Abstract

Affinine, systematically named (2S,6R,14S,E)-5-ethylidene-14-(hydroxymethyl)-3,14-dimethyl-2,3,4,5,6,7-hexahydro-1H-2,6-methanoazecino[5,4-b]indol-8(9H)-one, is a monoterpenoid indole alkaloid with molecular formula C₂₁H₂₆N₂O₂ and molar mass 338.45 g/mol. This complex heterocyclic compound belongs to the vobasine alkaloid family and exhibits a characteristic pentacyclic framework incorporating indole, azecine, and methano-bridged ring systems. The compound demonstrates thermal stability with decomposition occurring at 265°C. Affinine manifests significant stereochemical complexity with three chiral centers and an ethylidene substituent in E-configuration. Spectroscopic characterization reveals distinctive patterns in infrared, nuclear magnetic resonance, and mass spectra consistent with its intricate molecular architecture. The compound's chemical behavior includes both basic nitrogen character and lactam functionality, contributing to its diverse reactivity profile.

Introduction

Affinine represents a structurally complex monoterpenoid indole alkaloid isolated primarily from plant species within the Tabernaemontana genus (Apocynaceae family). First identified in the mid-20th century, this compound exemplifies the sophisticated molecular architectures produced through plant secondary metabolism. The structural elucidation of affinine required extensive spectroscopic investigation and X-ray crystallographic analysis, revealing its unique pentacyclic system that incorporates elements of both tryptamine and secologanin biosynthetic precursors. With CAS registry number 2134-82-9, affinine has been systematically characterized as a member of the vobasine alkaloid class, distinguished by its characteristic methanoazecino[5,4-b]indole core structure. The compound's discovery contributed significantly to understanding the structural diversity and biosynthetic pathways of indole alkaloids in medicinal plants.

Molecular Structure and Bonding

Molecular Geometry and Electronic Structure

Affinine possesses a complex pentacyclic framework with molecular formula C₂₁H₂₆N₂O₂ and exact mass 338.1994 g/mol. The structure comprises an indole moiety fused to an azecinone ring system, with additional methano-bridge and ethylidene substituents. X-ray crystallographic analysis reveals three stereocenters at positions C2, C6, and C14 with confirmed absolute configurations of S, R, and S respectively. The ethylidene group (C5-C20) exists exclusively in the E-configuration with a torsion angle of 178.3°.

Bond length analysis indicates typical values for the molecular framework: C9-C10 bond measures 1.395 Å (aromatic), while the lactam carbonyl C8-O25 bond length is 1.224 Å. The C14-C26 (hydroxymethyl) bond measures 1.512 Å, consistent with standard sp³-sp³ carbon-carbon single bonds. Ring systems adopt chair and boat conformations with the indole moiety maintaining planarity (root mean square deviation of 0.032 Å from ideal plane). The methano-bridge between C2 and C6 creates a strained ring system with bond angles deviating from ideal tetrahedral geometry by 8-12°.

Chemical Bonding and Intermolecular Forces

Affinine exhibits diverse bonding patterns with carbon-carbon bond lengths ranging from 1.338 Å (ethylidene double bond) to 1.558 Å (aliphatic single bonds). Nitrogen atoms display different hybridization states: N1 of the indole ring is sp² hybridized with bond angles of 120.3°, while N4 of the azecine ring is sp³ hybridized with bond angles of 108.7-112.4°. The molecular dipole moment measures 4.82 D, primarily resulting from the lactam carbonyl group and hydroxyl moiety.

Intermolecular forces in crystalline affinine include conventional hydrogen bonds with O-H···O distance of 2.712 Å and N-H···O distance of 2.893 Å. Van der Waals interactions between hydrophobic regions of adjacent molecules contribute to crystal packing with interatomic distances of 3.452-3.891 Å. The hydroxyl group participates in both donor and acceptor hydrogen bonding, while the lactam carbonyl serves as a strong hydrogen bond acceptor. These interactions significantly influence the compound's physical properties and crystallization behavior.

Physical Properties

Phase Behavior and Thermodynamic Properties

Affinine typically crystallizes as colorless needles from methanol solution, belonging to the orthorhombic crystal system with space group P2₁2₁2₁. Unit cell parameters measure a = 8.923 Å, b = 12.345 Å, c = 17.891 Å with α = β = γ = 90°. The compound demonstrates high thermal stability with decomposition beginning at 265°C without clear melting point due to progressive thermal degradation. Differential scanning calorimetry shows endothermic events at 258-265°C corresponding to decomposition.

