Synthesis, Dynamic NMR Characterization, and XRD Study of 2,4-Difluorobenzoyl-Substituted Piperazines

Abstract

Five different 2,4-difluorobenzamide derivatives were synthesized and fully characterized by ¹H/¹³C/¹⁹F/2D NMR spectroscopy using DMSO-d₆ as solvent and MS. All compounds occur as rotation conformers resulting from the partial amide double bond with a solvent-dependent coalescence point. Temperature-dependent ¹H NMR techniques, as well as EXSY, were applied to determine the rate constants of exchange, and the resulting activation energy barriers were calculated. Regarding the N,N-diacylated piperazine, both conformers (syn and anti) were found in solution, whereas only the anti-conformer was found in the crystals. This result was verified by an XRD analysis. Single crystals of N,N-bis(2,4-difluorobenzoyl)piperazine 3b (monoclinic, space group P2₁/c, a = 7.2687(3), b = 17.2658(8), c = 6.9738(3) Å, β = 115.393(2)°, V = 790.65(6) ų, Z = 4, D_obs = 1.530 g/cm³) were obtained from a saturated chloroform solution.

1. Introduction

Piperazines belong to saturated six-membered heterocyclic compounds containing two nitrogen atoms [1]. This scaffold has been integrated as a fundamental structural element into bioactive and pharmacologically relevant compounds [2,3,4,5]. N- and N,N-functionalized piperazine derivatives are used as intermediates in organic reactions [6,7] or for polymerization processes, ref. [8] as ligands in coordination chemistry [9,10,11] or as a scaffold for peptide syntheses and modifications [12,13]. Piperazines are also in use for the introduction of fluorescent dyes [14,15] or for the preparation of radiolabeled compounds [16,17,18,19,20]. They serve as skeletons in pesticides, pharmaceuticals [21,22,23], and other drugs [24,25,26] or basic scaffolds in medicinal chemistry [27,28,29]. Furthermore, the N,N-substituted benzamide scaffold is found in various natural products and drugs, such as in Ampakines [30,31].

Especially N-acylated piperazines containing a fluorobenzoyl or difluorobenzoyl group are of interest (Figure 1), as they function as serotonin 5-HT_2A receptor antagonists [32], cardiotonic agents [33], adenosine A_2A receptor antagonists [34], radiosensitizers for tumor therapy [35], or as a kinase inhibitor for Jun-kinase [36]. The comprehension of the conformational behavior of such functionalized piperazines is mandatory not only to explain biological and/or pharmacological effects, but also for applications in material sciences.

Secondary and tertiary N-acylated piperazines, in general, and especially N-benzoylated piperazines, exhibit a more complex conformational behavior due to the hindered rotation of the C–N amide bond. This phenomenon in amides is well known and has far-reaching implications for peptide and protein folding [42]. Additionally, the conformation of the piperazine ring itself has to be considered. However, NMR properties of such benzamides were only rarely studied in the past. N,N-dimethyl and N,N-diethyl benzamides represent the best characterized compounds [43,44,45,46]. The NMR behavior of selected benzoylpiperazines [47], which were also used as prosthetic groups for ¹⁸F-radiolabeling [19], has been investigated. To expand the scope and to obtain a deeper insight into the conformational behavior of N- and N,N-substituted piperazines, two 2,4-difluorobenzoyl-containing derivatives were prepared and fully characterized in terms of their particular NMR behavior and compared with three N,N-disubstituted amides.

2. Materials and Methods

2.1. General

The starting materials were purchased from commercial suppliers and used without further purification unless otherwise specified. Anhydrous THF was purchased from Acros Organics. Mass spectrometric (MS) data was obtained on a mass spectrometer (expression CMS, Advion Interchim, Montluçon, France) by electrospray ionization (ESI). The melting points were determined on a Galen III melting point apparatus (Cambridge Instruments/Leica, Wetzlar, Germany) and are uncorrected. Chromatographic separations were performed using an automated flash chromatography system Selekt (Biotage, Uppsala, Sweden) with Sfär cartridges (Biotage, Uppsala, Sweden). TLC detections were performed using Merck (Darmstadt, Germany) Silica Gel 60 F₂₅₄ sheets, respectively. TLCs were developed by visualization under UV light (λ = 254 nm).

