Mechanochemical Synthesis, Spectroscopic Characterization and Molecular Structure of Piperidine–Phenytoin Salt

Abstract

Phenytoin is an anticonvulsant drug that suffers from low aqueous solubility. The formation of phenytoin salts is a strategy employed to address this issue. A phenytoin–piperidine salt (PPD–PNT) was synthesized by solvent-assisted grinding and characterized by infrared (IR) spectroscopy, ¹H and ¹³C Nuclear Magnetic Resonance (NMR), and powder and single crystal X-ray diffraction. The IR and NMR spectra obtained differed from those of the starting compounds, showing shifts in the N-H and C=O group signals, as well as the appearance of NH⁺ signals, indicating proton transfer and salt formation. Powder X-ray diffraction confirmed the formation of a new solid phase corresponding to the salt. Single crystal X-ray diffraction showed the molecular structure of the PPD–PNT salt.

1. Introduction

Most active pharmaceutical ingredients used in the manufacture of medicines are solids in the form of powders and serve as raw materials for the production of solid, liquid, semi-solid, and gaseous dosage forms. The study of the physicochemical properties of these solid ingredients is of great importance because they directly impact the stability and therapeutic effect of the drug [1].

Solubility is one of the most important physicochemical properties of an active pharmaceutical ingredient. A drug’s bioavailability depends directly on its solubility in the gastrointestinal tract and its permeation through the cell membrane. The passage of the drug through biological membranes requires that it be dissolved; therefore, low solubility of a drug will decrease its absorption [2].

Preparation of pharmaceutical salts is the most used strategy to improve the solubility of water-insoluble drugs. A salt is formed by two or more components (drug and salt coformer), which are in ionized form and interact through ionic interactions. Another advantage of pharmaceutical salts is that they can generally be crystallized, which makes them easier to handle and process [3,4]. Salt formation typically involves acid–base reactions (HA ⇌ H⁺ + A⁻ and B + H⁺⇌BH⁺) and can be predicted by using the pKa rule, which states that “when the pKa difference between a cocrystallizing acid and base is greater than 2 or 3 (ΔpKa = pKa[protonated base] − pKa[acid]), salt formation is expected” [5].

Piperidine is a heterocyclic amine that has been used as a coformer of salts with pharmaceutical ingredients such as diclofenac, sulfapyrine, sulfamethazine, and fenofibric acid [6,7,8,9].

Phenytoin (5,5′-diphenylimidazolidine-2,4-dione) belongs to the hydantoin family and possesses carbonyl (C=O), amino (N-H), and benzene rings in its chemical structure (Figure 1). It is used as an anticonvulsant drug that acts by blocking calcium channels [10]. It has a low aqueous solubility (less than 0.1 g/mL), classifying it within class II of the biopharmaceutical classification system (low solubility and high permeability). The sodium salt of phenytoin also has low aqueous solubility (1 g/66 mL). Some strategies that have been developed to modify the solubility of phenytoin are: salt formation, crystal habit modification, complex formation with cyclodextrin, cosolvency with N-methylpyrrolidone, and mixtures with polymers [11,12,13,14,15,16].

The formation of pharmaceutical salts generally involves the use of chemical solvents that can harm the environment and health, and incur higher economic costs. Mechanochemical synthesis (by dry or solvent-assisted grinding) is an alternative to the synthesis of salts, which is based on the application of a mechanical force to modify the solid properties of the ingredients, resulting in the formation of a new solid phase [4,17,18]. Considering the solubility limitations of phenytoin and the few options available for its administration (phenytoin and phenytoin sodium), as well as the search for alternative solid forms, the objective of this work was the synthesis of a phenytoin salt with piperidine (PPD–PNT) (Figure 1), to generate chemical and structural information about another solid form of this pharmaceutical ingredient. The obtained salt was characterized by IR spectroscopy, ¹H and ¹³C NMR, powder X-ray diffraction, and single crystal X-ray diffraction.

2. Materials and Methods

2.1. Reagents

Piperidine and phenytoin were purchased from Aldrich; dichloromethane, methanol, and acetonitrile were purchased from Química Meyer. All the reagents and solvents were used as received.

