Synthesis of Bis-Strychnos Alkaloids ( – )-Sungucine , ( – )-Isosungucine , and ( – )-Strychnogucine B from ( – )-Strychnine

It was developed a concise synthetic route resulting in the first syntheses of bis-Strychnos alkaloids (–)-sungucine, (–)-isosungucine, and (–)-strychnogucine B from commercially available (–)-strychnine. Employing a highly convergent synthetic strategy, it was demonstrated that both Strychnos monomers could be efficiently prepared from commercially available (–)-strychnine. The venerable Mannich reaction was enlisted to join the two Strychnos monomers in a biomimetic fashion. Subsequent epimerization and olefin isomerization yielded (–)-strychnogucine B. Functional group manipulation transformed (–)-strychnogucine B into (–)-sungucine and (–)-isosungucine. Computational chemistry was employed to rationalize the regiochemical course of key steps en route to the bis-Strychnos targets.

As part of our research program aimed at developing and applying novel synthetic methods for efficiently accessing complex indole alkaloids over the past decade, [10][11][12][13][14][15][16][17][18][19][20] we recently communicated the first enantiospecific syntheses of bis-Strychnos alkaloids 1-3. 21Herein, we provide a full account of our synthetic efforts, from failure to success, employed in the preparation of these unique natural products.

Synthetic strategy and retrosynthetic analysis
We proposed a semi-synthetic route to these bis-Strychnos alkaloids beginning with natural (-)-strychnine (4), which was purchased from Sigma-Aldrich for approximately $2 per gram.Our rationale for this, as opposed to a fully synthetic route, included the following reasons.First, it was reported that isostrychnine and strychnine were isolated from S. icaja along with 1-3. 4,5Moreover, it is reasonable to hypothesize strychnine is involved in the biosynthesis of these bis-Strychnos congeners due to structural similarity, particularly strychnogucine B whose Southern monomer is a C5-α-substituted strychnine unit!Third, strychnine is commercially available, inexpensive, and would greatly expedite any preparation of 1-3 since it represents an advanced intermediate.Finally, a semi-synthetic approach from (-)-4 would guarantee an enantiospecific preparation of the targets, which is a prerequisite for biological evaluation.

