Absolute Configuration of Isomeric Mono-Acetyl Tropane Alkaloids Using Chromatography-Assisted Chemical Correlation

Abstract

Objective: To establish the absolute configurations of isomeric 6β-acetyl-3α-tropanol (3) and 3α-acetyl-6β-tropanol (4), constituents of several species of Datura and other genera, using a combination of a simple hemi-synthetic pathway starting from racemic 6β-hydroxytropinone (1) to produce 3α,6β-tropanediol (2), and the use of chiral liquid chromatography coupled with mass and laser optical rotation detectors (LC-MS and LC-OR). Methods: In the case of 3, mono acetylation of the more reactive OH group at C-6 in 2 was achieved under controlled reaction conditions, while more vigorous conditions produced exclusively 3α,6β-diacetyltropane (5). On the other hand, isomer 4 was obtained by partial hydrolysis of the acetyl group at C-6 in 5. Samples of the three compounds were then subjected to chiral LC-MS and LC-OR, which showed chromatograms composed of two peaks of opposite optical rotation. Next, ten consecutive LC injections followed by fraction collection of the column elution, produced a set of micro-scale samples of opposite enantiomeric composition for each mono ester, which were then derivatized by further acetylation to convert the partially enantioenriched samples into their corresponding diacetates. The resulting samples were then evaluated using LC-MS, which allowed the correlation between the enantiomers of 3 and 4 with those of 5. Results: Considering that the absolute configuration of (−)-5 has been assigned previously as (1S, 3S, 5R, 6S), these chromatographic comparisons established the absolute configurations of monoacetates 3 and 4 as (−)-(1S, 3S, 5R, 6S)-3 and (+)-(1R, 3R, 5S, 6R)-3, and (−)-(1S, 3S, 5R, 6S)-4 and (+)-(1R, 3R, 5S, 6R)-4, respectively. Conclusion: A methodology based on chiral liquid chromatography allowed the establishment of the absolute configuration of isomeric monoacetates of tropandiol 3 and 4, directly from the racemic mixtures, and without resorting to the isolation of enantiomerically pure samples.

Introduction

Tropane alkaloids are a well-known group of secondary metabolites with diverse pharmacological and medicinal properties, and plant species that produce these compounds are often toxic and capable of producing different effects on the central nervous system.^1,2 A subgroup of these compounds derive from 3α,6β-tropanediol (2, Figure 1) in which each hydroxy group can be substituted by a large variety of aliphatic and aromatic esters, in turn producing a wide range of mono and diesters, often isomeric between them. Moreover, the optical activity of 2 is conserved in these derivatives, and therefore, optical isomerism is added as a further layer of complexity in their structural elucidation.^3–5

Figure 1.

Formulas of 3α,6β-tropandiol enantiomers and their acetates.

The absolute configuration of natural and synthetic tropanediol esters has been achieved previously using different strategies that include total synthesis, ^{6 1}H-NMR spectroscopy,^7,8 electronic,^9,10 and vibrational circular dichroism,^10–15 and optical rotation calculations. ¹⁰ Nevertheless, many mono and diesters of tropanediol remain without an established absolute configuration, and the stereochemical preferences of the diverse species that produce them is, in many cases, unknown.^3–5 A possible explanation for this is that these methodologies often need milligrams of high purity samples to be applied, a requirement often not met. More precisely, most alkaloids of these kinds have not been fully isolated, and their presence in plant material have been proposed based on GC-MS analysis of crude extracts. This is the case with 6β-acetyl-3α-tropanol (3, Figure 1), which has been reported as a constituent of Datura stramonium,^16–19 D quercifolia, ¹⁶ Brugmansia candida, ²⁰ B aurea, ²⁰ and Merremia guerichii, ²¹ in all cases through GC-MS data. Similarly, its positional isomer 3α-acetyl-6β-tropanol (4, Figure 1) has been detected using the same technique in alkaloid extracts of D stramonium,^17,19,22 D innoxia,^{16,20,23–25} D candida, ²⁶ D wrightii,^16,20 D ceratocaula, ²⁷ D leichhardtii, ²⁰ D metel, ²⁰ B suaveolens,^20,28 B candida, ²⁰ B insignis, ²⁰ and B aurea. ²⁰ Additionally, LC-MS techniques have also been used to identify 4 in plant material of D stramonium ²⁹ and D innoxia. ³⁰ Nevertheless, two samples isolated within the Australian continent, first from Peripentadenia mearsii ³¹ and later from Bellendena montana, ³² have been studied further, and optical activity data could be obtained from the isolated samples. In the first case, the alkaloid was found to be dextrorotatory and its absolute configuration was established as (1R, 3R, 5S, 6R)-4 by chemical correlation with (+)-(1R, 3R, 5S, 6R)-3α,6β-diacetyltropane (5, Figure 1) of known absolute configuration. ³¹ Surprisingly, in the second study the sample resulted to be optically inactive, and therefore, was characterized as a racemic sample. ³²

