Abstract

Cipargamin, systematically named (1′''R'',3′''S'')-5,7′-Dichloro-6′-fluoro-3′-methyl-2′,3′,4′,9′-tetrahydrospiro[indole-3,1′-pyrido[3,4-''b'']indol]-2(1''H'')-one, represents a synthetic spiroindolone compound with molecular formula C₁₉H₁₄Cl₂FN₃O. This complex heterocyclic system features a central spiro carbon atom connecting indole and tetrahydropyridoindole ring systems. The compound exhibits significant stereochemical complexity with two chiral centers and demonstrates characteristic physicochemical properties including limited aqueous solubility and moderate lipophilicity. Cipargamin manifests notable stability under ambient conditions and displays distinctive spectroscopic signatures across multiple analytical platforms. The compound's intricate molecular architecture presents synthetic challenges that have been addressed through innovative multi-step organic synthesis methodologies.

Introduction

Cipargamin belongs to the spiroindolone class of synthetic organic compounds, characterized by their unique spirocyclic architecture connecting indole and heterocyclic systems. The compound emerged from systematic drug discovery efforts focused on identifying novel chemical entities with specific biological activities. Its molecular complexity arises from the fusion of multiple heterocyclic systems including indole, pyridine, and lactam functionalities. The structural design incorporates halogen substituents at strategic positions that significantly influence electronic distribution and molecular recognition properties. The compound's development represents a significant achievement in synthetic organic chemistry, requiring sophisticated stereochemical control and regioselective functionalization strategies.

Molecular Structure and Bonding

Molecular Geometry and Electronic Structure

The molecular architecture of cipargamin features a central spiro carbon atom (C-3) that connects two orthogonal ring systems: a 2-oxindole moiety and a tetrahydropyrido[3,4-b]indole system. X-ray crystallographic analysis reveals that the spiro center adopts a nearly tetrahedral geometry with bond angles approximating 109.5°. The indole system displays planarity with maximum deviation from mean plane measuring less than 0.05 Å, while the tetrahydropyridoindole system exhibits slight puckering with dihedral angles of 15.2° between ring systems. The absolute stereochemistry is established as (1′R,3′S) configuration, with the methyl substituent at position 3′ adopting equatorial orientation relative to the piperidine ring.

Electronic structure analysis indicates significant π-delocalization throughout the conjugated systems. The highest occupied molecular orbital (HOMO) primarily resides on the indole nitrogen and adjacent carbon atoms, while the lowest unoccupied molecular orbital (LUMO) shows localization on the pyridoindole system and carbonyl group. Density functional theory calculations at the B3LYP/6-31G* level predict HOMO-LUMO gap of 4.2 eV, indicating moderate electronic stability. The molecular electrostatic potential map reveals regions of electron deficiency near halogen substituents and electron richness around the lactam oxygen.

Chemical Bonding and Intermolecular Forces

Covalent bonding in cipargamin follows typical patterns for polycyclic heteroaromatic systems. Carbon-carbon bond lengths in aromatic regions range from 1.38 Å to 1.42 Å, consistent with sp² hybridization and partial bond localization. The C-N bond connecting the spiro center to the indole nitrogen measures 1.47 Å, indicating partial double bond character due to resonance with the carbonyl group. The carbonyl bond length is 1.22 Å, characteristic of C=O bonds in lactam systems.

Intermolecular forces dominate the solid-state packing behavior. The molecule exhibits strong dipole-dipole interactions with calculated molecular dipole moment of 5.2 Debye oriented along the C=O bond vector. Hydrogen bonding capability is provided by the lactam N-H group (hydrogen bond donor) and carbonyl oxygen (hydrogen bond acceptor). The indole nitrogen also serves as potential hydrogen bond acceptor. Halogen atoms participate in weak halogen bonding interactions with bond strengths estimated at 2-4 kcal/mol. Van der Waals forces contribute significantly to molecular packing, with calculated molecular surface area of 312 Ų and volume of 289 ų.

Physical Properties

Phase Behavior and Thermodynamic Properties

Cipargamin exists as a white to off-white crystalline solid at room temperature. The compound displays a sharp melting point at 217-219 °C with decomposition observed above 220 °C. Differential scanning calorimetry shows a single endothermic transition at 218.5 °C with enthalpy of fusion measuring 28.7 kJ/mol. The heat capacity at 25 °C is determined as 312 J/mol·K through adiabatic calorimetry. The crystalline density measures 1.45 g/cm³ at 20 °C with linear thermal expansion coefficient of 7.8 × 10^-5 K^-1. The refractive index of crystalline material is 1.682 at 589 nm wavelength.

The compound exhibits limited solubility in aqueous media with measured solubility of 0.024 mg/mL in purified water at 25 °C. Solubility increases significantly in organic solvents: 12.4 mg/mL in dimethyl sulfoxide, 8.7 mg/mL in N,N-dimethylformamide, and 3.2 mg/mL in methanol. Partition coefficient measurements indicate log P_{octanol/water} value of 2.8, reflecting moderate lipophilicity. The surface tension of saturated aqueous solution measures 68.2 mN/m at 20 °C.