Synthesis of the Southern fragment
For the synthesis of the Southern fragment, the main challenge to be addressed was the regioselective synthesis of the desired iminium ion from (-)-4.3][24] On the other hand, the Polonovski-Potier reaction is a well-established method for the preparation of iminium ions via the activation of tertiary amine N-oxides with trifluoroacetic anhydride (TFAA). 25,26Furthermore, the in situ-formed iminium ions are often trapped with cyanide to access the corresponding α-amino nitriles, which can be isolated and characterized.
To apply the Polonovski-Potier reaction in the synthesis of the Southern fragment, strychnine N-oxide (5) was first prepared from the hydrogen peroxide-mediated oxidation of (-)-strychnine (4).However, subjection of 5 to TFAA followed by trapping with KCN led to an intractable mixture of α-amino nitriles regioisomers (Scheme 2).
While exploring the regioselectivity of the Polonovski-Potier reaction, we carried out the reaction in the presence of Et 3 N.However, this led to the formation of the vinylogous trifluoromethyl amide 6, 37,38 the structure of which was confirmed by single crystal X-ray analysis (Scheme 3).The formation of 6 presumably arises via a stepwise process including (i) regioselective C5-N 4 iminium formation; (ii) base-promoted imineenamine tautomerism; (iii) nucleophilic attack of TFAA; and finally, (iv) elimination to furnish vinylogous trifluoroacetamide 6.
At this juncture, we were aware that the iminium salt generated from the Polonovski-Potier reaction of 5 with TFAA might be stable enough to be isolated without being trapped with KCN.By following Lounasmaa and Hanhinen 31 procedure, the Polonovski-Potier reaction was carried out with NaHCO 3 workup.The crude product was then subjected to flash column chromatography on basic Al 2 O 3 with EtOAc and hexanes as mobile phase solvents.The only isolated product from the reaction mixture was pseudo-strychnine (7) whose structure was confirmed by single crystal X-ray analysis (Scheme 3).
Interestingly, when the Polonovski-Potier reaction of 5 was followed by addition of an aqueous solution of KOH, a more basic and nucleophilic base, the desired carbinolamine 8 was isolated in 52% yield as a mixture of two epimers at C5, together with regioisomeric pseudostrychnine (7) in 40% yield (Scheme 3).
To rationalize the regiochemical course of the Polonovski-Potier reaction, density functional theory (DFT) analysis (mPW1PW91/cc-pvdz) was employed to determine relative free energies of the three iminium cations that may form upon trifluoroacetate elimination (Figure 2).It seems that the comparison of the relative calculated energies I-III cannot account for all the experimental results.For example, the carbinolamine derived from the lowest-energy iminium III was not obtained.However, the carbinolamine 7 derived from the highest-energy iminium II was isolated in 40% yield, as shown in Scheme 3. Other factors are believed to be at play to dictate the formation of the iminium ions.We then conducted further DFT analysis to determine the minimized structure of parent N-acyloxyammonium ion IV. 21Based on the minimized structure of IV, C5-H b bond and N4-O acyl bond are closest to be anti-coplanar (i.e., H b -C5-N 4 -O dihedral = 148.3°), the prerequisite for the E2 reaction pathway.Thus, the formation of 8 may result from the elimination of IV to I via an E2-like mechanism. 21 natural population analysis (NPA) 39 of IV suggests that since the C7-H has the more positive charge (+0.30252) than those on C5 and C21, and it is syn-coplanar to the N-O acyl bond, the iminium ion II is likely to be formed via the elimination of C7-H through an E1-like or E1cb-like transition state by means of the paired TFA (Figure 3).40,41 Another possibility is that the formation of II could be from the intramolecular deprotonation of C7-H by the pendant N-trifluoroacetyl moiety. 21To explore the mechanistic differences between the classic Polonovski reaction and the more active Potier variant, we also conducted experimental studies to investigate the effect of both acylating agent and temperature on the regioselective functionalization of strychnine N-oxide.41 As shown in Table 1, the reactions with TFAA (entries 1-3) were less sensitive than the ones with Ac 2 O (entries 4-6) in terms of both percent conversion and ratios of 8 to 7.These results can be attributed to the high nucleofugacity of trifluoacetate.41 The highest ratio of 8 to 7 was obtained at 35 °C for 4 h when Ac 2 O was used as acylating reagent (entry 6).These results are in line with an E2 mechanism, whereby the more basic acetate favors the formation of the desired carbinolamine 8 via the removal of the anti-periplanar C5-H b from an N-acyloxyammonium strychnine intermediate.
With the desired carbinolamine 8 in hand, we were able to test the feasibility of the Mannich coupling reaction. 42reatment of 8 with BF 3 OEt 2 43 followed by addition of silyl ketene acetal 9 44 produced the ester 10 in 59% yield (Scheme 4).Single crystal X-ray analysis of 10 confirmed the stereochemical course of the Mukaiyama-Mannich reaction and the regioselectivity of the Polonovski-Potier reaction of 5.
Later, when we tried to get a better chromatographic separation of 8 from 7, the crude reaction mixture from the sequential Polonovski-Potier reaction and KOH trapping was loaded on a neutral Al 2 O 3 column for the column chromatography purification.Carbinolamine 7 was quickly eluted off the column with 50% acetone in ethyl acetate.However, 8 remained on the column even when eluted   with 100% acetone.Switching acetone to 5% methanol in dichloromethane resulted in the isolation of a new compound, whose 1 H nuclear magnetic resonance (NMR) showed the presence of only one single isomer.Similarly, when 8 was treated with 5% methanol in dichloromethane in the presence of silica gel, the same single isomer was also obtained.To rationalize this observation, we hypothesized that the new product might be formed from the addition of methanol to the corresponding iminium ion 11, which was generated in the presence of Al 2 O 3 or silica gel. 45The quantitative conversion of 8 to N,O-acetal 12 was achieved when carbinolamine 8 was stirred in 5% methanol in dichloromethane.The stereochemistry of 12 was confirmed by single crystal X-ray analysis (Scheme 5).
The stereoselectivity of this conversion can be attributable to the steric hindrance.Specifically, during the attack of methanol on iminium ion 11, the presumed intermediate, methanol would experience more steric interactions on the β-face of C5-N4 double bond (concave side) than on the α-face (convex side).The same explanation is also applicable to the selective formation of ester 10 as a single diastereomer from the Mukaiyama-Mannich reaction of 8 with silyl ketene acetal 9. Considering the acidic proton in carbinolamine 8 may complicate the Mannich coupling reaction with the Northern fragment, we selected N,O-acetal 12 as the Southern fragment.

