Chirality is a key factor for the pharmacological activity and safety of therapeutic compounds. Enantiomers or chiral isomers tend to possess different biological activities; one enantiomer potentially has therapeutic activity, while its mirror image is less active or even toxic. 1 This fact supports the need for enantioselective synthesis and resolution for the development of pharmaceuticals.

Carbinoxamine is an antihistaminic drug often administered as a racemic mixture of equal quantities of its (R)- and (S)-enantiomers. Considering the possibility of differences in pharmacodynamics and pharmacokinetics between enantiomers, individual characterisation and resolution are of important interest. In the past, enantiomers from racemic mixtures have been separated using a variety of methods, such as chiral chromatography and diastereomeric salt crystallisation. The latter involves the creation of diastereomeric salts via a chiral resolving agent, taking advantage of the differences in their physical properties to allow separation. One naturally occurring chiral molecule, tartaric acid, has been widely used in such cases because it can form crystalline diastereomeric salts with many amines. Notably, Louis Pasteur's pioneering work in the 19th century demonstrated the resolution of racemic tartaric acid into its enantiomers by manual separation of their crystals, laying the foundation for stereochemistry. 2

The resolution of racemic carbinoxamine with tartaric acid derivatives is a promising method for obtaining individual enantiomers. The literature has documented the efficiency of di-p-toluoyl-D-tartaric acid in resolving the racemic mixtures of other amines, which results in the generation of diastereomeric salts separable due to their solubility differences. 3 Subsequent basification resulted in the generation of free enantiomerically pure amines. This process not only permits the separation of single enantiomers but also permits their detailed analysis with methods including melting point analysis, infrared spectroscopy, measurement of optical rotation, and nuclear magnetic resonance spectroscopy. 4

With carbinoxamine, resolution methods are important to understand the different pharmacological profiles of its enantiomers. Due to the possibility of one enantiomer having better antihistaminic activity, along with a better safety profile, the possibility of isolating and investigating each enantiomer separately is fundamental. Understanding the specific interactions of each enantiomer with biological targets will pave the way for the production of more effective and safer therapeutic compounds.

The present study aims to achieve the resolution of racemic carbinoxamine maleate using di-p-toluoyl-d-tartaric acid and the subsequent exhaustive characterisation of the product enantiomers. Through the application of mixed crystallisation methodologies and analytical techniques, this study revealed the structural and pharmacological differences between the (R)- and (S)-enantiomers of carbinoxamine. These findings are expected to lead to improved antihistaminic therapies and highlight the importance of chiral resolution in medicinal research.

Resolution and synthesis were conducted at the R&D Laboratory of R. L. Fine Chemicals, Yelahanka, Bangalore. Pharmacological screening was performed in the Department of Pharmacology, Krupanidhi College of Pharmacy, Bangalore.

The resolution of (±)-carbinoxamine maleate was successfully achieved through diastereomeric salt formation with O,O'-di-p-toluoyl-D-tartaric acid as the resolving agent. The crude racemic salt was crystallised with isopropyl alcohol (1:3 ratio). Crystals of the (S)(–)-isomer were isolated after two days of vacuum filtration, with a yield of 92%. After ethyl acetate treatment, the mother liquor gave the (R)(+)-isomer. The two isomers were basified with NaOH to provide free bases.

The melting points of the resolved compounds verified their successful resolution and revealed their purity. The racemic salt (R)(S)-carbinoxamine di-p-toluoyl-D-tartaric acid had melting points of 124–127°C, whereas the resolved salts of (S)- and (R)-carbinoxamine di-p-toluoyl-D-tartaric acid had melting points of 101–103°C and 95–98°C, respectively. The corresponding maleate and oxalate salts also have different melting points (Table 1).

The IR spectral information further validated the structural resolution and integrity. The racemic compound showed specific peaks like 3427.51 cm^-1 (O–H stretch) and 1718.58 cm^-1 (C=O stretch). On resolution, the (S)-isomer showed peaks at slightly shifted positions of 3423.65 cm^-1 and 1720.50 cm^-1, and the (R)-isomer showed peaks at 3398.57 cm^-1 and 1716.65 cm^-1, which indicate enantiomer-specific interactions with the resolving agent (Table 2).

Table 2

Compound	IR KBr λ cm-1
(R)(S)-Carbinoxamine di-p-toluoyl-D-tartaric acid	3427.51 cm-1 (OH str.), 3059.10 cm-1 (Ar. C-H str.), 2956.87 cm-1 (Alip. C-H str.), 2350 cm-1 (quaternary nitrogen atom (asymmetric str.)), 1718.58 cm-1 (C=O str.), 1612.49 cm-1 (Ar. C=C str.), 1267.23 cm-1 (C-N str.), 1018.41 cm-1 (C-Cl Ar.).
(S)-Carbinoxamine di-p-toluoyl-D-tartaric acid	3423.65 cm-1 (OH str.), 3037.89 cm-1 (Ar. C-H str.), 2960.73 cm-1 (Alip. C-H str.), 2370.51 cm-1 (quaternary nitrogen atom (asymmetric str.)), 1720.50 cm-1 (C=O str.), 1616.35 cm-1 (Ar. C=C str.), 1267.23 cm-1 (C-N str.), 1020.34 cm-1 (C-Cl Ar.).
(R)-Carbinoxamine di-p-toluoyl-D-tartaric acid	3398.57 cm-1 (OH str.), 3059.10 cm-1 (Ar. C-H str.), 2970.38 cm-1 (Alip. C-H str.), 2372.44 cm-1 (quaternary nitrogen atom (asymmetric str.)), 1716.65 cm-1 (C=O str.), 1616.35 cm-1 (Ar. C=C str.), 1265.30 cm-1 (C-N str.), 1020.34 cm-1 (C-Cl Ar.).