2.2. Mechanochemical Synthesis of the PPD–PNT Salt

The compounds (PPD and PNT in a 1:1 molar ratio) were placed in a mortar, 0.5 mL of dichloromethane was added before starting the grinding, and the mixture was ground with a pestle for 3 min. Upon completion of grinding, the resulting powder was placed in the center of the mortar, 0.5 mL of dichloromethane was added again, and the mixture was ground for an additional 3 min. The addition of 0.5 mL of dichloromethane and grinding for 3 min was repeated two more times to complete 12 min of grinding. The ground powder was stored in a glass vial.

2.3. IR Spectroscopy

Infrared spectra were collected on a Bruker Tensor 27 spectrophotometer equipped with a diamond ATR (Attenuated Total Reflection) accessory in a spectral range from 4000 to 600 cm⁻¹ with a resolution of 4 cm⁻¹ (16 scans).

2.4. Nuclear Magnetic Resonance

¹H (400 MHz) and ¹³C NMR (100 MHz) spectra were recorded on a Bruker Ultrashield plus 400 MHz instrument using DMSO-d₆ as solvent and TMS as the internal reference. The NMR spectra were processed with the MestReNova software version 14.2.0 [19].

2.5. X-Ray Diffraction

Powder diffraction patterns were recorded by a PANalytical X’pert Pro diffractometer with Cu Kα radiation (λ = 1.5405 Å, 45 kV, 40 mA) in a 2θ scanning range from 2° to 50°.

Single crystals suitable for monocrystal diffraction were obtained by dissolving 0.3 g of PPD–PNT_ground in 4 mL of a 1:1 methanol–acetonitrile mixture and leaving the solvent to evaporate.

Single crystal diffraction data of PPD–PNT were collected at 130 K with an Oxford Diffraction Gemini diffractometer (λMoKα = 0.71073 Å, monochromator: graphite) with a CCD-atlas area detector. Data collection and integration were performed with CrysAlisPro and CrysAlis RED software version 1.171.36.32 [20,21]. SHELXS and SHELXL software version 2018 were used for structure solution and refinement [22,23], and WinGX version 2023.1 [24] software was used to prepare material for publication. Full-matrix least-squares refinement was carried out by minimizing (Fo² − Fc²)². All non-hydrogen atoms were refined anisotropically. H atoms of the N-H group were located in a difference map and refined isotropically with Uiso(H) 1.2 Ueq for N-H. H atoms attached to C atoms were placed in geometrically idealized positions and refined as riding on their parent atoms, with C–H = 0.95 − 0.99 Å with Uiso(H) = 1.2Ueq(C) for aromatic and methylene groups. Graphic material was prepared using Mercury Software version 2020.3.0 [25]. Crystal data, data collection, and structure refinement details are summarized in

Table 1. Crystal data of PPD–PNT_crystal.

CCDC No.	2464266
Empirical formula	C5H12N·C15H11N2O2
Formula weight	337.41
Crystal system	Triclinic
Space group	P-1
a (Å)	8.3364(10) Å
b (Å)	9.7349(10) Å
c (Å)	11.4086(14) Å
α (◦)	83.854(9)°
β (◦)	77.735(10)°
γ (◦)	76.844(10)
Wavelength	0.71073 Å
V (Å3)	879.30(18) Å3
Crystal size	0.360 × 0.340 × 0.270 mm3
Z	2
T	130(2) K
Dcalc. (g/cm3)	1.274
μ (mm−1)	0.084
F(000)	360
Reflections collected	9816
Independent reflections	4176
R (int)	0.0228
R1/wR2 [I > 2σ(I)]	0.0417/0.0952
R1/wR2 (all data)	0.0536/0.1042
GooF on F2	1.038

.

3. Results

3.1. Spectroscopic Characterization

The ΔpKa value between PPD (pKa = 11.1) [26] and PNT (pKa = 8.3) [27] is 2.8; therefore, the expected new solid phase is a salt. IR-ATR spectroscopy is a rapid method to determine the formation of new solid forms by the shift of the characteristic bands of the functional groups of a solid phase compared with the starting compounds (IR assignments of the characteristic frequencies of PNT, PPD, and the PPD–PNT mixtures are listed in

Table 2. IR frequencies (cm⁻¹) of the NH, NH⁺, and C=O stretching frequencies of the starting compounds, PNT_Na and the PPD–PNT salt.