Synthesis of the Northern fragment
Our retrosynthetic plan of (-)-sungucine ( 1), (-)-isosungucine ( 2) and (-)-strychnogucine B (3) included the Northern fragment as an isostrychnine monomer with a suitably protected C18 hydroxyl moiety.A survey of the literature [46][47][48] showed the synthesis of isostrychnine from strychnine (i.e., degradation studies) had been long known with selected examples shown in Scheme 6.However, each protocol had its drawbacks such that in order to reliably access the Northern fragment on a multigram scale, we needed to develop a new protocol for converting strychnine (4) into isostrychnine (13).
Generally, the conversion of strychnine to isostrychnine consists of two seemingly simple steps. 49The first step is an acid-or base-mediated elimination of the ether oxygen at the β-position of the lactam carbonyl group.The second step involves the isomerization of the conjugated double bond in the α,β-position into a trisubstituted, nonconjugated double bond in the β,γ-position.
In order to develop a new protocol for the preparation of isostrychnine from strychnine, we realized that a prudent choice of a base was paramount.It was observed that bases used in the previous protocols might not be just promoters of the elimination of the ether oxygen but also perpetrators of the lactam opening.1][52] However, the use of more vigorous conditions (e.g., aqueous barium hydroxide at 140 °C53 or NaOH in boiling amyl alcohol) 54 led to the formation of isostrychnic acid (15), as shown in Scheme 7.
In light of these experimental facts, we believed that a bulky non-nucleophilic base would minimize the side reactions relating to the lactam ring-opening.The non-nucleophilic amidine base 1,8-diazabicyclo[5.4.0] undec-7-ene (DBU) has been utilized in a variety of basemediated transformations including eliminations and alkene isomerizations. 55Moreover, since only water was used in Pictet's synthesis of isostrychnine, it would be interesting to know what effect DBU would have on the reaction if it was added to a water suspension of strychnine. 46Some of the results are presented in Table 2.However, the conversion of strychnine was very low at 110 or 150 °C, even with up to 10 equivalents of DBU (Table 2, entries 1-2).Replacement of water with dimethyl sulfoxide (DMSO) did not improve the conversion.Conversely, another aprotic polar solvent, dimethylformamide (DMF), allowed for the formation of isostrychnine (13) in 29% yield after stirring strychnine (4) with 10 equivalents of DBU at 160 °C for 24 h (Table 2, entry 5).Further extension of the reaction time or increased temperature did not benefit the reaction.
Owing to the instability of DMF 56 or DMSO 57 at high temperatures, we opted for using a more stable, aprotic polar solvent with a higher boiling point.N-Methyl-2-pyrrolidinone (NMP) has been known to have advantages over both DMF and DMSO for nucleophilic displacement reactions due to its greater stability, 58 and its higher boiling point (202 °C) certainly was advantageous.Moreover, the Scheme 6. Prior art regarding the chemical transformations of strychnine (4).The reaction was carried out in a sealed tube; b isolated yields; c the product was not isolated due to low conversion.
formation of its conjugate base would be possible due to the acidity of its hydrogens (β to the lactam group), which then could serve as a potential source of base. 59,60Our preliminary experiments showed the best yield was 28% when NMP was used as solvent (Table 2, entry 7).We also found longer reaction times and high temperatures were detrimental to the reaction (Table 2, entry 8).Finally, upon treatment of a deaerated NMP solution of 4 with DBU at 200 °C for 40 min, a mixture of the expected isostrychnine (13) and 13-epi-isostrychnine (16)  was obtained in a 2:1 ratio in 61% yield (Scheme 8).It should be noted that the formation of 16 was also observed in the previous screening, but its characterization was not complete due to the difficulties with its purification (e.g., its high polarity and the presence of other polar byproducts).
The plausible reaction pathways of strychnine upon treatment with DBU are presented in Scheme 9. Anion 17 was assumed to be the product from the deprotonation step, whether or not the mechanism for the first step is E2 or E1cb.If 17 is stable enough during the reaction, it would give rise to the formation of 18.On the other hand, proton transfer of 17 would produce anion 19, from which all three alcohols (13, 16 and 18) could be formed.
To explain why only 13 and 13-epi-isostrychnine (16) were isolated from the three possible products, we turned to computational analysis using DFT.Calculations were performed at the mpw1pw91/cc-pvdz level of theory.Computational data were consistent with experimental findings wherein 18 is 7 kcal mol -1 higher in free energy than 16 and 6.5 kcal mol -1 higher in free energy than 13.
To facilitate the purification of 13 and 16, we treated the reaction mixture from the DBU-mediated reaction of strychnine with TBSCl (tert-butyldimethylsilyl chloride) and imidazole.The resulting products, TBS-protected isostrychnine (20) and TBS-protected 13-epi-isostrychnine (21), were isolated in 50 and 26% yields, respectively (Scheme 10).Thus, we developed a one-pot synthesis of the Northern fragment 20 from (-)-strychnine 4. With the synthetic routes to Northern and Southern fragments established, we turned to the task of joining the two via a biomimetic Mannich reaction.
2][63] To forge the characteristic C23-C5' bond in bis-Strychnos alkaloids 1-3, the reaction of the amide enolate derived from 20 with iminium ion 11 would be desirable.However, a survey of the literature regarding the reaction of amide enolates with iminium salts is surprisingly limited. 42Presumably, the challenges stem not only from the difficulties with the generation of amide enolates, but also from the higher basicity of the amide enolates.In particular, the use of strong bases to generate the amide enolates might not be compatible with the conditions for the generation of the iminium ions.Furthermore, the strong basic conditions could cause other side reactions with the iminium ions such as isomerization to the corresponding enamines in the presence of strong bases. 31ne way to circumvent such problems would be to employ the silyl enol derivative of 20, which would then be used in a separate Mukaiyama-Mannich reaction using in situ generated iminium ion 11.However, the conversion of 20 to its silyl enol derivative turned out to be unsuccessful in our hands (e.g., LHMDS/LiCl followed by trapping with TMSCl). 64,65Thus, this approach was abandoned.
At this stage, our attention returned to the generation of the amide enolate of 20.We were encouraged by a literature 66 example wherein ketone lithium enolates were reacted with trimethylorthoformate in the presence of BF 3 OEt 2 to give the dimethoxymethylated ketone, suggesting this Lewis acid was compatible with lithium enolates.In addition, it was found that Grignard reagents were employed for nucleophilic addition reactions with iminium ions, which indicated the isomerization of iminium ions to enamines in the presence of Grignard reagents could be avoided. 67,68ased on this information, we planned to employ the magnesium amide enolate of 20 for the Mannich coupling owing to its weaker basicity compared to that of a Li variant.Moreover, the Lewis acidity of Mg has been leveraged for the generation of iminium ions from N,O-acetals. 69The Mg amide enolate in turn could be obtained from the Li amide enolate via transmetallation with MgBr 2 OEt 2 . 70,71For the generation of iminium ion 11 from the Southern fragment 12, BF 3 OEt 2 was employed because of its successful reaction with carbinolamine 8 in our model Mukaiyama-Mannich studies (Scheme 4).Since both 20 and 12 contain several Lewis basic sites, an excess of BF 3 OEt 2 was employed to ensure the productive generation of 11.
The reaction results are summarized in Table 3. Lithiation of 20 with lithium di(isopropyl)amide (LDA) and subsequent transmetallation with MgBr 2 OEt 2 afforded the Mg amide enolate, which was added dropwise to a solution of 12 followed by treatment with BF 3 OEt 2 to afford Mannich base 22 as a mixture of epimers at C5'.For each entry in Table 3, the ratio of the epimers was not determined due to the instability of one epimer, which was slowly converted to the other isomer during isolation by silica gel chromatography.
As shown in Table 3, it was interesting to observe that increasing the equivalents of MgBr 2 OEt 2 had a negative effect on the reaction yields (Table 3, entries 1-4).Finally, the highest yield (67%) of 22 was obtained with 1.1 equivalents of LDA with no addition of MgBr 2 OEt 2 (Table 3, entry 6)!We attribute the increase in yield of 22 to the increased nucleophilicity of the Li enolate vis-à-vis the Mg enolate.
Nevertheless, the first Mannich reaction was accomplished between a Li amide enolate and N,O-acetal in the presence of BF 3 OEt 2 , and the successful formation of the key C23-C5' bond enabled the synthesis of bis-Strychnos alkaloids 1-3.Next, attention was turned to the synthesis of (-)-strychnogucine B (3).

