Structure and physical properties of salcaprozoic acid supporting SNAC-based drug delivery

Details of the crystal structure refinement and refinement statistics for HNAC are given in Table 1▸. The sample of HNAC (≥98% purity) was procured from Synlyfe Research Lab­or­a­tory (Gujarat, India). Crystals of HNAC were obtained by slow evaporation of a methano­lic solution at room tem­per­a­ture (≃ 298 K). A 5 ml sample of the com­pound dissolved in methanol was left undisturbed, and solvent evaporation over a period of 5–6 d yielded crystals suitable for analysis.

H atoms were positioned with idealized geometry, with the exception of those bound to heteroatoms, the positions of which were located using peaks in the Fourier difference map, with their coordinates allowed to refine freely. The displacement parameters of the H atoms, U_iso, were constrained using a riding model, with U_iso(H) set to be an appropriate multiple of the U_eq value of the parent atom.

Powder X-ray diffraction patterns were recorded using an Empyrean diffractometer (Malvern Panalytical, UK) in Bragg–Brentano θ/θ geometry, equipped with a PIXcel 3D detector and a reflection–transmission spinner stage. The instrument utilized monochromatic Cu Kα₁ radiation (λ = 1.54184 Å) generated at 40 kV and 40 mA, with a take-off angle of 6.0°. Data were collected at room tem­per­a­ture over a 2θ range of 5 to 40°, with a step size of 0.01° and 20 s per step. The obtained diffractograms were analyzed using HighScore Plus (Malvern Panalytical, UK) software (Degen et al., 2014▸). Rietveld refinement was performed using the structural model derived from single-crystal data, allowing accurate determination of lattice parameters, phase identification and confirmation of the sample purity.

Differential scanning calorimetry (DSC) was carried out using a TA Instruments Q2000 system (Cheshire, UK) over a tem­per­a­ture range of 30–300 °C. Approximately 2–5 mg of each sample were weighed into hermetically sealed aluminium pans and heated at a rate of 10 °C min⁻¹ under a constant nitro­gen purge (50 ml min⁻¹) to prevent oxidative degradation. The resulting thermograms were analysed using TA Instruments Universal Analysis software (https://www.tainstruments.com/↗).

Thermogravimetric analysis (TGA) was also performed to assess the decom­position behaviour of the samples. Mea­surements were conducted under a nitro­gen atmosphere using a TA Instruments Q500 system (Cheshire, UK). 2–5 mg of samples were weighed into aluminium pans and heated from 30 to 500 °C at a constant heating rate of 10 °C min⁻¹. The resulting thermograms were analysed using TA Instruments Universal Analysis software.

IR spectra were recorded using an Agilent Cary 630 FT–IR spectrometer equipped with a diamond attenuated total re­flectance (ATR) accessory. The instrument was operated with MicroLab FT–IR software (https://www.agilent.com/en/product/mol­ecular-spectroscopy/ftir-spectroscopy↗) for spectral acquisition and processed by OriginPro software (Origin­Lab Corporation, 2022▸). Samples were analysed in their solid form without further preparation. Each spectrum was obtained over the range 4000–650 cm⁻¹. The obtained spectra were pro­cessed to identify characteristic functional group vibrations.

HNAC (100 mg) was transferred to a 25 ml round-bottomed flask equipped with a magnetic stirrer bar. Iso­propanol (≥99.5% purity, 5.0 ml) was added and the mixture was heated to 50 °C with constant stirring until a clear solution was obtained. An aqueous solution of sodium hydroxide (1 M) was added dropwise over a period of 5 min under continuous stirring until the pH reached 9–10, at which point a white precipitate of SNAC began to form. The reaction mixture was stirred at room tem­per­a­ture for an additional 30 min, and the precipitate was collected by vacuum filtration and dried at ambient tem­per­a­ture. PXRD (Fig. S1 in the supporting information) and DSC analysis confirmed the material as polymorphic form II (Levchik et al., 2016▸). However, despite multiple crystallization attempts from various solvent systems, we were unable to obtain single crystals suitable for X-ray diffraction analysis.

HNAC crystallizes in the monoclinic space group P2₁/c (Fig. 1▸), with two crystallographically independent mol­ecules in the asymmetric unit (Z′ = 2) that are related by approximate inversion symmetry. Overlaying the two mol­ecules further demonstrates this relationship, revealing two distinct conformations.