Optical Rotation

Optical rotation analysis was used to establish enantiomeric purity. The racemic base demonstrated 0° rotation, whereas the resolved (S)-carbinoxamine had a specific rotation of − 2°, indicating that it was levorotatory in nature. (R)-Carbinoxamine had a rotation of +4° and was thus determined to be dextrorotatory. These findings clearly establish successful chiral resolution (Table 3).

The antihistaminic activity of maleate and oxalate salts of the resolved isomers was evaluated in the guinea pig ileum. (S)(–)-Carbinoxamine maleate and oxalate showed 95.83% inhibition of histamine, but the (R)(+)-isomers showed only 9.09% and 12.5% inhibition, respectively. This clearly showed that the (S)-isomer was pharmacologically more active than the (R)-isomer (Figure 1).

The ¹H NMR spectra provided detailed structural information. In the racemic mixture (Figure 1), N,N-dimethyl groups existed as two singlets at 5.450–5.600 ppm, indicating the coexistence of both enantiomers. The resolved (S)-carbinoxamine spectrum (Figure 2) showed a single singlet at 5.4517 ppm for the 6H of the dimethyl groups, proving the enantiomeric purity. (R)-Carbinoxamine (Figure 3) displays a singlet at 5.5690 ppm, reflecting its purity. These spectral variations further confirmed the effective separation and identification of enantiomers.

The present study effectively showed the resolution of (±)-carbinoxamine maleate to its enantiomers by O,O'-di-p-toluoyl-D-tartaric acid through diastereomeric salt formation, a tried and tested chiral resolution method. 5 Crystallization from isopropyl alcohol provided the (S)(–)-isomer with high purity and yield (92%), and the (R)(+)-isomer was then recovered from the mother liquor. This method is in line with earlier studies that employed tartaric acid derivatives to resolve simple pharmaceutical compounds. 6, 7 Melting point analysis confirmed successful resolution, with the resolved enantiomers having different melting ranges from one another and from the racemic mixture.

This heating pattern heating was in accordance with expectation of the formation of pure diastereomeric salts, consistent with literature accounts on enantiomer separation with tartaric acid where melting point discrimination has provided a principal basis for purity determination. 8

Infrared spectral data of the present study also confirmed the structural integrity of the resolved enantiomers. Minor differences in O–H and C=O stretching frequencies reflected minor variations in hydrogen bonding geometries and molecular interactions with the resolving agent, a process widely observed in earlier spectroscopic work on resolved drug isomers. 9 Optical rotation measurements clearly demonstrated successful chiral resolution. As predicted, the racemic mixture was not optically active; however, the resolved (S)- and (R)-enantiomers exhibited specific rotations of -2° and +4°, respectively. These observations were consistent with previous characterizations of resolved carbinoxamine and other H1-antihistamines, which have similar optical properties upon resolution. 10

Pharmacological screening also supported enantiomeric discrimination. The (S)(–)-carbinoxamine maleate and oxalate salts exhibited significantly higher antihistaminic activities than the (R)(+)-enantiomers, suggesting that the biological activity was largely confined to the (S)-enantiomer. These findings are consistent with earlier reports, where one enantiomer of chiral antihistamines was found to be much more potent through selective binding to H1-receptors. 11, 12 ¹H NMR spectroscopy provided unequivocal proof of the enantiomeric purity. The separated isomers exhibited single sharp singlets for the N,N-dimethyl protons, whereas the racemic mixture exhibited overlapping peaks. This was consistent with previous study, using NMR as a confirmatory technique for resolution of chiral amines, specifically in detecting differences in chemical shifts between enantiomers. 13

Overall, the resolution of carbinoxamine by O,O'-di-p-toluoyl-D-tartaric acid was not only effective but also produced enantiomers whose physical, spectral, and pharmacological characteristics are well known. (S)-Enantiomer exhibited superior antihistaminic activity, affirming its potential use as a single-enantiomer drug, a pattern increasingly followed in contemporary drug development for enhanced efficacy and fewer side effects. 14

The present study achieved the resolution of racemic carbinoxamine maleate with O,O'-di-p-toluoyl-D-tartaric acid, yielding enantiomerically pure (S)- and (R)-isomers. Characterisation by melting point, IR, NMR, and optical rotation analyses validated the purity and identity of the resolved products. The pharmacological activity revealed that the (S)-enantiomer had a much greater antihistaminic activity, indicating its therapeutic value. These findings support the development of enantiomerically pure (S)-carbinoxamine as a potent and targeted antihistamine drug.