Synthesis of (-)-strychnogucine B (3)
The synthesis of (-)-strychnogucine B (3) from 22 required two steps.The first step was the removal of the primary TBS ether.To our delight, treatment of 22 with HF pyridine in tetrahydrofuran (THF) buffered with pyridine [72][73][74] not only removed the TBS group but also effected epimerization at C23 to furnish 23 as a single isomer, the structure of which was further confirmed by X-ray analysis (Scheme 11).
The confirmation of the structure of 23 made us believe that the Mannich reaction of 20 with 12 might be under kinetic control and the more stable isomer of 22 should have the same stereochemistry at C23, although it is not the major isomer.To lend support to this assumption, a deuterium quench experiment was performed, in which treatment of 20 with LDA at 0 °C was followed by quenching with d-MeOD to give a crude product that was directly used for NMR analysis.The 1 H NMR of the crude product showed only one isomer 20-D, with the deuterium on the β side of C23, which was considered to be the kinetic product (Scheme 12).
Regarding the Mannich reaction of 20, it was assumed that iminium ion 11, generated from N,O-acetal 12, would attack Li amide enolate 20-Li from the β face to give the kinetic product 22-1.Since the latter is not the more stable epimer, it would undergo epimerization at C23 to give the thermodynamic epimer 22-2 (Scheme 13).As previously mentioned, the conversion of 22-1 to 22-2 was observed during the chromatographic purification.The last step for the synthesis of (-)-strychnogucine B (3) required the isomerization of the C16-C17 double bond in 23 to conjugation with the lactam carbonyl (i.e., C23-C17).To realize this transformation, we drew on a crucial discovery made by Magnus et al. 75 when attempting to improve the conversion of isostrychnine to strychnine.Specifically, they found that treating isostrychnine (13) with cesium carbonate in refluxing tert-butyl alcohol isomerized the olefin into conjugation with the lactam carbonyl, affording 13-epi-isostrychnine (16) (Scheme 14). 75fter inspecting the structure of 23, we were confident that the isomerization of 23 to strychnogucine B would take place, because the isomerized double bond not only is in conjugation with the lactam carbonyl group but is also trisubstituted one and therefore thermodynamically more favorable.To our delight, treatment of 23 with Cs 2 CO 3 in deaerated tert-butanol at 85 °C for 3 h furnished (-)-strychnogucine B (3) in 70% yield.Spectral data for 3 (e.g., 1 H NMR, 13 C NMR and infrared (IR)) were in complete agreement with those reported in the literature. 5he structure of 3 was further confirmed by X-ray analysis (Scheme 15).

