of the O-Boc moiety. The relative stereochemistry of 15a was determined by extensive 2D-nuclear magnetic resonance (NMR) spectroscopic analysis. It is currently unknown whether the cleavage of the carbonate occurs prior to, during, or after cycloaddition, but it should be noted that Jacobsen and coworkers also observed outstanding diastereoselectivity for a related Lewis acid-catalyzed IMDA reaction using a more stable terminal O-benzoate group on their diene component (Scheme 2A). 6b Although the Sc(OTf) 3 -mediated IMDA of triene 4a always proceeded with high diastereoselectivity, it proved particularly challenging to completely remove the sodium triflate formed during the workup of the reaction from the resulting product 15a, thus impacting our ability to accurately determine the yield of the transformation. The removal of the lingering NaOTf could be achieved after acidic hydrolysis of the Boc carbamate on the indole nitrogen, however. Accordingly, the treatment of the product of the IMDA cycloaddition of 4a with trifluoroacetic acid afforded the single cycloadduct 16a in 59% isolated yield over the two steps. Alternatively, Sc(OTf) 3 -mediated IMDA of unprotected indole 4a′ directly afforded triflate-free cycloadduct 16a as a single diastereomer in 65% isolated yield.
The other IMDA precursors 4b and 4c were obtained following a process similar to the route used to generate 4a in Scheme 3 (see the Supporting Information for more details). Mixing 3-chloroindole 4b with Sc(OTf) 3 in acetonitrile afforded the endo-chair cycloadduct 16b as a single diastereomer in 72% yield (Scheme 4). The IMDA cycloaddition of the N,O-di-Boc-protected precursor 4c was more complicated, however. Treatment of 4c with Sc(OTf) 3 in acetonitrile at 0°C and workup with saturated aqueous sodium bicarbonate at this temperature led to the isolation of the IMDA cycloadduct 15b (72%), in which both O-Boc groups in the starting material were removed under the reaction conditions. Quenching the IMDA reaction mixture with NaHCO 3 at a slightly elevated temperature (35°C), however, resulted in the exclusive formation of the cyclic urea 17. As with the IMDA cycloadduct 15a, cyclic urea 17 retained up to 0.5 equiv of sodium triflate, even after extraction and silica gel chromatography, thus complicating the accurate calculation of the yield. This phenomenon proved to be advantageous for structure confirmation, however. A single crystal of 17 cocrystallized with NaOTf (2:1 ratio) and water crashed out of the NMR sample (CDCl 3 solvent) and X-ray crystallographic analysis of this crystal verified the presence of the novel pentacyclic core and the relative stereochemistry in the IMDA product ( Figure 2, CCDC deposition number: 2082133). It is presumed that the carbonyl group linking the two nitrogens in 17 comes from the indole Boc group on the IMDA precursor 4c, but this was not confirmed definitively.
As detailed in Table 3, the IMDA cycloadducts 15 and 16 were converted to the corresponding decahydroisoquinolines 18 by catalytic hydrogenation. The Boc-protected indoles 15a and 15b and the free indole 16a were readily converted to the corresponding hydrogenated products 18a−c, respectively, in good yields (65−83%) using methanol as the solvent. When chloride 16b was subjected to these conditions, the hydrogenated and dechlorinated product 18c was obtained exclusively (not shown). Simply changing the solvent for the catalytic hydrogenation of 16b from MeOH to EtOAc afforded the corresponding chloride 18d in 70% isolated yield. . ORTEP diagram of (±)-17 cocrystallized with NaO 3 SCF 3 (2:1 ratio) and water (from CDCl 3 ) obtained by single-crystal X-ray crystallography (CCDC-2082133, C = gray, H = white, F = green, N = blue, Na = cyan, O = red, and S = yellow).
Decahydroisoquinoline 18c was further elaborated in two steps to yohimbine analogue 19 (Scheme 5). Specifically, reductive amination between amine 18c and O-TBDPSprotected hydroxyacetaldehyde, 13 followed by the removal of the silyl-protecting group with tetrabutylammonium fluoride, afforded the tertiary amine 19 in 27% yield over the two steps. It is worth noting that alcohols 19 and 18b both contain all of the carbons and heteroatoms present in yohimbine (1). While our goal in this project was always to generate and evaluate the biological activity of novel conformationally liberated analogues of 1, it is tempting to consider using alcohols 18b or 19 as late-stage intermediates in the total synthesis of this venerable alkaloid. Preliminary attempts to convert either of these two alcohols into racemic 1, however, have not borne fruit.
2.2. Biological Activity. As mentioned in the Introduction, the α2-AR antagonist yohimbine (1) exhibits proapoptotic activity against two different pancreatic cancer cell lines (PC-2 and PC-3). 3b The synthetic strategies discussed above afforded us access to novel structural analogues to 1, and we sought to determine whether these analogues also possessed appreciable cytotoxic activity against similar cancer cells. Toward this goal, an MTT colorimetric assay was employed to determine the cytotoxicity against one pancreatic cancer cell line (PATU-8988) and one gastric cancer cell line (SGC-7901) for four of the novel yohimbine analogues obtained in our study (15a, 16a, 16b, and 17). The general cytotoxicity of these four compounds was evaluated using the same assay against a normal human gastric mucosal cell line (GES-1). The results from these assays are summarized in Table 4. The four compounds all demonstrated modest (sub-millimolar) cytotoxicity against all three cell lines. The Boc-protected indole 15a proved to be an average of 2 times more toxic than the corresponding free indole 16a, but essentially no difference was observed between 16a and the corresponding chloride 16b. While 15a, 16a, and 16b did not display any significant selectivity between the different cell lines, the more conformationally constrained cyclic urea 17 appeared to be more active (potentially 4-fold) against the gastric cancer cell line SGC-7901. To the best of our knowledge, the tetrahydro-6H, 8Hpyrido[1′,2′:3,4]imidazo [1,5-a]indol-6-one core of 17 is unique to this compound; no other examples of molecules containing this heterotetracyclic core were detected in multiple online database searches. Given the structural novelty of this core and the potential for selective cytotoxicity against cancer cells, future investigations will focus on the synthesis and detailed evaluation of libraries of small molecules inspired by cyclic urea 17.