	NH	NH+	C=O	Ar/alkyl	C-N
PNT	3263, 3200	---	1770, 1738, 1715	3070, 1598, 1494, 1449	1400
PNTNa [28]	3318	---	1674, 1687	1592, 1492	N.R.
PPD	3284	---	---	2928–2735, 1444	1149
PPD–PNTpowder	3153	3070	1703, 1643	2945, 2863, 1602, 1577	1355, 1126
PPD–PNTcrystal	3153	3065	1702, 1644	1599, 1575, 2940, 2862	1353, 1125

N.R. = Not reported.

).

The IR spectra of PNT and PPD were assigned as previously reported [28,29,30]. The IR spectra of PPD–PNT (ground and single crystal) showed differences with respect to the starting compounds (Figure 2 and Table 2). To confirm the salt formation, the IR frequencies of PPD–PNT_{ground/crystal} were compared with the IR frequencies of phenytoin sodium salt (PNT_Na) [28]. In PNT_Na (a deprotonated form of PNT), the C=O signal shifts to a lower wavenumber appearing at 1674 cm⁻¹ and 1687 cm⁻¹, while in PPD–PNT_ground they were observed at 1703 cm⁻¹ (ΔνC=O = −67 cm⁻¹) and 1643 cm⁻¹ (ΔνC=O = −72 cm⁻¹) (in PPD–PNT_crystal they appeared at 1702 cm⁻¹ and 1644 cm⁻¹, ΔνC=O = −68 cm⁻¹ and −71 cm⁻¹ respectively), which is in agreement with the deprotonation of carboxylic acids, where the C=O band shifts to a lower wavenumber due to the electron delocalization to form the carboxylate ion [31]. The N-H signal of PNT_Na is reported at 3318 cm⁻¹, while in PPD–PNT_{ground/crystal}, it was observed at 3153 cm⁻¹ (ΔνN-H = −47 cm⁻¹). When theophylline is deprotonated to form salts, the NH band shifts by 30–40 cm⁻¹ [32].

The signal at 3070 cm⁻¹ in the IR spectrum of PPD–PNT_ground (3065 cm⁻¹ in PPD–PNT_crystal) belongs to the NH₂⁺ group of piperidine, which is another signal indicating the proton transfer to form the salt, in a similar way to that reported for piperidine–sulfamethazine salt (3076 cm⁻¹) [8], and piperidinium–4-carboxylic acid hydrogen squarate (3073 cm⁻¹) [33].

The frequencies of the aromatic/alkyl and C-N groups also appeared shifted in the IR spectra of PPD–PNT_{ground/crystal} (Table 2).

The NMR spectroscopy study (the chemical shifts of which are listed in

Table 3. NMR chemical shifts (ppm in DMSO-d₆) of PNT (phenytoin), PPD (piperidine), PPD–PNT_powder (piperidine–phenytoin ground powder), and PPD–PNT_crystal (piperidine–phenytoin single crystal). (N.O. = not observed).

1H NMR (δ = ppm)
	NH1		NH3		H7–H9			NHa		Hb		Hc		Hd
PNT	9.33		11.13		7.42–7.34			2.86		---		---		---
PPD	---		---		---			---		2.63		1.41		1.46
PPD–PNTground	9.24		N.O.		7.41–7.33			5.14		2.65		1.40		1.45
PPD–PNTcrystal	9.19		N.O.		7.38–7.33			4.89		2.66		1.42		1.42
13C NMR (δ = ppm)
	C2	C4		C5		C6	C7		C8	C9	Cb		Cc		Cd
PNT	156.44	175.28		70.68		140.38	127.04		128.97	128.50
PPD											47.29		26.80		25.16
PPD–PNTground	157.27	175.90		70.74		140.62	127.07		128.91	128.38	47.00		27.14		25.38
PPD–PNTcrystal	157.60	176.13		70.78		140.69	127.08		128.89	128.35	46.87		26.66		25.07