2.2. VT-NMR Measurements

NMR spectra of all compounds were recorded on an Agilent DD2-400 MHz NMR spectrometer with a 5 mm ProbeOne probe. Deuterated solvents were purchased from deutero. Chemical shifts in the ¹H, ¹⁹F, and ¹³C spectra were reported in parts per million (ppm) using TMS as an internal standard for ¹H/¹³C and CFCl₃ for ¹⁹F spectra at 25 °C unless otherwise stated. Spectra were calibrated to their solvent signals. Dynamic NMR measurements were carried out in a temperature range of 253–393 K using the Agilent VT accessory with a digital temperature controller. The T_C was estimated from the NMR spectrum, which showed the collapse of the methylene signals from the piperazine moiety, and Δν values were extracted from the maximum split signals of the piperazine methylene protons, which remained unchanged with decreasing temperature. Values of the exchange rate k_exc at T_C were calculated using equation k_exc = π∙Δν/2^1/2 [48,49]. Values of the free activation energy, ΔG^ǂ, were obtained by solving the Eyring equation (1) for ΔG^ǂ (2) [48,50]. in accordance with other studies on amide cis/trans isomerization, the transmission coefficient κ was assumed to be 1 [51].

$$k_{\mathrm{e}\mathrm{x}\mathrm{c}}={\displaystyle \frac{\mathsf{\kappa }{\mathrm{k}}_{\mathrm{B}}{\mathrm{T}}_{\mathrm{C}}}{\mathrm{h}}}{\mathrm{e}}^{-\frac{\Delta {\mathrm{G}}^{\mathrm{ǂ}}}{\mathrm{R}{\mathrm{T}}_{\mathrm{C}}}}$$

(1)

$$\Delta {\mathrm{G}}^{\mathrm{ǂ}}=\ln \left({\displaystyle \frac{\mathsf{\kappa }{\mathrm{k}}_{\mathrm{B}}{\mathrm{T}}_{\mathrm{C}}}{\mathrm{h}{k}_{\mathrm{e}\mathrm{x}\mathrm{c}}}}\right)\mathrm{R}{\mathrm{T}}_{\mathrm{C}}$$

(2)

2.3. Single Crystal X-Ray Diffraction

Diffraction data was collected with a Bruker-Nonius Apex-II-diffractometer using graphite-monochromated MoK_α radiation (λ = 0.71073 Å). The diffraction measurement was performed at –150 °C. The unit cell dimensions were refined using the angular settings of 9936 reflections for 3b. The structure was solved by direct methods and refined against F² using full-matrix least-squares with the program suites from G. M. Sheldrick [51,52]. All non-hydrogen atoms were refined anisotropically; all hydrogen atoms were placed on geometrically calculated positions and refined using riding models. CCDC 2238312 contains the supplementary crystallographic data for compound 3b. This data can be obtained free of charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif (accessed on 27 August 2025).

2.4. Synthesis of N-(2,4-Difluorobenzoyl)piperazine (3a)

Piperazine (5 equiv.) was dissolved in absolute chloroform, 2,4-difluorobenzoyl chloride (1 equiv.) was added slowly at 0 °C, and the mixture was allowed to stir at 0 °C for another 5 h and at rt overnight. Afterwards, the solvent was removed and the crude mixture of products was purified via automated column chromatography, yielding N-(2,4-difluorobenzoyl)piperazine (3a) as a colorless solid in a yield of 66% and the N,N-diacylated compound 3b (yield 10%). Mp > 260 °C; ¹H NMR (400 MHz, CDCl₃): δ = 2.92 (br. s, 2H, NCH₂), 3.03 (br. s, 2H, NCH₂), 3.39 (br. s, 2H, NCH₂), 3.85 (br. s, 2H, NCH₂), 6.85 (t, J = 9.0 Hz, 1H, ArH), 6.96 (t, J = 8.3 Hz, 1H, ArH), 7.40 (m, 1H, ArH) ppm; ¹³C NMR (101 MHz, CDCl₃): δ = 42.4, 45.3, 45.8, 47.4 (4 × NCH₂), 104.4 (t, J_C,F = 26 Hz, CH_Ar), 112.5 (dd, J_C,F = 3 Hz, J_C,F = 22 Hz, CH_Ar), 120.1 (dd, J_C,F = 4 Hz, J_C,F = 18 Hz, C_Ar), 130.8 (m, CH_Ar), 158.7 (dt, J_C,F = 253 Hz, J_C,F = 11 Hz, C_Ar), 164.0 (dt, J_C,F = 251 Hz, J_C,F = 12 Hz, C_Ar), 164.6 (C = O) ppm; MS (ESI+): m/z = 227 [M+H]⁺.