The asymmetric unit is held together by hy­dro­gen bonds (Table 2▸) in which the amide proton donates to the carbonyl O atom of the carb­oxy­lic acid group to form a ring motif with the graph set (22) (Etter, 1990▸). These rings are linked by further hy­dro­gen bonds, where the carb­oxy­lic acid group acts as a donor to the carbonyl O atom of the amide, forming a 2D network coplanar with the crystallographic (101) plane (Fig. 2▸). There do not appear to be any salient inter­molecular inter­actions between these hy­dro­gen-bonded layers.

In addition, an intra­molecular hy­dro­gen bond forms between the hydroxyl group and the amide carbonyl group, as would be anti­cipated according to Etter's second rule of hy­dro­gen bonding (Etter, 1990▸).

In the absence of an experimentally resolved structure for SNAC, despite several attempts, the structure of HNAC can instead be com­pared with known examples in the Cambridge Structural Database (CSD; Groom et al., 2016▸) that share similar fragments and functionalities. The structures of other medium-chain fatty acids which acts as permeation enhancers like hepta­noic acid (Bond, 2004▸; CSD refcode ISENOJ) and suberic acid (Mishra et al., 2015▸; SUBRAC12) exhibit the same anti­periplanar arrangement along the length of the carbon chain as that observed in HNAC, though in these instances hy­dro­gen bonding is observed between carb­oxy­lic acid groups forming (22) ring motifs. This is likely attributable to the absence of alternative donor or acceptor sites in these structures, unlike in HNAC, where multiple options are available.

In terms of the 2-hy­droxy­benzoyl­amino moiety, few examples are available that possess a similar saturated carbon chain with terminal hy­dro­gen-bond donors or acceptors, with the closest being 2-hy­droxy-N-(2-hy­droxy­eth­yl)benzamide (CSD refcode family EVIWIQ). Among the two available examples of this structure in the CSD (Betz et al., 2011▸; Wanke et al., 2011▸), the entry EVIWIQ01 reported by Wanke and co-authors was selected for com­parison, as its data were collected at the same tem­per­a­ture as HNAC (150 K).

Considering the structural fragment that both HNAC and EVIWIQ01 share, the two exhibit relatively similar conformations (Fig. 3▸). Where they do differ is in the orientation of the benzene ring relative to the amide moiety. Where in HNAC the N1—C7—C1—C6 torsion angles are 4.43 (15) and 6.45 (15)°, the equivalent torsion in EVIWIQ01 is 12.2 (2)°, representing a significant deviation from planarity.

This can be rationalized by considering the relative directions of the hy­dro­gen bonds in each structure. In HNAC, the bonds are all essentially in the plane of the hy­dro­gen-bonding network [i.e. the (101) plane]. However, in EVIWIQ01, though the structure ultimately forms a 2D network in the (100) plane, the hy­dro­gen bonds are not in this plane (e.g. O3—H4⋯O2 forms an angle of ca 22° with the b axis). These attractive forces therefore act at an angle to the plane of the mol­ecule and lead to greater distortions of the otherwise planar geometry. The absence of similar inter­actions in HNAC allows it to form the almost perfectly planar sheets observed in the structure.

The crystalline structure of HNAC was confirmed by Rietveld refinement of the PXRD data using HighScore Plus (Fig. 4▸). The experimental diffraction pattern was measured using monochromatic Cu Kα₁ radiation (λ = 1.54184 Å) and refinement was performed against the single-phase structural model derived from the CIF. Closer inspection of the diffraction profile revealed weak shoulder features, notably near 7.5° 2θ. These could be accounted for within the Rietveld model as overlapping reflections from the same monoclinic phase, combined with low-angle axial divergence effects. The refinement converged successfully with good agreement between the calculated and experimental patterns. The goodness-of-fit (GoF) was 2.541, with the weighted profile R factor (R_wp) = 6.295%, profile R factor (R_p) = 4.708% and Bragg R factor = 2.001%. The expected R factor was 2.477%, indicating a well-fitted model with no overfitting. All Bragg reflections could be indexed to a single monoclinic phase, P2₁/c, and no additional peaks were detected. The refined unit-cell parameters from the PXRD data (a = 10.23430, b = 23.73930, c = 12.04533 Å and β = 100.0874°) closely match those from the SCXRD data. The Rietveld refinement confirms that the sample is crystalline and structurally consistent with the reference CIF which supports the reasoning that the shoulders arise from intrinsic profile broadening rather than from impurities or an additional polymorph.