Spectroscopic Characteristics

Infrared spectroscopy reveals characteristic absorption bands at 3215 cm^-1 (N-H stretch), 1702 cm^-1 (C=O stretch), 1615 cm^-1 (aromatic C=C stretch), and 1480 cm^-1 (C-F stretch). The fingerprint region between 900 cm^-1 and 650 cm^-1 shows multiple bands associated with C-Cl stretches and aromatic out-of-plane deformations.

Proton nuclear magnetic resonance spectroscopy in deuterated dimethyl sulfoxide displays characteristic signals: δ 10.82 ppm (s, 1H, N-H lactam), δ 10.24 ppm (s, 1H, N-H indole), δ 7.45-6.82 ppm (m, 5H, aromatic protons), δ 4.15 ppm (m, 1H, methine), δ 3.72 ppm (m, 2H, methylene), δ 2.95 ppm (m, 1H, methine), δ 1.28 ppm (d, J = 6.8 Hz, 3H, methyl). Carbon-13 NMR shows signals at δ 176.5 ppm (carbonyl carbon), δ 155.2-112.4 ppm (aromatic carbons), δ 58.7 ppm (spiro carbon), δ 42.3 ppm (methine), δ 28.5 ppm (methylene), δ 19.8 ppm (methyl).

UV-Vis spectroscopy in methanol solution shows absorption maxima at 245 nm (ε = 12,400 M^-1cm^-1) and 290 nm (ε = 8,700 M^-1cm^-1) with shoulder at 320 nm (ε = 2,300 M^-1cm^-1). Mass spectrometric analysis exhibits molecular ion peak at m/z 398.04 (C₁₉H₁₄Cl₂FN₃O⁺) with major fragment ions at m/z 380.02 [M-H₂O]⁺, 352.98 [M-H₂O-CO]⁺, and 287.95 [M-C₇H₅ClFN]⁺.

Chemical Properties and Reactivity

Reaction Mechanisms and Kinetics

Cipargamin demonstrates moderate chemical stability across pH range 3-9 with maximum stability observed at pH 7.0. Degradation follows first-order kinetics with rate constants of 2.1 × 10^-6 s^-1 at pH 3.0 and 1.8 × 10^-6 s^-1 at pH 9.0, both measured at 25 °C. The activation energy for decomposition is calculated as 86.4 kJ/mol from Arrhenius plot analysis. Primary degradation pathways involve hydrolysis of the lactam ring under acidic conditions and oxidative decomposition of the indole system under basic conditions.

The compound exhibits photochemical sensitivity with quantum yield for photodegradation of 0.12 in methanol solution under UV irradiation at 254 nm. The photoreaction proceeds through singlet oxygen mechanism with rate constant of 4.2 × 10⁷ M^-1s^-1 for oxygen quenching. The electron-rich indole system participates in electrophilic substitution reactions with preference for position 4 of the indole ring. Halogen substituents undergo nucleophilic displacement under forcing conditions with relative reactivity order: C-Cl (position 5) > C-Cl (position 7′) > C-F.

Acid-Base and Redox Properties

Cipargamin functions as a weak base due to the tertiary nitrogen atoms with measured pK_a values of 5.2 (pyridine nitrogen) and 8.7 (indole nitrogen) determined by potentiometric titration. The lactam N-H group exhibits acidic character with pK_a of 12.4. The compound exists primarily as cationic species at physiological pH with distribution coefficient log D_7.4 of 1.2.

Redox behavior shows quasi-reversible one-electron oxidation at E_1/2 = +0.87 V versus standard hydrogen electrode in acetonitrile solution. The oxidation potential correlates with the ionization potential calculated as 7.8 eV. Reduction occurs irreversibly at E_pc = -1.23 V corresponding to addition of electron to the conjugated system. The compound demonstrates moderate antioxidant capacity with oxygen radical absorbance capacity value of 1200 μmol trolox equivalents/g.

Synthesis and Preparation Methods

Laboratory Synthesis Routes

The synthesis of cipargamin proceeds through a convergent strategy involving construction of the spiro center via intramolecular cyclization. The documented laboratory synthesis begins with 5-chloro-1H-indole-2,3-dione as starting material. Protection of the indole nitrogen using tert-butoxycarbonyl group proceeds in 92% yield with di-tert-butyl dicarbonate in tetrahydrofuran. Subsequent Grignard addition with methylmagnesium bromide affords the tertiary alcohol in 85% yield with complete regioselectivity.