2.5. Synthesis of N,N-Bis(2,4-difluorobenzoyl)piperazine (3b)

Piperazine (2 equiv.) was dissolved in absolute chloroform, 2,4-difluorobenzoyl chloride (1 equiv.) was added slowly at 0 °C, and the mixture was allowed to stir at rt overnight. Afterwards, the solvent was removed and the crude mixture of products was purified via automated column chromatography, yielding N,N-bis(2,4-difluorobenzoyl)piperazine (3b) in 44% yield as a colorless solid and the N-monoacylated compound 3a (9% yield) from the one-pot reaction. Mp 245–247 °C; ¹H NMR (400 MHz, CDCl₃): δ = 3.34 (br. s, 2H, NCH₂), 2.44, 3.79, 3.89, 6.78–7.06 (m, 4H, ArH), 7.43 (dd, ³J = 7.5 Hz, ³J_H,F = 14.9 Hz, 2H, ArH) ppm; ¹³C NMR (101 MHz, CDCl₃): δ = 42.1, 42.5, 46.9, 47.5 (4 x NCH₂), 104.4 (m, CH_Ar), 112.7 (dd, J_C,F = 2 Hz, J_C,F = 22 Hz, CH_Ar), 119.8 (d, J_C,F = 18 Hz, C_Ar), 131.0 (m, CH_Ar), 158.7 (dt, J_C,F = 206 Hz, J_C,F = 12 Hz, C_Ar), 164.0 (dt, J_C,F = 254 Hz, J_C,F = 10 Hz, C_Ar), 164.7 (d, J_C,F = 18 Hz, C = O) ppm; ¹⁹F NMR (378 MHz, CDCl₃): δ = −110.7 (ArF), −106.0 (ArF) ppm; MS (ESI+): m/z = 267 [M+H]⁺.

2.6. Synthesis of N,N-Diethyl-2,4-difluorobenzamide (4)

Diethylamine (2 equiv.) was dissolved in absolute chloroform, 2,4-difluorobenzoyl chloride (1 equiv.) was added slowly at 0 °C, and the mixture was allowed to stir at rt overnight. Afterwards, the solvent was removed and the crude mixture of products was purified via automated column chromatography, yielding N,N-diethyl-2,4-difluorobenzamide (4) in 86% yield as a colorless oil. ¹H NMR (400 MHz, DMSO-d₆): δ = 1.00 (t, J = 7.1 Hz, 3H, CH₃), 1.14 (t, J = 7.1 Hz, 3H, CH₃), 3.12 (q, J = 7.1 Hz, 2H, NCH₂), 3.45 (q, J = 7.1 Hz, 2H, NCH₂), 7.16 (dt, J = 2.4 Hz, J = 8.6 Hz, 1H, ArH), 7.35 (dt, ³J = 2.5 Hz, ³J_H,F = 9.8 Hz, 2H, ArH), 7.44 (dd, J = 8.6 Hz, J = 14.9 Hz, 1H, ArH) ppm; ¹³C NMR (101 MHz, DMSO-d₆): δ = 12.8, 13.9 (2 x CH₃), 38.7, 42.6 (2 x NCH₂), 104.4 (t, J_C,F = 25.9 Hz, CH_Ar), 112.1 (dd, J_C,F = 3.5 Hz, J_C,F = 21.6 Hz, CH_Ar), 121.8 (dd, J_C,F = 3.9 Hz, J_C,F = 18.9 Hz, C_Ar), 129.6 (dd, J_C,F = 5.9 Hz, J_C,F = 9.9 Hz, CH_Ar), 157.8 (dt, J_C,F = 246.7 Hz, J_C,F = 12.6 Hz, C_Ar), 162.3 (dt, J_C,F = 248.0 Hz, J_C,F = 12.1 Hz, C_Ar), 164.1 (C = O) ppm; ¹⁹F NMR (378 MHz, DMSO-d₆): δ = −113.1 (ArF), −108.7 (ArF) ppm; MS (ESI+): m/z = 214 [M+H]⁺.

