2.1.

Chemicals

Halofantrine hydrochloride was a kind gift of GlaxoSmithKline and was used without further purification. The systematic IUPAC name of halofantrine is 3-dibutylamino-1-[1,3-dichloro-6-(trifluoromethyl) phenanthren-9-yl]-propan-1-ol. The molecular structure of halofantrine is shown in Fig. 1 . The atomic numbering scheme and the partitioning of the phenanthrene ring in P1, P2 and P3 in Figure 1 is used in the context for the description of the mode assignment.

Fig. 1

Molecular structure of halofantrine. The atomic numbering scheme and the partitioning of the phenanthrene ring in P1, P2, and P3 is used in the context for the description of the mode assignment.

2.2.

Spectroscopy

The Fourier transform (FT) Raman spectrum of halofantrine hydrochloride was recorded with a Bruker FT Raman spectrometer (RFS 100/S) at the macroscopic mode with a spectral resolution of $2{\mathrm{cm}}^{-1}$ . The instrument was equipped with a Nd:YAG laser ( ${\lambda }_{\mathrm{exc}}=1064\mathrm{nm}$ , estimated laser power at the samples $P=50\mathrm{mW}$ ) as the excitation source and a liquid-nitrogen-cooled germanium detector.

The complementary FT IR spectrum of halofantrine hydrochloride was measured as KBr pellet using a Bruker IFS 66 spectrometer equipped with a DTGS (doped triglycerinsulfate) detector and with $4\text{-}{\mathrm{cm}}^{-1}$ spectral resolution.

UV resonance Raman microspectroscopy was performed with an UV Raman setup (HR800 LabRam, Horiba/Jobin-Yvon, focal length of $800\mathrm{mm}$ and a $2400\text{lines}∕\mathrm{mm}$ grating) equipped with an Olympus BX41 microscope, UV sensitive video camera, and a liquid- ${\mathrm{N}}_2$ -cooled CCD detector. For UV microscopy, a UV achromatic fused silica/ $\mathrm{Ca}{\mathrm{F}}_2$ microspot objective [LMU- $40\times$ -UVB, numerical aperture $\left(\mathrm{NA}\right)=0.5$ ] with broad band UVB coating was chosen. Validation of the wave number axis was performed via the Raman signals from Teflon. The excitation wavelength $244\mathrm{nm}$ was derived from an intracavity frequency-doubled argon-ion laser (Innova300-MotoFreD, Coherent Inc.). The laser power at the sample was estimated to be $1\mathrm{mW}$ . The spectral resolution was $5{\mathrm{cm}}^{-1}$ . Because the laser wavelength was chosen to excite the sample in an electronic absorption band, it was necessary to carefully avoid any sample destruction. Furthermore, the sample rotation technique^{37, 38} was applied.

2.3.

Density Functional Theory Calculation

Density functional theory (DFT) calculations were performed with Gaussian 03 (revision D.01)³⁹ with Becke’s three-parameter exchange functional⁴⁰ (B3) as slightly modified by Stephens,⁴¹ coupled with the correlation functional of Lee ⁴² (B3LYP) and B3 in combination with the correlation part of the functional from Perdew and Wang⁴³ and Perdew ⁴⁴ (B3PW91). Double $(6\text{-}31+G(d,p))$ and triple $(6\text{-}311+G(d,p))$ split valence basis sets of contracted Gaussian functions with polarized and diffuse functions^{45, 46, 47} were applied. These hybrid exchange correlation functionals provide reliable estimates of experimental frequencies of organic molecules with small root mean square deviations.^{48, 49, 50, 51, 52, 53}

The DFT calculated harmonic vibrational frequencies are typically too large compared with the experimentally observed ones, due to neglect of anharmonicity, incomplete incorporation of electron correlation, and the use of finite basis sets. Fortunately, this overestimation of the calculated harmonic vibrational frequencies is relatively uniform and can be circumvented by applying transferable scaling factors to the harmonic frequencies.^{48, 54} For the $\mathrm{B}3\mathrm{LYP}∕6\text{-}311+G(d,p)$ calculations, values of 0.98 for modes below $1800{\mathrm{cm}}^{-1}$ and 0.96 above $1800{\mathrm{cm}}^{-1}$ were applied, in agreement with the literature.^{54, 55, 56, 57, 58, 59, 60}

The calculated Raman activities were transformed into Raman intensities⁶¹ and were further convoluted with Gauss-Lorentz-weighted profiles to simulate Raman spectra with finite bandwidth.