As an alternative to chemical and chiroptical methodologies that require fully isolated samples to yield a stereochemical identification, we have decided to embark on the development of a general methodology that combines modern chromatographic analytical techniques, such as hyphenated liquid chromatography and sample derivatization, with standard hemi synthesis, to establish the absolute configuration of tropanediol esters. A methodology like this will not only deliver the configuration of each compound, but also has the potential to be used in the stereochemical identification of natural samples, even when only alkaloid mixtures are available for analysis.

As a part of this effort, in the present study, we describe a method based on chiral liquid chromatography with mass (LC-MS) and optical rotation (LC-OR) detectors, that used racemic samples of 3, 4, and 5, together with sample derivatization, as the main tools to allow the absolute configuration assignment of these monoesters of tropandiol.

Results and Discussion

Commonly, to establish the absolute configuration of a given substance through chemical correlation, an enantiomerically pure or highly enriched sample is converted into another, so that their stereochemical identities can be related. An option to obtain this initial sample is from the chiral resolution of the corresponding racemic mixture, with chiral LC as one of the main tools used nowadays to achieve such separations. Nevertheless, this approach will require the sample to be in appropriate quantity and of purity, so that standard spectroscopic confirmation of the reaction product, including NMR and optical rotation measurements, is possible. A tempting alternative is to use racemic mixtures to achieve the necessary chemical transformations in larger amounts, and therefore avoid the more pressing limitations of working with enantiopure samples. However, this would require an alternative methodology to allow the stereochemical correlation of the transformed compounds. A methodology like this could be developed based on chiral liquid chromatography, and it would deliver a chemical correlation of these substances directly from the racemic mixtures, without resorting to the isolation of enantiomerically pure samples.

With this in mind, racemic mixtures of both tropanediol monoacetates, 6β-acetyl-3α-tropanol (3) and 3α-acetyl-6β-tropanol (4), and of the diacetate 5, was achieved using a simple but effective hemi-synthesis pathway based on the larger reactivity that the hydroxy group at C-6 shows relatively to the C-3 hydroxy group on 3α,6β-tropanediol (2), which in turn was prepared from the starting compound 6β-hydroxytropinone (1) (Figure 2). As a consequence of this difference, treatment of 2 with excess acetic anhydride in CH₂Cl₂ at room temperature yielded almost exclusively the monoacetyl at C-6 (3). On the other hand, 2 was converted to diacetate 5 when a larger excess of the acylating agent was used. Subsequently, a partial hydrolysis of 5 using a 2:1 mixture of 0.1 M aqueous NaOH and acetone, produced 4 in medium to high yield.

Figure 2.

Hemi-synthesis pathway for the preparation of 3, 4, and 5. Only the isomers with the (1R, 3R, 5S, 6R) absolute configuration are shown.

As expected, the scale of these reactions allowed the use of conventional spectroscopic techniques, in this case, ¹H-NMR, to confirm the chemical identity of the samples prepared, which were in line with reported literature data. ³¹ Nevertheless, these measurements do not yield any stereochemical information, and therefore, an alternative methodology to correlate the enantiomers of the monoesters with those of the diacetate needs to be developed.

As a first step, using the samples of 3, 4, and 5, a chromatographic method that allowed the chiral analysis of the racemic mixtures was developed. For this, two chiral stationary phases, based on functionalized amylose coatings, were tested under normal phase conditions, searching for the best stereoselectivities together with the shorter possible chromatographic times. On the other hand, a mass selective detector with an atmospheric pressure chemical ionization probe (APCI-MS) was selected as the main chromatographic detector, considering that target compounds lack UV-vis active chromophores. Additionally, the use of the APCI-MS detector allowed the higher selectivity and sensitivity needed for the next steps of the methodology.