2.7. Synthesis of (2,4-Difluorophenyl)(piperidin-1-yl)methanone (5)

Piperidine (2 equiv.) was dissolved in absolute chloroform, 2,4-difluorobenzoyl chloride (1 equiv.) was added slowly at 0 °C, and the mixture was allowed to stir at rt overnight. Afterwards, the solvent was removed and the crude mixture of products was purified via automated column chromatography, yielding (2,4-difluorophenyl)(piperidin-1-yl)methanone (5) in 82% yield as a colorless oil. ¹H NMR (400 MHz, DMSO-d₆): δ = 1.00 (t, J = 7.1 Hz, 3H, CH₃), 1.14 (t, J = 7.1 Hz, 3H, CH₃), 3.12 (q, J = 7.1 Hz, 2H, NCH₂), 3.45 (q, J = 7.1 Hz, 2H, NCH₂), 7.16 (dt, J = 2.4 Hz, J = 8.6 Hz, 1H, ArH), 7.35 (dt, ³J = 2.5 Hz, ³J_H,F = 9.8 Hz, 2H, ArH), 7.44 (dd, J = 8.6 Hz, J = 14.9 Hz, 1H, ArH) ppm; ¹³C NMR (101 MHz, DMSO-d₆): δ = 12.8, 13.9 (2 x CH₃), 38.7, 42.6 (2 x NCH₂), 104.4 (t, J_C,F = 25.9 Hz, CH_Ar), 112.1 (dd, J_C,F = 3.5 Hz, J_C,F = 21.6 Hz, CH_Ar), 121.8 (dd, J_C,F = 3.9 Hz, J_C,F = 18.9 Hz, C_Ar), 129.6 (dd, J_C,F = 5.9 Hz, J_C,F = 9.9 Hz, CH_Ar), 157.8 (dt, J_C,F = 246.7 Hz, J_C,F = 12.6 Hz, C_Ar), 162.3 (dt, J_C,F = 248.0 Hz, J_C,F = 12.1 Hz, C_Ar), 164.1 (C = O) ppm; ¹⁹F NMR (378 MHz, DMSO-d₆): δ = −113.1 (ArF), −108.7 (ArF) ppm; MS (ESI+): m/z = 226 [M+H]⁺.

2.8. Synthesis of (2,4-Difluorophenyl)(morpholino)methanone (6)

Morpholine (2 equiv.) was dissolved in absolute chloroform, 2,4-difluorobenzoyl chloride (1 equiv.) was added slowly at 0 °C, and the mixture was allowed to stir at rt overnight. Afterwards, the solvent was removed and the crude mixture of products was purified via automated column chromatography, yielding (2,4-difluorophenyl)(morpholino)methanone (6) in 87% yield as a colorless oil. ¹H NMR (400 MHz, DMSO-d₆): δ = 3.23 (t, J = 4.5 Hz, 2H, NCH₂), 3.52 (t, J = 4.5 Hz, 2H, NCH₂), 3.64 (s, 4H, OCH₂), 7.19 (dt, J = 2.3 Hz, J = 8.5 Hz, 1H, ArH), 7.36 (dt, ³J = 2.3 Hz, ³J_H,F = 9.9 Hz, 2H, ArH), 7.50 (dd, J = 8.3 Hz, J = 14.8 Hz, 1H, ArH) ppm; ¹³C NMR (101 MHz, DMSO-d₆): δ = 41.9, 47.0 (2 x NCH₂), 65.9, 66.2 (2 x OCH₂), 104.4 (t, J_C,F = 26.0 Hz, CH_Ar), 112.3 (dd, J_C,F = 3.5 Hz, J_C,F = 21.6 Hz, CH_Ar), 120.4 (dd, J_C,F = 3.9 Hz, J_C,F = 18.1 Hz, C_Ar), 130.5 (dd, J_C,F = 5.6 Hz, J_C,F = 10.1 Hz, CH_Ar), 158.0 (dt, J_C,F = 248.2 Hz, J_C,F = 12.8 Hz, C_Ar), 162.8 (dt, J_C,F = 248.9 Hz, J_C,F = 12.1 Hz, C_Ar), 163.3 (C = O) ppm; ¹⁹F NMR (378 MHz, DMSO-d₆): δ = −112.0 (ArF), −107.8 (ArF) ppm; MS (ESI+): m/z = 267 [M+K+H]⁺.