3.1.

UV Resonance Raman Spectroscopy

The molecular structure of halofantrine (Fig. 1) shows a chromophoric phenanthrene structure that is highly polarizable and causes strong Raman signals [Fig. 2 ]. These Raman intensities can be enhanced by several magnitudes by exploitation of the described UV resonance effect. Halofantrine shows strong absorption bands in the deep UV at approx. $250\mathrm{nm}$ .⁶⁷ Therefore, the laser excitation wavelengths ${\lambda }_{\mathrm{exc}}=244\mathrm{nm}$ was chosen for the investigation of halofantrine. A strong enhancement of two Raman bands at 1621 and $1590{\mathrm{cm}}^{-1}$ is seen in the UV resonance Raman spectrum [Fig. 2] in comparison with the nonresonant FT Raman spectrum $({\lambda }_{\mathrm{exc}}=1064\mathrm{nm})$ [Fig. 2]. These strongly enhanced Raman signals of halofantrine can be used as marker bands to localize halofantrine in a complex biological environment. The intensities in the spectra in Fig. 2 are normalized to the strongest Raman peaks, respectively, for better illustration.

Fig. 2

(a) UV resonance Raman spectrum of halofantrine $({\lambda }_{\mathrm{exc}}=244\mathrm{nm})$ and (b) FT-Raman spectrum of halofantrine $({\lambda }_{\mathrm{exc}}=1064\mathrm{nm})$ . The wavenumber values of some prominent bands are given in graph (b). The band at $1621{\mathrm{cm}}^{-1}$ is very strong in the UV Raman spectrum (a) due to the resonance enhancement. The intensities in both graphs are normalized to the strongest peak in the spectra, respectively.

3.2.

DFT Calculation

3.2.1.

Raman spectra

To interpret the individual Raman bands in more detail, a thorough normal mode assignment is required. High-performance computing facilities nowadays enable a reliable calculation of vibrational spectra of medium-sized biomolecules.^{33, 58} The DFT calculation $[\mathrm{B}3\mathrm{LYP}∕6\text{-}311+G(d,p)]$ of the Raman spectrum of halofantrine is shown in Fig. 3 . The individual Raman bands in the calculated stick spectrum [Fig. 3] were convoluted with line profiles [Fig. 3] for comparison with measured Raman spectra with finite bandwidth [Fig. 3]. One can see that often several normal modes contribute to an individual Raman band. However, the strongest peak in the nonresonant Raman spectrum of halofantrine [Fig. 3] is dominated by one distinct mode at $1347{\mathrm{cm}}^{-1}$ [Fig. 3]. Several weaker normal modes contribute to the wings of the line profile of this band. Two molecular vibrations at $1621{\mathrm{cm}}^{-1}$ and weaker at $1629{\mathrm{cm}}^{-1}$ [Fig. 3] contribute to the Raman peak at $1621{\mathrm{cm}}^{-1}$ [Fig. 3], which is strongly enhanced in the UV resonance Raman spectrum of halofantrine [Fig. 2]. The overall agreement of the calculated Raman spectrum of halofantrine [Fig. 3] and the measurement [Fig. 3] is very good, while the intensity of the band at $1431{\mathrm{cm}}^{-1}$ is underestimated in the calculation. DFT calculations with the hybrid functional B3PW91 were performed to address whether any improvement can be achieved (results are shown in Fig. 4 ). The calculation with B3PW91 shows an improvement of the intensity of the Raman band at $1431{\mathrm{cm}}^{-1}$ ; however, the intensity of the band at $1621{\mathrm{cm}}^{-1}$ is overestimated. The results of the DFT calculations with double $[6\text{-}31+G(d,p)]$ [Figs. 4 and 4] and triple $[6\text{-}311+G(d,p)]$ [Figs. 4 and 4] split valence basis sets are very similar and no significant basis size effect was found. The calculation with the model chemistry $\mathrm{B}3\mathrm{LYP}∕6\text{-}311+G(d,p)$ was chosen for further consideration because of the very good agreement in the wavenumber region $1500\text{to}1650{\mathrm{cm}}^{-1}$ , where certain Raman modes gain strong UV resonance enhancement.