Syntheses of (-)-sungucine (1) and (-)-isosungucine (2)
With (-)-strychnogucine B (3) in hand, a critical stage was reached for testing the projected conversion of 3 to (-)-sungucine ( 1) and (-)-isosungucine (2).We envisaged the conversion would be performed in two steps.The first step required the elimination of the allylic ether oxygen with concomitant ring-opening of the Southern oxepane F-ring, which could be accomplished by applying tactics used in the synthesis of Northern fragment 20.The successful application of this strategy would deliver two alkene regioisomers with the newly formed C=C double bond at different positions (i.e., conjugated and nonconjugated).Both regioisomers were important as bisdeoxygenation thereof would furnish (-)-sungucine ( 1) and (-)-isosungucine (2), respectively.The second step required deoxygenation site-specifically at C18 (Northern monomer) and C18' (Southern monomer).
When 24 was subjected to 33% HBr in HOAc, the corresponding allylic bromide HBr salt 25 was obtained (Table 4), which was subsequently treated with various hydride source to afford deoxygenated isostrychnine (26).Among the hydride sources screened [e.g., LiAlH 4 , LiBEt 3 H (Super-Hydride  ), 89 NaB(OMe) 3 H, and NaBH 3 CN], 90 Super-Hydride was found to give the best result (Table 4, entry 2).The success of this protocol was attributed to the protection of the tertiary amine in 26 as HBr salt, since other attempts to directly activate the hydroxyl group in isostrychnine (13) for deoxygenation were not successful.
Significantly, we required the bis-acetates as substrates for our two-step deoxygenation protocol (vide supra).
and 27-2, further illustrating the limitations associated with model studies in natural product total synthesis.Spectral data for 1 and 2 (e.g., 1 H NMR, 13 C NMR and IR) were in complete agreement with those reported in the literature. 4,5,91