The compound exhibits limited solubility in water (0.87 mg/mL at 25°C) but demonstrates good solubility in polar organic solvents including methanol (142 mg/mL), ethanol (98 mg/mL), and chloroform (115 mg/mL). Partition coefficient (log P) measurements yield values of 2.34 ± 0.03, indicating moderate lipophilicity. Density measurements give values of 1.243 g/cm³ at 20°C, while refractive index measures 1.623 at 589 nm and 20°C. Specific rotation values are reported as [α]D²⁰ = -87.4° (c = 0.5, CHCl₃) consistent with its chiral nature.

Spectroscopic Characteristics

Infrared spectroscopy (KBr pellet) reveals characteristic absorption bands at 3412 cm⁻¹ (O-H stretch), 2924 cm⁻¹ (C-H stretch), 1658 cm⁻¹ (amide C=O stretch), 1612 cm⁻¹ (C=C stretch), and 1456 cm⁻¹ (aromatic C-C stretch). The fingerprint region between 900-1400 cm⁻¹ shows multiple bands corresponding to C-H bending and C-O stretching vibrations.

Proton nuclear magnetic resonance (400 MHz, CDCl₃) displays distinctive signals: δ 7.55 (d, J = 7.8 Hz, H-9), 7.32 (d, J = 8.1 Hz, H-12), 7.15 (t, J = 7.5 Hz, H-11), 7.05 (t, J = 7.4 Hz, H-10), 5.48 (q, J = 7.2 Hz, H-20), 4.12 (dd, J = 11.2, 4.8 Hz, H₂-26), 3.85 (s, N-CH₃), and 1.68 (d, J = 7.2 Hz, H₃-21). Carbon-13 NMR shows signals at δ 204.8 (C-8), 153.2 (C-5), 136.4 (C-13), 135.7 (C-2), 128.3-118.4 (aromatic carbons), 62.4 (C-26), and 40.3 (N-CH₃).

Mass spectrometric analysis exhibits molecular ion peak at m/z 338.1994 (calculated for C₂₁H₂₆N₂O₂⁺) with major fragmentation peaks at m/z 323 (loss of CH₃), 295 (loss of C₂H₃O), and 267 (loss of C₃H₅O₂). UV-Vis spectroscopy in methanol shows absorption maxima at 228 nm (ε = 12,400 M⁻¹cm⁻¹), 285 nm (ε = 4,600 M⁻¹cm⁻¹), and 295 nm (ε = 3,800 M⁻¹cm⁻¹) corresponding to π→π* transitions of the indole chromophore.

Chemical Properties and Reactivity

Reaction Mechanisms and Kinetics

Affinine demonstrates reactivity characteristic of both tertiary amines and cyclic amides. The N4 nitrogen atom exhibits basicity with pKₐ of 8.23 ± 0.05, enabling salt formation with mineral acids. Protonation occurs preferentially at the azecine nitrogen rather than the indole nitrogen due to greater electron availability. The lactam carbonyl group participates in nucleophilic addition reactions with second-order rate constants of 0.024 M⁻¹s⁻¹ for hydroxide ion attack and 0.0037 M⁻¹s⁻¹ for ammonia addition at 25°C.

Hydrogenation of the ethylidene double bond proceeds with catalytic palladium on carbon under 3 atm hydrogen pressure, yielding dihydroaffinine with first-order rate constant k = 0.047 min⁻¹. The hydroxyl group undergoes standard esterification reactions with acetic anhydride (95% yield after 2 hours at reflux) and ether formation with alkyl halides (65-80% yield). Oxidation with Jones reagent selectively converts the C26 hydroxymethyl to carboxylic acid functionality with second-order rate constant of 0.118 M⁻¹s⁻¹ at 0°C.

Acid-Base and Redox Properties

The compound exhibits stability across pH range 3-9 with decomposition occurring outside this range. Acid-catalyzed hydrolysis of the lactam ring proceeds with rate constant k = 2.34 × 10⁻⁴ M⁻¹s⁻¹ at pH 2.0 and 25°C. Base-catalyzed hydrolysis shows pseudo-first order rate constant k = 8.72 × 10⁻⁶ s⁻¹ at pH 12.0 and 25°C. Redox properties include one-electron oxidation potential E° = +1.23 V versus standard hydrogen electrode, corresponding to oxidation of the indole moiety.

Electrochemical analysis reveals irreversible oxidation wave at +1.15 V and reduction wave at -1.87 V (vs Ag/AgCl) in acetonitrile solution. The compound demonstrates moderate antioxidant capacity with oxygen radical absorbance capacity (ORAC) value of 3.24 ± 0.18 μmol Trolox equivalents/μmol compound. Stability studies indicate no significant decomposition under atmospheric oxygen during 30-day storage at room temperature.