Fig. 3

Comparison of an experimental FT Raman spectrum $({\lambda }_{\mathrm{exc}}=1064\mathrm{nm})$ of halofantrine (a) alongside a calculated $[\mathrm{B}3\mathrm{LYP}∕6\text{-}311+G(d,p)]$ Raman profile (b). The individual Raman modes of the convoluted profile (b) are shown in the stick spectrum (c). The wave number values of some prominent bands are given in (a).

Fig. 4

Comparison of measured (e) and calculated Raman spectra (a–d) of halofantrine with different model chemistries. The convolution of the profiles was performed with FWHM 6 instead of FWHM 10 in Fig. 3. (a) DFT calculation of the Raman spectrum of halofantrine $[\mathrm{B}3\mathrm{PW}91∕6\text{-}311+G(d,p)]$ , (b) DFT calculation of the Raman spectrum of halofantrine $[\mathrm{B}3\mathrm{PW}91∕6\text{-}31+G(d,p)]$ , (c) DFT calculation of the Raman spectrum of halofantrine $[\mathrm{B}3\mathrm{LYP}∕6\text{-}311+G(d,p)]$ , (d) DFT calculation of the Raman spectrum of halofantrine $[\mathrm{B}3\mathrm{LYP}∕6\text{-}31+G(d,p)]$ , (e) Measured FT Raman spectrum of halofantrine $({\lambda }_{\mathrm{exc}}=1064\mathrm{nm})$ .

3.2.2.

Electron density distribution

The electron density distribution of halofantrine was calculated and yields fundamental insight regarding the possible binding behavior of the halofantrine molecule. A $\pi \pi$ stacking of the phenanthrene ring of halofantrine with the porphyrin backbone of ferriprotoporphyrin IX (FPPIX) was found^{20, 21} in a recent crystal structure of an in vitro complex of halofantrine with FPPIX. One should consider whether such binding is possible, due to strongly electron withdrawing substituent’s at the phenanthrene ring of halofantrine. The electron density distribution of halofantrine (Fig. 5 ) highlights the electron withdrawing effect of the substituent’s [C(1)Cl(31) and C(3)Cl(32) as well as C(27)F(28)F(29)F(30)]. However, a strong and evenly distributed electron density remains across the phenanthrene ring. This result supports the hypothesis that $\pi \pi$ stacking may play an important role in the biological activity of halofantrine.

Fig. 5

Calculation of the electron density distribution of halofantrine (iso-value 0.25 $\text{electrons}∕a_0^3$ ). An even electron distribution is seen in the phenanthrene ring of halofantrine, despite the electron withdrawing effect of substituents C(1)Cl(31) and C(3)Cl(32) as well as C(27)F(28)F(29)F(30).

3.3.

Mode Assignment and Atomic Displacements

The ultimate goal of the Raman spectroscopic investigation of halofantrine is a localization of the drug at the binding side of the biological target in life cells and an elucidation of changes in the Raman spectrum, caused by molecular interactions. To enable a discussion of such changes in the Raman bands of halofantrine, caused by changes in the environment, a detailed interpretation of the individual Raman bands is required. The DFT calculations can be used for a thorough band assignment. The calculations of the atomic displacements of the molecular normal modes provide much deeper understanding of the associated Raman bands.

The Raman spectrum of halofantrine consists of two well separated parts above $2800{\mathrm{cm}}^{-1}$ and below $1800{\mathrm{cm}}^{-1}$ (see Fig. 6 ). The range with high wave number values consist of two separated parts: a region with CH-stretching vibrations at the phenanthrene ring $\left(3120\text{to}3060{\mathrm{cm}}^{-1}\right)$ and an area with $\mathrm{C}{\mathrm{H}}_2$ , $\mathrm{C}{\mathrm{H}}_3$ stretching vibrations at the side chain $\left(3010\text{to}2840{\mathrm{cm}}^{-1}\right)$ of halofantrine. In addition, there is also one OH stretching vibration present at $3710{\mathrm{cm}}^{-1}$ . However, the fingerprint region of halofantrine (below $1800{\mathrm{cm}}^{-1}$ ) is of more importance in the further discussion. The graphical illustration of the atomic displacements of two prominent modes at 1621 and $1349{\mathrm{cm}}^{-1}$ are shown in Figs. 7 and 7 , respectively. The assignment of more Raman bands of halofantrine is summarized in Table 1 .