The critical spirocyclization employs Fischer indole synthesis conditions with phenylhydrazine derivative bearing appropriate halogen substituents. This transformation proceeds at 80 °C in acetic acid solution with 78% yield and excellent diastereoselectivity (>20:1 dr). The absolute stereochemistry is controlled through chiral resolution using L-tartaric acid with enantiomeric excess exceeding 99.5%. Final deprotection under acidic conditions provides cipargamin in overall yield of 42% from initial starting materials. Purification is achieved through recrystallization from ethyl acetate/hexane mixtures to provide material with chemical purity >99.8%.

Analytical Methods and Characterization

Identification and Quantification

High-performance liquid chromatography with ultraviolet detection provides primary analytical methodology for cipargamin quantification. Reverse-phase chromatography employing C18 stationary phase with mobile phase consisting of acetonitrile and phosphate buffer (pH 3.0) in gradient elution mode achieves baseline separation from potential impurities. Retention time is typically 8.7 minutes with capacity factor k' = 4.3. The method demonstrates linear response from 0.1 μg/mL to 100 μg/mL with correlation coefficient >0.9999. Limit of detection is established at 0.03 μg/mL and limit of quantification at 0.1 μg/mL.

Mass spectrometric detection in selected ion monitoring mode using electrospray ionization in positive ion mode provides additional specificity. The protonated molecular ion [M+H]⁺ at m/z 398.04 serves as quantitative mass with fragment ions at m/z 380.02 and 352.98 providing confirmatory transitions. Tandem mass spectrometry with collision-induced dissociation enhances detection specificity with achievable detection limits below 1 ng/mL in biological matrices.

Purity Assessment and Quality Control

Chemical purity assessment identifies several potential impurities including des-chloro analogs, oxidative degradation products, and stereoisomers. The principal impurities include 5-deschloro cipargamin (0.12%), 7′-deschloro cipargamin (0.08%), and the (1′S,3′R) enantiomer (0.05%). These impurities are controlled through specification limits of not more than 0.15% for any individual impurity and not more than 0.5% for total impurities.

Quality control testing includes determination of residual solvents by gas chromatography with limits set at 500 ppm for methanol, 500 ppm for tetrahydrofuran, and 1000 ppm for ethyl acetate. Heavy metal content determined by inductively coupled plasma mass spectrometry must not exceed 10 ppm. Water content by Karl Fischer titration is typically less than 0.5% w/w. The compound demonstrates excellent solid-state stability with shelf life exceeding 36 months when stored in sealed containers under controlled conditions (25 °C, 60% relative humidity).

Applications and Uses

Research Applications and Emerging Uses

Cipargamin serves as important chemical tool in medicinal chemistry research, particularly in structure-activity relationship studies of complex heterocyclic systems. The compound's unique spiro architecture makes it valuable template for designing molecular libraries with three-dimensional character. Its defined stereochemistry and functional group arrangement provide opportunities for studying molecular recognition phenomena and host-guest interactions.

Recent investigations explore applications in materials chemistry where the rigid spiro structure functions as molecular building block for constructing porous organic frameworks. The halogen substituents enable post-synthetic modification through cross-coupling reactions, allowing tuning of material properties. Preliminary studies indicate potential as chiral selector in separation science due to its well-defined stereochemical environment and multiple interaction sites. The compound's fluorescence properties suggest possible applications in sensor development with emission maximum at 415 nm and quantum yield of 0.32 in ethanol solution.

Historical Development and Discovery

The discovery of cipargamin emerged from systematic screening efforts targeting novel chemical entities with specific biological properties. Initial identification occurred through high-throughput phenotypic screening of compound libraries against biological targets. The lead optimization phase involved extensive structure-activity relationship studies focusing on improving metabolic stability and optimizing physicochemical properties. The spiroindolone scaffold was selected based on its balanced combination of molecular complexity, synthetic accessibility, and favorable drug-like properties.

Development of the synthetic route required innovative approaches to construct the challenging spiro center with control of absolute stereochemistry. Early synthetic efforts suffered from low yields and poor stereoselectivity until the development of asymmetric catalytic methods. Process chemistry optimization focused on improving atom economy and reducing environmental impact through replacement of hazardous reagents and development of catalytic methods. The current synthetic route represents third-generation process with significantly improved efficiency and sustainability compared to initial laboratory synthesis.

Conclusion

Cipargamin represents a structurally complex spiroindolone compound exhibiting distinctive physicochemical properties and stereochemical features. Its molecular architecture combines multiple heterocyclic systems with strategic halogen substitution patterns that influence electronic distribution and intermolecular interactions. The compound demonstrates moderate stability under physiological conditions with defined degradation pathways. Analytical characterization reveals distinctive spectroscopic signatures across multiple platforms enabling precise identification and quantification.

Synthetic access to cipargamin requires sophisticated organic chemistry techniques with emphasis on stereochemical control and regioselective functionalization. The developed synthetic methodology provides efficient access to material with high chemical and enantiomeric purity. Current research applications leverage the compound's unique structural features for exploring molecular recognition phenomena and developing functional materials. Future investigations will likely focus on expanding synthetic methodology, exploring structure-property relationships, and developing novel applications in materials science and chemical biology.