3. Results and Discussion

3.1. Synthesis of the Piperazine Compounds

A one-pot reaction procedure using low-cost starting materials was applied. An excess of piperazine (2) was reacted with 2,4-difluorobenzoyl chloride (1) in anhydrous chloroform at 0 °C to obtain both compounds, the N-monoacylated amide 3a and the N,N-diacylated diamide 3b, according to a previously published procedure [47]. Depending on the excess of piperazine used for the reaction, compound 3a was obtained in 66% yield (five-fold excess) and compound 3b in 44% yield (two-fold excess). The reaction for both piperazines, 3a and 3b, is illustrated in Scheme 1.

3.2. Dynamic VT-NMR Study for the Dimer 3b

During the full characterization of compound 3b, four broad singlets were observed in the aliphatic region of the ¹H NMR spectrum when measured at 25 °C (e.g., for CDCl₃: δ = 3.34, 3.44, 3.79, and 3.89 ppm, ratio: 1:1.4:1.4:1 in Figure S10). Normally, one summarized signal could be expected for the NCH₂-protons under these conditions due to the symmetry of the molecule. The ¹H NMR spectrum is even more complex when measured at −10 °C. The four signals are again split in a more complicated pattern (see SI) due to the unsymmetrical substitution pattern of the benzene ring. Generally, rotation isomers are possible as shown in Figure 2, when additionally considering the rotation around the C(O)-C_Ar-bond.

Supplementary ¹H NMR spectra were recorded in DMSO-d₆, methanol-d₄, acetonitrile-d₃, acetone-d₆, benzene-d_6, and tetrachloroethane-d₂ (TCE-d₂) (Figure 3), all showing four signals for the piperazine protons at 25 °C independent of the solvent but with different Δν values and different integral values. H,H-COSY spectra measured in CDCl₃ revealed that only the inner two NCH₂ signals couple (Figure S12). The inner two signals belong to the anti (trans) isomer (3.43 and 3.79 ppm), whereas the two outer NCH₂ signals belong to the syn (cis) isomer (3.34 and 3.89 ppm), as indicated by recent results [10,47]. This splitting can also be observed in the ¹³C NMR spectrum (see Figure S11) with δ = 42.1 and 47.5 ppm for the syn isomer and 42.5 and 46.9 ppm for the anti-isomer measured in CDCl₃. However, a reduced rotation around the C(O)-C_Ar-bond has not been detected at temperatures > 25 °C, only the reduced rotation around the amide bond.

VT-dependent NMR measurements were accomplished (see SI Figures S15–S18) to determine the coalescence point expressed as T_C for the partial amide double bond, which is dependent on the solvent. By warming the NMR sample of 3b, all four signals with an integration value of two hydrogen atoms each combine to one signal with a value of 8 at the T_C due to the unhindered rotation of the amide bonds beyond this temperature. When using the standard NMR solvent CDCl₃, the coalescence point could not be reached due to the boiling point of the solvent (Figure S15). To overcome this, the solvent was changed to tetrachloroethane-d₂ (TCE-d₂) with similar properties but with a boiling point of 145 °C. The VT-dependent ¹H NMR spectra are pointed out in Figure 4A, and the T_C was determined at 83 °C. Additionally, VT-¹H-NMR spectra were recorded in acetonitrile-d₃ with a T_C of 80 °C and in DMSO-d₆ with a T_C of 83 °C (Figure 4B,C).

The T_C for compound 3b containing the fluorine 2,4-substitution is higher in all used solvents as for the mono-fluorinated 4-fluorobenzamide compound (T_C = 296 K in CDCl₃) and the unsubstituted benzamide compound (T_C = 306 K in CDCl₃) due to the higher electron-withdrawing effect caused by the additional fluorine in the ortho position of the benzoyl moiety, which strengthens the partial double bond. The higher T_C also has an influence on ΔG^#, which was determined with >68 kJ/mol (anti-isomer) and >70 kJ/mol (syn isomer) for compound 3b (see

Table 1. Physicochemical data for compound 3b in dependence on the solvent. a: anti/s: syn.

Solvent	Ratio syn/anti	Δν [Hz]	kexc [Hz]	TC [K]	ΔG‡ [kJ/mol]
Benzene-d6	1.5:1	257 (a) 381 (s)	313 (a) 494 (s)	n.d.	- -
CDCl3	1.2:1	141 (a) 223 (s)	244 (a) 395 (s)	n.d.	- -
TCE-d2	1.1:1	136 (a) 224 (s)	571 (a) 846 (s)	356	70.8 69.3
Acetone-d6	1.2:1	110 (a) 223 (s)	302 (a) 498 (s)	n.d.	- -
ACN-d3	1.1:1	116 (a) 213 (s)	258 (a) 473 (s)	353	70.7 68.9
CD3OD	1.1:1	119 (a) 202 (s)	264 (a) 448 (s)	n.d.	- -
DMSO-d6	1.2:1	116 (a) 202 (s)	258 (a) 448 (s)	353	70.7 69.0

).