, and the atom numbering is according to Figure 1) was performed to characterize the PPD–PNT salt in solution. The ¹H NMR spectrum of starting PNT (Figure 3) showed the N-H characteristic signals [34] at 11.13 ppm and 9.33 ppm, and the aromatic signals in the 7.42–7.34 ppm range. PPD showed the N-H signal at 2.86 ppm, and the rest of the alkyl signals in the 2.63–1.41 ppm range. The absence of the NH3 signal of PNT (11.13 ppm) in the ¹H NMR spectra of PPD–PNT_{ground/crystal} (Figure 3) is evidence of the salt formation and proton transfer from PNT to PPD to form the PPD–PNT salt. The NH1 signal appeared as a broad signal at 9.24 ppm in PPD–PNT_ground and at 9.19 ppm in PPD–PNT_crystal (similar to the ¹H chemical shifts reported for phenytoin derivatives [35,36]); the aromatic signals remained in the same range.

Another proof of salt formation is the shift of the NHa signal (the piperidinium signal in PPD–PNT) from 2.86 ppm in the starting PPD to 5.14 ppm in PPD–PNT_ground (Δδ = 2.28 ppm), and 4.89 ppm in PPD–PNT_crystal (Δδ = 2.03 ppm).

The ¹³C NMR signals (Figure 4 and Table 3) of C2 and C4 carbonyls were shifted from 156.44 ppm and 175.28 ppm in the starting PNT to 157.27 ppm (Δδ = 0.83 ppm) and 175.90 ppm (Δδ = 0.62 ppm) in PPD–PNT_ground, and 157.60 ppm (Δδ = 1.16 ppm) and 176.13 ppm (Δδ = 0.85 ppm) in PPD–PNT_crystal, due to the deprotonation causing a higher deshielding effect over C2 and C4 carbonyls, as occurs in the deprotonation of carboxylic acids [37]. PPD carbons also were sensitive to the salt formation, showing shifts from 47.29 ppm, 26.80 ppm, and 25.16 ppm in the starting PPD to 47.00 ppm, 27.14 ppm, and 25.38 ppm in PPD–PNT_ground (Δδ = −0.29 ppm, 0.34 ppm, and 0.22 ppm, respectively), and 46.87 ppm, 26.66 ppm, and 25.07 ppm in PPD–PNT_crystal (Δδ = −0.42 ppm, 0.14 ppm, and 0.09 ppm, respectively).

3.2. X-Ray Diffraction

The production of the solid form of the PPD–PNT salt was confirmed because the powder X-ray diffraction pattern of PPD–PNT_ground (Figure 5) was different from the pattern of PNT and similar to the simulated pattern of PPD–PNT_crystal. Distinctive signals of the PPD–PNT_ground diffraction pattern were observed at 2θ = 9.2° (0 1 0), 13.4° (1 1 1), 15.8° (0 0 2), 22.0° (2 0 1) and 25.1° (9.3°, 13.6°, 15.9°, 22.1° and 25.1° in the simulated pattern of PPD–PNT_crystal). Signals of residual PNT appeared at 2θ = 11.4°, 20.4°, and 27.0° in the PPD–PNT_ground diffraction pattern.

There are a few reports about crystal structures of multicomponent forms of phenytoin. Only the following crystal structures have been reported: the cocrystal of PNT-1-(4-bromophenyl)-4-dimethylamino-2,3-dimethyl-3-pyrazolin-5-one (CCDC REFCODE: DPHPZL [38]) where the PNT molecules adopt the R²₂(8) imide type pattern (Figure 6), as well as the cocrystal of PNT-9-ethyladenine 2,4-pentanedione solvate (CCDC REFCODE: DPHEAD10 [39]), and PNT-choline salt (REFCODE: COXYUK [11]) where, in both crystal structures, the PNT molecules form the R²₂(8) urea type pattern (Figure 6).

Single crystal X-ray diffraction showed the molecular structure (triclinic, P-1 space group) and confirmed the proton transfer from PNT to PPD to form the PPD–PNT_crystal salt because the PPD molecule showed the NH₂⁺ group and the PNT molecule showed the O=C-N⁻ group (Figure 7a). PPD and PNT are connected by a N-H⁺···N⁻ hydrogen bond, forming the 1:1 salt (hydrogen bond details are listed in

Table 4. Hydrogen bond geometry (Å, °).