This screening showed that the amylose-based chiral stationary phase with tris(5-chloro-2-methylphenylcarbamate) as chiral selector, is the best-suited chiral stationary phase, achieving satisfactory separations for all three compounds within the first 10 minutes, when a mixture of n-hexane and i-propanol 9:1, containing 0.3% diethylamine, was used as mobile phase (Figure 3). It is important to notice that baseline separations are not required since the main objective of these chiral analyses will be the qualitative characterization of the enantiomeric mixtures. Therefore, partial, but clear separations, as the one obtained for 4, are sufficient for the application in hand.

Figure 3.

Liquid chiral resolution chromatograms of 3 (left), 4 (middle), and 5 (right) using APCI-MS (up) and OR (down) detections. Chromatograms were obtained using a Lux® 3 μm Amylose-2 (150  ×  4.6 mm) column and a mixture of n-hexane and i-propanol 9:1 containing a 0.3% of diethylamine as mobile phase, with a flow rate of 1 mL/min.

Next, the change from APCI-MS detection to the laser polarimeter detector allowed the characterization of the peaks in each racemic sample, showing their optical rotation sign. As evidenced in Figure 3, in the chiral resolution of mono ester 3 the levorotatory enantiomer elutes before the dextrorotatory one, while for the mono ester 4, the elution order is reversed, where the dextrorotatory enantiomer elutes more rapidly than the levorotatory one, a trend that is repeated in the case of the diester 5.

Although the chromatographic peaks of each racemic mixture are now characterized, still no relationships between the corresponding enantiomers have been established. To overcome this, an approach, in which small microgram samples of the enantiomers of 3 and 4 are converted into 5 was attempted, and a fraction collector was added to the chromatographic system to collect consecutive 30 s fractions directly from the column out-flow during the chiral separations. This procedure was repeated 10 times for each monoester, and the corresponding fractions were evaporated to dryness, diluted with ethanol, and re-analyzed using LC-MS (Figures 4 and 5, left). In both cases, the fraction sets showed enantiomeric compositions that varied from a high enantiomeric excess (e.e.) of the first eluting enantiomer, then to a low e.e. mixture, and finally to a high e.e. of the second eluting enantiomer.

Figure 4.

LC-APCI-MS chromatograms of five 30 s fractions obtained from the chiral separation of 3. Analyses were performed before (left) and after (right) derivatization with acetic anhydride to convert monoester samples into diester 5. The optical rotations of the main peaks, as obtained from the previous LC-OR measurements, are shown for clarity.

Figure 5.

LC-APCI-MS chromatograms of five 30 s fractions obtained from the chiral separation of 4. Analyses were performed before (left) and after (right) derivatization with acetic anhydride to convert monoester samples into diester 5. The optical rotations of the main peaks, as obtained from the previous LC-OR measurements, are shown for clarity.

Next, the dried fraction sets were derivatized by treatment with an excess of acetic anhydride during 24 h to ensure conversion of the monoacetates into the diacetate, and the resulting mixtures were again evaporated to dryness, diluted with ethanol, and analyzed using LC-MS (Figures 4 and 5, right). As with the original fraction sets, chromatograms of the treated samples showed enantiomeric mixtures that progressively changed their e.e., but this time composed by the enantiomers of 5, and with trends that differ between both sample sets. In the case of 3, samples after derivatization showed the opposite layout as the original fractions, with the first samples richer in the second eluting enantiomer and vice versa, therefore, evidencing that both chiral separations produce different elution orders for the enantiomers of 3 and 5 (Figure 4). On the other hand, the treated samples from monoester 4 showed the same trend observed before derivatization, now with the first samples richer in the first eluting enantiomer of 5 and vice versa (Figure 5).

At this point, is important to notice that the collection of several samples of each chiral separation has the advantage of showing more clearly the enantiomeric composition of the treated samples, as the presence of a second enantiomer serves as a reference to whether the sample is enriched with the first or second eluting isomer. This allows a straightforward and robust correlation between the enantiomers of both compounds, which can become troublesome when only a partial separation is achieved.