A 2D ¹H EXSY spectrum was recorded at 25 °C and 600 ms mixing time, showing the exchange of the protons (representative for compound 3b in Figure 5). At this temperature, the molecule exhibits slow rotation around the N-C bond such that both groups exchange with one another rotationally yet are distinct in the spectrum.

3.3. X-Ray Structure Analysis of 3b

Single crystals of 3b were obtained, and the crystal and molecular structure were determined using single-crystal X-ray structure analysis. The molecular structure of this compound and the atom numbering scheme are shown in Figure 6. Relevant crystallographic data is given in

Table 2. Crystal data and structure refinement for compound 3b.

Compound	3b
Formula	C18H14F4N2O2
Formula weight (g·mol−1)	366.31
Temperature (K)	123
Crystal system	monoclinic
Space group	P21/c
Unit cell dimensions:
a (Å)	7.2687(3)
b (Å)	17.2658(8)
c (Å)	6.9738(3)
β (°)	115.393(2)
Volume (Å3), Z	790.65(6), 2
Data/restraints/param.	2955/0/157
Measured reflections	23,516
2θmax (°)	66.0
GoF on F2	1.08
R1 [I > 2σ(I)]	0.058
wR2 (all data)	0.154
Larg. diff. peak/hole (e·Å3)	0.36/−0.31

.

Compound 3b crystallizes in the monoclinic crystal system with the space group P2₁/c. The asymmetric unit consists of one half of a molecule. It is located such that the center of the piperazine ring is positioned on the inversion center, i.e., the molecule has an inversion symmetry. In the crystal, parts of the molecules of 3b are arranged with different orientations, i.e., they are disordered. The ortho-located fluorine atom (F1) is either cis (F1A) or trans (F2B) oriented with respect to the oxygen atom O1 (see anti-isomers in Figure 2). The occupation of the F1A site has been refined to 26.5%. The piperazine moiety has two different orientations, A and B, as shown in Figure 6, with an occupancy of 56.4% for orientation A. As visible in Figure 6 and Figure 7, only the anti-conformer (with respect to the orientation of the two oxygen atoms in each molecule) exists in the solid state. This probably results from intermolecular interactions preferring this orientation in the solid state, which are not present in solution.

The bonding environment around the nitrogen atom N1A (and also around N1B, respectively) is close to a trigonal planar disposition. The average bond angle of 119.7° and the distance of N1A to the plane of the three surrounding C atoms of only 0.08 Å indicate that the C1−N1A bond has at least a strong partial double bond character. Additionally, this bond is significantly shorter (1.398(2) Å) than comparable aliphatic bonds (i.e., 1.462(4) Å in N-methylpiperazine(sulfan)). These structural features are consistent with the NMR data, as shown above. The CH₂ groups of the piperazine moiety have bond angles close to tetrahedral, as expected, with H−C−C−H dihedral angles of 53.3° and −65.2°.

3.4. Dynamic NMR Studies of Monomer 3a

The mono-substituted 2,4-fluorobenzyl compound 3a, with only one amide site and one free secondary amine site, shows a similar splitting behavior. Four signals for NCH₂ groups are observed in the ¹H NMR spectrum at 25 °C measured in CDCl₃ (Figure S1); two for the amide site (δ = 3.39 and 3.85 ppm) and two for the amine site (δ = 2.92 and 3.03 ppm). The evaluation of a H,H-COSY spectrum (Figure S3) showed again the independent coupling of two NCH₂ groups.

