(T1530-08-49) Preparation and Characterization of Cocrystals and Salts of an Indole-2-Carboxamide Anti-Mycobacterial Agent

Purpose: Tuberculosis and Nontuberculous mycobacteria (TM and NTM, respectively) primarily infect the lungs and target patients with existing pulmonary diseases and/or compromised/congenital immune response. N-(1-Adamantyl)-1H-indole-2-carboxamide (N2), synthesized in our lab, is a derivative of indole-2-carboxamide exhibiting potent activity against TM and NTM with low MIC values and acts by inhibiting the essential transporter MmpL3 (1). However, its poor aqueous solubility significantly limits its pharmacokinetic profiles and therapeutic outcomes (2). Cocrystallization and salt formation are some of the strategies used to improve the physicochemical properties of the drug, including solubility and pharmacokinetic properties. This study investigates the preparation and characterization of various cocrystals and salt forms of N2 with suitable coformers/counterions, respectively.

Methods: N-(1-Adamantyl)-1H-indole-2-carboxamide (N2) was synthesized in the lab according to the previously published method (1). For the screening of coformers, high occurrence probability in the Cambridge Structural Database for carboxylic acid–amide heterosynthons led to the focus on carboxylic acid coformers (3). Screening was conducted based on the pKa difference of less than 3 between the API and coformer, ensuring incomplete proton transfer (4) and solubility parameter difference of less than 7 MPa^0.5, ensuring miscibility with the API (5). The solubility parameter of the drug was calculated using the group contribution method (6). Basic counterions for the salt preparation were screened based on the acidic nature of N2(2) and the pKa difference between drug and the counter ions of more than 3, required for complete proton transfer (7). A series of cocrystals/salts of N2 were investigated in equimolar ratios using the liquid-assisted grinding and solvent evaporation method. For solvent evaporation, N2 and coformer/counterion were dissolved in a suitable solvent/cosolvent system separately, followed by gradual mixing and solvent evaporation at 40°C. During liquid-assisted grinding (LAG), equimolar quantities of the API and selected coformer/counterions were physically triturated in a mortar and pestle, with intermittent addition of 10 µL solvent/cosolvent at five equal intervals (3 minutes each), over a period of 15 minutes. The formation of cocrystal/salt was characterized by FTIR, PXRD and hot stage microscopy (HSM). FTIR analysis was performed using the infrared spectrophotometer (IRSpirit, Shimadzu, Japan). PXRD patterns were obtained by using X-ray diffractometer (Rigaku SmartLab, Japan) equipped with a cross-beam optics, HyPix-3000 detector and SmartLab Studio II software. Samples were scanned in the range of 3° - 80° 2Θ values at the scanning rate of 4° per minute. Cu-kβ radiation filter was used with a voltage of 40kV and 45 mA. HSM analysis was performed using an optical microscope (Leica 2500M, Leica Microsystems) to evaluate any physical changes in the sample, including melting point, as a function of temperature. Geometry-optimized structure and the surface electron density of the N2 were generated by ADF COSMO calculations using ADF COSMO-RS GUI (2025.103) under Amsterdam Modelling Suite. ADF COSMO calculations were performed using TZP basis set, BP86 density functional and the relativistic scalar ZORA method.

Results: The weak acidic nature of N2 is due to deprotonation primarily at the indole nitrogen, which is indicated in the FTIR spectra at 3400cm^-1 for the indole N-H bond (8). A shift in the FTIR spectra at this region, as observed in the case of compounds formed using sodium hydroxide, triethylamine and L-arginine by solvent evaporation method, may indicate salt formation (Figure 1). No such shifts were observed in other cases. PXRD patterns display peaks for the salt formulation, distinct from the peaks for their parent components. The PXRD patterns of N2, coformer/counterions and their solvent evaporated products are shown in Figure 2. The diffractogram for the product by liquid-assisted grinding showed peaks corresponding to both N2 and the coformer/counterion, indicating a physical mixture without interaction. In contrast, for example, in the case with sodium hydroxide, the solvent evaporated product demonstrated new peaks at 2θ values of 7.5, 12.5 and 17.5, with reductions or disappearance of peaks at 15.5 and 32.5. HSM images revealed that N2 and solvent evaporated products had a similar melting point between 252°C and 256°C. Surface electron potential distribution analysis was computed to understand the interaction sites of N2 (Figure 3). Electron-rich (on oxygen) and electron-deficient (on hydrogen bonded to the indole nitrogen) sites were observed. Moreover, due to resonance in the indole moiety and the presence of the adamantyl group, the N2 structure is predominantly neutral, lacking partial charge.

Conclusion: The present study highlights the challenges in cocrystal/salt formation of the indole-2-carboxamide derivative N2. Despite similar melting points, FTIR and PXRD indicate interaction between the parent components in selected cases. Surface electron density analysis explains the observed interactions and may aid in rational design for improved cocrystal/salt formulations.

References: 1. Franz ND, Belardinelli JM, Kaminski MA, Dunn LC, Calado Nogueira de Moura V, Blaha MA, et al. Design, synthesis and evaluation of indole-2-carboxamides with pan anti-mycobacterial activity. Bioorg Med Chem. 2017 July 15;25(14):3746–55.
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Acknowledgements: The authors thank Erik Moore, Lab Manager, and the Drug Development and Characterization Core (DDCC), Department of Pharmacy Sciences, School of Pharmacy and Health Professions for the PXRD studies. Funding Source: The authors thank the Department of Pharmacy Sciences, Creighton University, for the financial support. Conflict of interest statement: We confirm that there are no conflicts of interest associated with this work, and there has been no significant financial support for this work that could have influenced its outcome.

Figure 1. Comparison of the FTIR Spectra of N2 with various salt forms of N2, a) N2 and sodium salt of N2 b), N2 and triethylamine salt of N2, and c) N2 and L-arginine salt of N2

Figure 2. PXRD patterns of, a) N2, sodium hydroxide, and sodium salt of N2, b) N2, and triethylamine salt of N2, c) N2, L-Arginine, and L-Arginine salt of N2

Figure 3. a) Chemical Structure of N2, b) Electron Potential Density distribution on geometry-optimized structure of N2