The thermal behaviours of HNAC and SNAC were investigated by DSC and TGA (Fig. 5▸). HNAC exhibits a single sharp endotherm at approximately 119 °C, indicative of the melting point, which is consistent with the existing literature reports (Rebollo et al., 2025▸). The TGA curve of HNAC shows a rapid and substantial weight loss immediately following melting, suggesting that decom­position is triggered directly after the phase transition. SNAC demonstrates a minor endothermic peak at approximately 150 °C and a sharp endothermic peak at approximately 198 °C, characteristic of polymorphic form II (Levchik et al., 2016▸). The TGA profile confirms that SNAC undergoes a two-stage decom­position process, with initial mass loss beginning after 198 °C, followed by progressive degradation at higher tem­per­a­tures. The thermal profiles are consistent with the mol­ecular structures of the HNAC and SNAC. For HNAC, the neutral carb­oxy­lic acid form, melting at 119 °C, appears to assist rapid volatilization and breakdown of the mol­ecular framework, possibly through deca­rboxylation and cleavage of the amide linkage. Whereas the ionic stabilization of the carboxyl­ate group of SNAC delays the decom­position to higher tem­per­a­tures. The stepwise mass losses observed for SNAC suggest that the initial stage may involve fragmentation of more labile substituents, followed by degradation of the aliphatic chain and aromatic core.

The IR spectra of HNAC and SNAC provide valuable insights into the structural and electronic differences induced by deprotonation and salt formation. These changes are particularly evident in the regions corresponding to O—H, C=O, C—N and aromatic functionalities.

In the IR spectrum of HNAC (Fig. 6▸), a broad and intense band centred around 3352 cm⁻¹ is attributed to the O—H stretching vibration of the carb­oxy­lic acid group (Shen et al., 2024▸). This broadness is indicative of strong hy­dro­gen bonding, which is characteristic of carb­oxy­lic acids (Langner & Zundel, 1995▸). Additionally, the phenolic –OH group may also contribute to this band, but its involvement in inter­molecular hy­dro­gen bonding with neighbouring mol­ecules could influence its precise position and intensity (Yamashita & Takatsuka, 2007▸). Upon the formation of the sodium salt, SNAC, the O—H stretching band is significantly diminished or absent, indicating deprotonation of the carb­oxy­lic acid and the formation of the carboxyl­ate anion (COO⁻). The disappearance of this peak is consistent with ionic salt formation, where the H atom is replaced by a sodium ion (Na⁺), resulting in altered vibrational characteristics. Moreover, the phenolic –OH group may undergo extensive inter­molecular hy­dro­gen bonding, especially in the solid state, which can either shift the absorption to lower frequencies or render it IR-inactive due to reduced dipole change during vibration (Dai et al., 2023▸).

The C=O stretching vibration is another critical diagnostic region. In the free acid, a strong sharp absorption at 1727 cm⁻¹ corresponds to the carbonyl (C=O) stretch of the carb­oxy­lic acid group. This peak is notably absent or shifted in the SNAC spectrum. Instead, a prominent band appears around 1589 cm⁻¹, which is characteristic of the asymmetric stretching of the carboxyl­ate anion (COO⁻) (Max & Chapados, 2004▸). This shift to lower wavenumbers reflects the delocalization of negative charge across the two O atoms in the carboxyl­ate group, reducing the bond order and thus lowering the stretching frequency (Dey et al., 2025▸). Additionally, the amide functional group is evident in both spectra. The amide II band, arising from N—H bending coupled with C—N stretching, is observed in the range 1597–1554 cm⁻¹. Hydrogen bonding or conjugation with the aromatic ring may influence the exact position and intensity of this band. The C—N stretching vibration of the amide is detected near 1299 cm⁻¹, further confirming the presence of the amide moiety.

Aromatic features are clearly present in both spectra. The C=C stretching vibrations of the aromatic ring are observed as multiple medium-intensity bands in the range 1450–1600 cm⁻¹, consistent with a substituted benzene ring system. The C—O stretching vibrations, arising from both phenolic and carb­oxy­lic acid groups, occur near 1230 cm⁻¹, although these can sometimes overlap with C—N stretches depending on the local environment and substitution pattern. The aliphatic C—H stretching vibrations are represented by strong bands around 2945 and 2924 cm⁻¹, common to both the acid and salt forms. These arise from the symmetric and asymmetric stretching modes of CH₂ groups in the aliphatic chain. Furthermore, aromatic C—H out-of-plane bending vibrations appear around 870 cm⁻¹, supporting the presence of mono- or disubstituted benzene rings, which are key structural motifs in HNAC and SNAC (Lin-Vien et al., 1991▸).