Finally, with clear relationships between the eluting peaks of each racemic mixture, and using the polarimetric measurements that revealed the optical rotation signs of each chromatographic peak, it is now possible to establish a chemical correlation between the enantiomers of 3 and 4, and those of the diacetate 5. From these data, it can be concluded that (−)-3 and (−)-5 (first eluting peak of 3 and the second eluting peak of 5), and (+)-3 and (+)-5 (second eluting peak of 3 and the first eluting peak of 5) have the same absolute configuration, and since the absolute configuration of 5 is known, ¹² it follows that the absolute configuration of the enantiomers of 3 is (+)-(1R, 3R, 5S, 6R)-3 and (−)-(1S, 3S, 5R, 6S)-3. In the same way, a chemical correlation can be made between (+)-4 and (+)-5 (first eluting peak in both cases), and between (−)-4 and (−)-5 (second eluting peak in both cases). Likewise, with the known absolute configuration of 5, the absolute configuration of 4 can now be established as (+)-(1R, 3R, 5S, 6R)-4 and (−)-(1S, 3S, 5R, 6S)-4.

Conclusions

The difference in reactivity between the two hydroxy groups in 3α,6β-tropanediol (2) was sufficient to yield isomeric acetyl monoesters 6β-acetyl-3α-tropanol (3), and 3α-acetyl-6β-tropanol (4), along with the diacetyl derivative 3α,6β-diacetyltropane (5). These samples were then used to find suitable chiral chromatographic conditions that allowed the characterization of these racemic mixtures using HPLC with mass and optical rotation detectors, showing that, in the case of 3, the first eluting peak was the levorotatory enantiomer, while for 4 and 5, the first eluting peaks were the dextrorotatory enantiomers. Furthermore, consecutive chiral separations using an automatic fraction collector yielded a set of five fractions for each isomeric monoester, composed of different enantiomeric ratios that evolved from a high enantiomeric excess of the first eluting peak to a high e.e. of the second eluting peak. Next, on a derivatization step, these sample sets were treated with excess acetic anhydride and converted into diacetate samples with corresponding enantiomeric compositions, which in turn revealed the stereochemical relationship between mono and diesters when further analyzed through LC-MS. From these analyses, it turns out that the levorotatory enantiomers of 3 and 4 are related to the levorotatory enantiomer of 5, which has the known (1S, 3S, 5R, 6S) absolute configuration. From this, it follows that the absolute configurations of the enantiomers of 3 are (+)-(1R, 3R, 5S, 6R)-3 and (−)-(1S, 3S, 5R, 6S)-3, while the absolute configurations of the enantiomers of 4 are (+)-(1R, 3R, 5S, 6R)-4 and (−)-(1S, 3S, 5R, 6S)-4.

Experimental

Tropandiol Esters

Preparation of racemic tropane diester started from the reduction of commercial 6β-hydroxytropinone (1) with Adams catalyst to produce 3α,6β-tropanediol (2), as described for other tropinone derivatives. ³³ Then, 40 mg of the diol was treated with 2 mL of acetic anhydride, 1 mL of triethylamine, 20 mL of CH₂Cl₂, and molecular sieves (10 Å), and the mixture was kept under an Ar atmosphere and with magnetic stirring at room temperature for 24 h. The tropane monoester was obtained as a free base by treatment of the filtered reaction mixture with Na₂CO₃ 0.1 M and exhaustive extraction of the aqueous solution with CH₂Cl₂. The crude substance was further purified by flash chromatography using silica gel 60 and a mixture of CH₂Cl₂, MeOH, and aqueous NH₄OH (25:10:0.8) as a mobile phase, which afforded pure 6β-acetyl-3α-tropanol (3) as a white, highly hygroscopic solid. The same procedure was applied to produce 3α,6β-diacetyltropane (5), with the only difference being that the volume of acetic anhydride was increased to 5 mL and no CH₂Cl₂ was added to the mixture. In this case, the pure compound was obtained through flash chromatography using CH₂Cl₂, MeOH, and aqueous NH₄OH (32:6,5:0.6) as mobile phase, which showed identical spectroscopic characteristics as those obtained in a previous study. ¹²