D-H···A (Å)	Symmetry Code	D-H (Å)	H···A (Å)	D···A (Å)	D-H···A (°)
N1C-H1N···O1	x,y,z	0.925(17)	1.839(17)	2.7523(14)	169.0(15)
N1C-H3N···N1	-x,1-y,1-z	0.961(17)	1.836(17)	2.7959(16)	175.9(15)
N2-H2N···O2	1-x,1-y,-z	0.899(17)	1.926(17)	2.8221(14)	174.6(16)
C1-H1CA···Cg(3)	1-x,1-y,1-z	---	2.44	---	168

). A 1D supramolecular tape involving a R²₂(8) urea type PNT–PNT interaction (Figure 7b), and an R⁴₄(12) PNT₂-PPD₂ interaction is extended along the (16 17 16) plane. The 2D supramolecular architecture showed a supramolecular tape where one PNT molecule is interlinked by a C-H···π interaction (C1-H1CA···Cg(3) = 2.44 Å; Cg(3) = C7/C8/C9/C10/C11/C12) to one PPD molecule and extended along the a axis by the N1C-H3N···N1 hydrogen bond. Free PNT forms a supramolecular tape where three molecules of PNT are interlinked by N-H···O=C hydrogen bonds, depicting an R³₃(12) motif (Figure 7c). Formation of PPD–PNT_crystal involves the breaking of this pattern of hydrogen bond interactions and the molecular rearrangement to include PNT, now showing the characteristic R²₂(8) urea pattern between PNT molecules and the R⁴₄(12) pattern that connects PPD with PNT.

Deprotonation causes changes in the bond distances of the imidazolidinedione ring of PNT. An analysis of the bond distances of PPD–PNT_crystal (Figure 8) compared with the phenytoin sodium methanol hydrate (PNT_Na-MeOH-H₂O, REFCODE: COMREE [40]) (deprotonated form) and the starting PNT (protonated form) revealed that the imidazolidinedione fragment in PPD–PNT_crystal is similar to PNT_Na-MeOH-H₂O (Figure 8). The C-N imide distances become shorter in PPD–PNT_crystal (1.338(1) Å and 1.386(1) Å) with respect to PNT (1.350(4) Å and 1.394(4) Å), and similar to what was observed in PNT_Na-MeOH-H₂O (1.339(3) Å and 1.371(2) Å). Also, the C-O distances in PPD–PNT_crystal became longer (1.238(1) Å and 1.237(1) Å) compared with PNT (1.224(4) Å and 1.204(4) Å), similar also to PNT_Na-MeOH-H₂O (1.239(2) Å and 1.247(3) Å). The rest of the bond distances of the imidazolidinedione ring are shown in Figure 8.

4. Conclusions

The PPD–PNT salt was obtained by the solvent-assisted grinding method and characterized by IR spectroscopy, ¹H and ¹³C NMR, powder X-ray diffraction, and single crystal X-ray diffraction.

Formation of the PPD–PNT salt and the proton transfer from PNT to PPD was evidenced by: (a) the shift of the C=O band (similar to PNT_Na), and the appearance of the NH₂⁺ band in the IR spectrum of PPD–PNT_{ground/crystal}; (b) the absence of the NH3 proton, and the shift of the NHa proton in the ¹H NMR spectrum of PPD–PNT_{ground/crystal}; (c) the shift of the ¹³C NMR signals of the carbonyls of PPD–PN_{ground/crystal}; (d) a different powder diffraction pattern of PPD–PNT_ground compared with the powder diffraction pattern of PNT; and (e) the appearance of the NH₂⁺ and O=C-N⁻ groups, and the changes in the bond distances (similar to the PNT_Na-MeOH-H₂O) in the molecular structure obtained by single crystal X-ray diffraction of PPD–PNT_crystal.

The crystal structure of PPD–PNT_crystal showed a 1:1 stoichiometry, forming a N-H⁺···N⁻ hydrogen bond between PPD and PNT and depicting 1D and 2D supramolecular tapes.