CONCLUSIONS
We demonstrated that the transition-metal-catalyzed DcA of 2,2-diphenylglycinate imino esters, a process championed by our group, 7−9 can be combined with a Lewis acid-mediated endo-selective IMDA cycloaddition to afford a relatively rapid and modular strategy for the racemic synthesis of novel conformationally liberated analogues of the alkaloid natural product yohimbine. The synthesis of the necessary imino ester precursors 8 and 9 required employment of a nontraditional solvent-free imine condensation under reduced pressure. Although modest enantioselectivity was observed using chiral bisphosphine 12 in the Pd-and Ni-catalyzed DcA of these indole-containing imino esters, more extensive studies are still required for this strategy to reliably generate the novel yohimbine analogues disclosed herein as single enantiomers. Nevertheless, we demonstrated that four racemic yohimbine 15a Reaction conducted in EtOAc instead of MeOH.
Scheme 5. Synthesis of Conformationally Liberated Yohimbine Analogue 19 Moreover, the structurally unique cyclic urea 17, which was an unexpected product from the IMDA cycloaddition of the corresponding triene 4c, appears to exhibit selectivity in its cytotoxicity against the gastric cancer cell line SGC-7901 versus a pancreatic cancer cell line (PATU-8988) and a normal gastric mucosal cell line (GES-1). We intend to further explore the novel heterocyclic core present in 17 as a scaffold for other potentially selective cytotoxic small molecules.
4. EXPERIMENTAL SECTION 4.1. General Methods. All nonaqueous reactions were performed in oven-dried or flame-dried flasks or vials under an atmosphere of dried and deoxygenated argon with dry solvents and magnetic stirring, unless stated otherwise. All solvents were dried by storing over activate 4 Å molecular sieves for at least 48 h and sparged with dried and deoxygenated Ar gas for at least 30 min. 14 Unless noted otherwise, all reagents were purchased from commercial sources and used as received. Triethylamine (Et 3 N) was distilled and stored over potassium hydroxide before use. Allyl bromide was filtered through basic alumina immediately prior to use. Ester 7 and O-allyl 2,2diphenylglycinate were obtained by following the methods previously reported by our group. 7 The synthesis of imine 9a and its conversion into scalemic (S)-11a under Ni catalysis was reported previously by our group. 8 1H-Indole-2-carbaldehyde, 15 N-Boc-indole-2-carbaldehyde (6a), 16 3-chloro-1Hindole-2-carbaldehyde, 17 3-chloro-1-Boc-indole-2-carbaldehyde, 17 3-(2-hydroxyethyl)-1H-indole-2-carbaldehyde, 18 and O-Boc-glutaconaldehyde (5) 6a were all prepared by following literature procedures. All chromatography techniques were performed with indicated solvents over 60 Å 230−400 mesh silica gel (solvent abbreviation: PE = petroleum ether). Reaction progress was monitored using a TLC plate under UV light (254 nm) and ninhydrin or potassium permanganate stains. Unless otherwise noted, all yields in the main text refer to average isolated yields after column chromatography of at least two separate runs at different scales. Accordingly, these yields may differ from the specific examples provided below. Melting points were determined with INESA melting point apparatus and were uncorrected. The compound characterization (NMR spectroscopy, infrared (IR) spectroscopy, highresolution mass spectrometry (HRMS), and X-ray crystallography) was performed by the Comprehensive Specialized Laboratory Training Platform, College of Chemistry, Sichuan University. IR spectra were obtained with a NEXUS 670 FTIR using a thin film deposited on freshly made KBr disks; only strong and functional group-specific peaks were reported (in cm −1 ). All 1 H and 13 C NMR spectra were recorded using a Bruker Ascend 400 at 300 K, as indicated. Chemical shifts are reported in δ (ppm) units using residual solvent peaks ( 1 H/ 13 C: CDCl 3 δ 7.26/77.16, CD 3 CN δ 1.94/1.32, CD 3 OD δ 3.31/49.00, DMSO δ 2.50/39.52) as a standard or an internal standard of tetramethylsilane ( 1 H/ 13 C δ 0.00/0.00) if the residual solvent peak overlapped with compound signals. Initial diastereomeric ratios were determined from the 1 H NMR analysis of the crude reaction mixtures. Er values were determined from samples separated on a Chiral Technologies Daicel OD-H or AD-H chiral column and comparison to the racemic standard. HRMS were obtained using an LCMS−IT− TOF. Single-crystal X-ray crystallography was performed on a New Gemini, Dual, Cu at home/near, EosS2 diffractometer.
The crystal was kept at 160(15) K during data collection. Using OLEX2, 19a the structure was solved with the ShelXT 19b structure solution program using direct methods and refined with the ShelXL 19c refinement package using least-squares minimization.