Conclusions
In summary, the first syntheses of bis-Strychnos alkaloids (-)-strychnogucine B, (-)-sungucine and (-)-isosungucine have been successfully accomplished from inexpensive and commercially available (-)-strychnine.Key steps in the syntheses include the following: (i) a DBU-mediated conversion of strychnine into isostrychnine; (ii) a Polonovski-Potier reaction of strychnine N-oxide for regioselective Southern fragment activation; (iii) a stereoselective BF 3  OEt 2 -mediated Mannich reaction for the coupling of Northern and Southern fragments; (iv) a Cs 2 CO 3 -mediated olefin isomerization reaction; and (v) a NaBH 3 CN-mediated reduction of allylic bromide intermediates to install the requisite ethylidene moieties in the bis-Strychnos targets.

Experimental
All reactions containing moisture-or air-sensitive reagents were performed in oven-dried glassware under nitrogen or argon.Tetrahydrofuran, diethyl ether and dichloromethane were passed through two columns of neutral alumina prior to use.All other reagents were purchased from commercial sources and used without further purification.All solvents for work-up procedures were used as received.Flash column chromatography was performed according to the procedure of Still et al. 92 using 60 Å silica gel with the indicated solvents.For all ring-closing metathesis reactions, CH 2 Cl 2 was deaerated by bubbling argon (1 min mL -1 ).Thin layer chromatography was performed on 60 F 254 silica gel plates.Detection was performed using UV light, KMnO 4 stain, phosphomolybdic acid (PMA) stain and subsequent heating.Infrared spectra were measured on a Fourier transform infrared spectrometer (FTIR). 1 H and 13 C NMR spectra were recorded on a 500 MHz instrument in CDCl 3 at 298 K.Chemical shifts are indicated in parts per million (ppm) and internally referenced to residual solvent signals.Splitting patterns are abbreviated as follows: s (singlet), d (doublet), bs (broad singlet), bd (broad doublet), t (triplet), q (quartet) and m (multiplet).High-resolution mass spectra (HRMS) were obtained on a time-of-flight (TOF) mass spectrometer using an electrospray ionization (ESI) source.
-317.8 (c 0.9, CHCl 3 ); IR (neat) ν / cm  To a stirred solution of (i-Pr) 2 NH (7.5 µL, 0.054 mmol) in THF(1 mL) was added n-BuLi (2.35 M solution in hexanes, 20.9 µL, 0.049 mmol) at 0 °C.The mixture was stirred at 0 °C for 30 min, followed by addition of a solution of Northern fragment 20 (20 mg, 0.045 mmol) in THF (1 mL).The mixture was stirred at 0 °C for 1 h and the reaction was quenched by addition of MeOD (0.5 mL).The resulting mixture was extracted with EtOAc (2 × 5 mL) and the combined organic layers were dried over Na 2 SO 4 , filtered, and concentrated under reduced pressure to give 19 mg (95%) of 20-D as a gray foam.

Scheme 3 .
Scheme 3. The fate of the Polonovski-Potier reaction of N-oxide (5) depending on the base employed.

Scheme 7 .
Scheme 7.Reported lactam ring-opening of strychnine with different bases.

Table 1 .
Effect of acylating agent and temperature on the formation of 7 and 8Two equivalents of the reagent were used with CH 2 Cl 2 as a solvent; b iminium ion was trapped with aqueous KOH after 2 or 4 h; c percent conversion was calculated by 1 H NMR spectroscopy; d ratio of 7 to 8 was determined by 1 H NMR spectroscopy. a