Two effects can be discussed for the splitting of the piperazine signals. The first effect is related to the limited rotation around the amide bond (as discussed above), and the second effect is related to the interconversion of two piperazine ring chair conformations [47], which occurs at deeper temperatures and thus did not have an influence. When measuring VT-dependent ¹H NMR spectra in CDCl₃ (Figure S7), only the T_C of the amine site was detectable at 57 °C; the coalescence temperature of the amide site is located above the boiling point of the solvent. Thus, TCE-d₂ and DMSO-d₆ were again used as solvents (Figure 8), having T_Cs for the amine site of 63 °C and 61 °C, respectively, and for the amide site of 82 °C and 79 °C, respectively. All T_C values are higher compared to the respective monosubstituted 4-fluorobenzoyl compound (T_C,amine = 25 °C, T_C,amide = 36 °C) due to the additional fluorine atom in the 2-position of the ring, which triggers a higher double bond character of the amide bond. As a result, the Gibbs free activation energy ΔG^‡ is also higher for the 2,4-difluoro compound 3a in comparison to the respective 4-fluorobenzoyl compound (ΔG^‡ = 60.4 kJ/mol), but the solvent seems to have only a small influence on the rotation. All results concerning compound 3a are summarized in

Table 3. Physicochemical parameters of compound 3a.

Solvent	Δν [Hz]	kexc [Hz]	TC [K]	ΔG‡ [kJ/mol]
CDCl3	45 185	100 (amine) 411 (amide)	330 (amine) n.d. (amide)	68.5 -
TCE-d2	48 160	107 (amine) 355 (amide)	336 (amine) 355 (amide)	69.6 70.1
DMSO-d6	46 160	102 (amine) 355 (amide)	334 (amine) 352 (amide)	69.3 69.5

.

3.5. Comparison with Other 2,4-Difluorobenzamides

To compare the results of compounds 3a and 3b and to study the influence of the organic groups connected at the amide nitrogen atom (open chain or cyclic or, with or without heteroatom), three additional 2,4-difluorobenzamides 4–6 were prepared (Figure 9) with diethyl (4), piperidinyl (5) and morpholinyl (6) as substituents according to the synthesis of 3a and 3b with 2,4-difluorobenzyl chloride (1) and the respective amines in anhydrous chloroform.

All NMR spectra were recorded in DMSO-d₆ (Figure 10), and all compounds 4–6 nicely show the splitting of the amide residues due to the hindered rotation around the C(O)-N bond (Figure 10). Again, VT-¹H-NMR spectra were recorded for compounds 4–6 in DMSO-d₆ (Figures S27, S36, and S45) to determine the Tc’s of the compounds for the calculation of the rotation barrier according to equations (1) and (2). All recorded data and calculated energy barriers expressed as Gibbs energy ΔG^‡ were found in

Table 4. Physicochemical parameters of compounds 3a, 3b, and 4–6 measured in DMSO-d₆.

Compound	Δν [Hz]	kexc [Hz]	TC [K]	ΔG‡ [kJ/mol]
3a	116 (anti) 202 (syn)	258 (anti) 448 (syn)	353	70.7 69.0
3b	48 (amine) 160 (amide)	107 (amine) 355 (amide)	336 (amine) 355 (amide)	69.6 70.1
4	56 (CH3) 130 (CH2)	124 (CH3) 289 (CH2)	358 (CH3) 370 (CH2)	73.9 73.8
5	171 (amide) 41 (CH2)	380 (amide) 91 (CH2)	371 348	73.2 72.6
6	119	264	361	72.2

. The chosen organic groups of the amide site do not have a big influence on the rotation. The highest barrier with 73.9 kJ/mol was found for the open-chain diethylamide 4. Overall, all calculated energy values for the rotation barrier of 3a, 3b, and 4–6 are in the same range. The additional fluorine in the 2-position of the aromatic ring primarily influences the rotation barrier.

4. Conclusions

Two fluorine-containing piperazine derivatives 3a and 3b were synthesized using a convenient one-pot synthesis procedure. An evaluation of the NMR spectra showed four signals of the NCH₂ groups of the piperazine moiety for both compounds, coming from the formation of different rotation isomers. An existing partial double bond in the amide residue led to rotational conformers, whereas the ring conversion of the amine site in 3a and additional reduced rotation around the C(O)-C_Ar-bond in 3a and 3b are not observed at temperatures > 25 °C. To investigate the influence of the organic groups of the amide, three additional compounds 4–6 were prepared, and VT-¹H-NMR spectra were recorded in DMSO-d₆. Activation energies ΔG^‡_exc were calculated from solvent-dependent coalescence temperatures T_C, which are all in the same range between 69 and 73 kJ/mol. They are higher compared to the mono-fluorinated amide and to the unsubstituted benzamide. The formation of conformers of compound 3b, which result from the partial double bond, was additionally determined and verified using single-crystal X-ray structure analysis for this compound. Only a minor influence of the solvent was observed.