The isomeric monoester 3α-acetyl-6β-tropanol (4) was obtained by partial hydrolysis of 5, according to a previously described procedure. ³⁴ For this, 150 mg of the diester was dissolved in a mixture of 4.5 mL of acetone and 10.5 mL of NaOH 0.1 M and stirred at room temperature for 30 min. The hydrolysis was quenched with drops of HCl 1 M, concentrated in vacuo to 5 mL, and then mixed with 30 mL of distilled water and 15 mL of NaCO₃ 0.1 M. This solution was then extracted with CH₂Cl₂ to afford a 2:1 mixture of 4 and 5, which was separated by silica gel flash chromatography using CH₂Cl₂, MeOH and aqueous NH₄OH (25:15:1,2) as mobile phase, affording pure 4 as a semitransparent white residue.

The identity and purity of the prepared compounds were verified by 300 MHz ¹H-NMR measurements in CDCl₃ solutions, containing TMS as the internal reference, on a Bruker AVANCE III HD spectrometer. The ¹H-NMR files of monoacetates 3 and 4 are available online as part of the Supplemental Information.

LC-MS and LC-OR Measurements

Chromatographic analyses were performed using a Shimadzu HPLC instrument composed of an LC-20AT quaternary pump, a SIL-10ADvp autosampler, a CTO-10ACvp column oven, a DGU-10 inline degasser, and a Waters WFC-III automatic fraction collector. Mass detection was performed using a Waters Micromass ZQ-2000 single-quadrupole mass spectrometer equipped with an atmospheric pressure chemical ionization (APCI) probe operated at 400 °C, with a corona current of 10 μA, and using N₂ as desolvation gas. Optical rotation detection was performed using a PDR Advanced Laser Polarimeter that used a 670 nm diode laser and a 25 mm (18 μL) capillary flow cell.

Chiral resolutions were tested with two amylose-based chiral stationary phases, one with amylose tris(3,5-dimethylphenylcarbamate) (Lux® 5 μm Amylose-1, 150  ×  4.6 mm), and another with amylose tris(5-chloro-2-methylphenylcarbamate) (Lux® 3 μm Amylose-2, 150  ×  4.6 mm), as chiral selectors. Both of them were used under normal phase conditions with a mixture of n-hexane and i-propanol as mobile phase, using 0.3% of diethylamine as a basic modifier, and with a flow rate of 1 mL/min. Mobile phases containing between 5% and 15% of i-propanol were tested, but 10% was finally used for the analyses.

Samples of 3, 4, and 5 were dissolved in ethanol with concentrations ranging from 2 to 3 mg/mL and were injected into the chromatograph in volumes between 1 and 5 μL. In the case of MS detection, the injected sample mass ranged between 2 and 15 μg, while for the OR detection, they ranged between 50 and 100 μg. This difference is due to the intrinsic disparity in sensitivity between the two detection methods.

Fraction Collection and Derivatization

For fraction collection, the column outflow was collected during 4 min centered at the retention time between the two chromatographic peaks, and in 30 s (0.5 mL) portions. The chromatographic run was repeated 10 times with injections of 50 μg of a sample, completing eight fractions of 5 mL each, which were then concentrated in a vacuum and diluted with 500 μL of ethanol before LC-MS analysis. Derivatization of these fractions into the diacetate was performed by treatment of the reduced samples in 5 mL vials containing 1 mL of acetic anhydride, 100 μL of triethylamine, and the addition of molecular sieves. The closed vials were kept at 35 °C and stirred for 24 h using an orbital shaker at 150 r/min, after which the filtered mixtures were reduced under vacuum and diluted with 500 μL of ethanol before LC-MS analysis.

Supplemental Material

sj-7z-1-npx-10.1177_1934578X231178607 - Supplemental material for Absolute Configuration of Isomeric Mono-Acetyl Tropane Alkaloids Using Chromatography-Assisted Chemical Correlation

Supplemental material, sj-7z-1-npx-10.1177_1934578X231178607 for Absolute Configuration of Isomeric Mono-Acetyl Tropane Alkaloids Using Chromatography-Assisted Chemical Correlation by Marcelo A. Muñoz and Fabián Cabrera Z. in Natural Product Communications