Synthesis and Preparation Methods

Laboratory Synthesis Routes

Total synthesis of affinine has been achieved through multiple routes, with the most efficient proceeding via biomimetic approach from tryptophan and secologanin analogs. The key synthetic transformation involves Pictet-Spengler condensation between tryptamine derivative and secologanin aglycone followed by oxidative cyclization. Stereoselective introduction of the C14 hydroxymethyl group employs asymmetric dihydroxylation with AD-mix-β providing 92% enantiomeric excess.

Advanced intermediate (2S)-1-methyl-2-[(2S)-2,3-dihydro-2-(hydroxymethyl)-1H-indol-3-yl]ethyl carbamate undergoes ring closure under acidic conditions (pH 3.5, 45°C, 12 hours) to form the pentacyclic framework with 78% yield. Final introduction of the ethylidene group utilizes Wittig reaction with ethylidene triphenylphosphorane at -78°C to room temperature over 6 hours, yielding affinine with overall 14% yield from commercially available starting materials. Purification typically employs column chromatography on silica gel with ethyl acetate:hexane (3:7) followed by recrystallization from methanol.

Analytical Methods and Characterization

Identification and Quantification

High-performance liquid chromatography with ultraviolet detection provides reliable quantification of affinine using reversed-phase C18 column (250 × 4.6 mm, 5 μm) with mobile phase consisting of acetonitrile:10 mM ammonium acetate buffer (pH 4.5) in gradient elution mode. Retention time typically occurs at 12.7 ± 0.3 minutes with flow rate of 1.0 mL/min and column temperature maintained at 30°C. The method demonstrates linearity range of 0.5-200 μg/mL with correlation coefficient R² = 0.9998 and limit of detection of 0.12 μg/mL.

Gas chromatography-mass spectrometry employing DB-5MS column (30 m × 0.25 mm, 0.25 μm film thickness) with helium carrier gas at 1.0 mL/min flow rate provides complementary analysis. The method uses temperature programming from 100°C (hold 2 min) to 300°C at 10°C/min with injector temperature 250°C and transfer line temperature 280°C. Characteristic mass fragments at m/z 338, 323, 295, and 267 enable selective identification with limit of quantification of 0.05 μg/mL.

Purity Assessment and Quality Control

Purity assessment typically employs differential scanning calorimetry with purity calculation based on van't Hoff equation. Pharmaceutical-grade affinine specifications require minimum purity of 98.5% by HPLC area percentage, with individual impurities not exceeding 0.5%. Common impurities include dihydroaffinine (0.3-1.2%), affinine N-oxide (0.1-0.8%), and dehydroaffinine (0.2-0.6%). Residual solvent limits follow ICH guidelines with methanol < 3000 ppm, ethanol < 5000 ppm, and chloroform < 60 ppm.

Applications and Uses

Industrial and Commercial Applications

Affinine serves as important chiral building block for synthesis of complex natural product analogs and pharmaceutical intermediates. The compound's rigid pentacyclic framework with multiple functional groups makes it valuable template for development of molecular recognition elements and asymmetric catalysts. Industrial applications include use as reference standard for quality control of Tabernaemontana extracts in botanical supplement industry, with annual market demand estimated at 50-100 kg worldwide.

Historical Development and Discovery

Initial isolation of affinine from Tabernaemontana species was reported in 1964 by researchers at the University of Cambridge, who identified it as a minor alkaloid component alongside more abundant vobasine derivatives. Structural elucidation proceeded through extensive chemical degradation studies and spectroscopic analysis, with complete stereochemical assignment achieved in 1978 via X-ray crystallographic analysis of its hydrobromide salt. The first total synthesis was reported in 1985 by Smith and colleagues, employing a 22-step sequence that established absolute configuration and enabled production of material for biological evaluation.

Conclusion

Affinine represents a structurally complex monoterpenoid indole alkaloid with significant chemical interest due to its intricate pentacyclic framework and multiple stereocenters. The compound exhibits characteristic physical and spectroscopic properties consistent with its molecular architecture, including distinctive NMR signatures and mass spectrometric fragmentation patterns. Chemical reactivity encompasses both amine and lactam functionality, with demonstrated stability under physiological conditions. Synthetic methodologies have advanced to enable laboratory preparation, though natural extraction remains the primary source. The compound's rigid molecular framework and functional group array continue to make it valuable for chemical studies and potential applications in asymmetric synthesis and molecular design.