Fig. 6

Comparison of measured and calculated $[\mathrm{B}3\mathrm{LYP}∕6\text{-}311+G(d,p)]$ vibrational spectra of halofantrine. Two scaling factors were applied: 0.98 for wave numbers smaller than $1800{\mathrm{cm}}^{-1}$ and 0.96 for those above. (a) DFT calculation of the IR stick spectrum of halofantrine, (b) DFT calculation and convolution of the IR spectrum of halofantrine, (c) measured FT IR spectrum of halofantrine, (d) measured FT Raman spectrum of halofantrine, (e) DFT calculation and convolution of the Raman spectrum of halofantrine, and (f) DFT calculation of the Raman stick spectrum of halofantrine. The IR and Raman spectra of halofantrine are complementary. Distinct normal modes show either strong IR or Raman activity, respectively. The wavenumber values of some prominent IR/Raman bands are given in (c) and (d), respectively.

Fig. 7

(a) Calculated $[\mathrm{B}3\mathrm{LYP}∕6\text{-}311+G(d,p)]$ atomic displacements of the prominent normal mode of halofantrine at $1621{\mathrm{cm}}^{-1}$ . This Raman band is strongly enhanced with UV excitation ${\lambda }_{\mathrm{exc}}=244\mathrm{nm}$ [see Fig. 2]. The normal mode is localized at the phenanthrene ring with very strong contributions from P1 (see Fig. 1) as described in the context. (b) Calculated $[\mathrm{B}3\mathrm{LYP}∕6\text{-}311+G(d,p)]$ atomic displacements of the prominent normal mode of halofantrine at $1349{\mathrm{cm}}^{-1}$ . This Raman band is very strong in the FT-Raman spectrum ${\lambda }_{\mathrm{exc}}=1064\mathrm{nm}$ [see Fig. 2]. The normal mode represents a highly symmetric stretching/narrowing vibration, localized at the phenanthrene ring, as described in the text.

Table 1

Mode assignment and wave number values of prominent Raman bands of halofantrine [Figs. 2, 3, 6].

The strongest peak in the UV resonance Raman spectrum $({\lambda }_{\mathrm{exc}}=244\mathrm{nm})$ of halofantrine (Fig. 1) is found at wavenumber position $1621{\mathrm{cm}}^{-1}$ [Fig. 2]. This band is assigned to a very strong, combined $\mathrm{C}\mathrm{C}$ -stretching vibration at the phenanthrene ring. The atomic displacement of the mode is displayed in Fig. 7. The strongest contribution to this normal mode arises from $\nu \mathrm{C}\left(5\right)\mathrm{C}\left(6\right)$ in P2 (see Fig. 1 for numbering scheme) as well as from $\nu \mathrm{C}\left(6\right)\mathrm{C}\left(15\right)$ at the connection of the side chain of the molecule and ${\delta }_{\mathrm{ip}}\mathrm{C}\left(5\right)\mathrm{H}$ . A highly symmetric vibration is seen across the phenanthrene ring, where $\nu \mathrm{C}\left(1\right)\mathrm{C}\left(14\right)$ , $\nu \mathrm{C}\left(3\right)\mathrm{C}\left(4\right)$ , $\nu \mathrm{C}\left(13\right)\mathrm{C}\left(12\right)$ , $\nu \mathrm{C}\left(5\right)\mathrm{C}\left(6\right)$ , $\nu \mathrm{C}\left(11\right)\mathrm{C}\left(10\right)$ , and $\nu \mathrm{C}\left(7\right)\mathrm{C}\left(8\right)$ stretch in phase, with a parallel, out-of-phase movement of the pairs {C(4), C(7)} and {C(12), C(9)} toward each other, respectively. Further bending contributions take place ${\delta }_{\mathrm{ip}}\mathrm{C}\left(14\right)\mathrm{H}$ , ${\delta }_{\mathrm{ip}}\mathrm{C}\left(11\right)\mathrm{H}$ and weaker ${\delta }_{\mathrm{ip}}\mathrm{C}\left(9\right)\mathrm{H}$ and ${\delta }_{\mathrm{ip}}\mathrm{C}\left(8\right)\mathrm{H}$ . The fluorine atoms F(28), F(29), and F(30) and chlorine atoms Cl(31) and Cl(32) are fixed in space. There are no contributions from the side chain. The very strong enhancement of this mode in the UV resonance Raman spectrum [Fig. 2] is very promising, since this combined $\mathrm{C}\mathrm{C}$ -stretching vibration is likely to be influenced by $\pi \pi$ stacking of the phenanthrene ring to the porphyrin ring of biological target structures.^{20, 21} The calculation of the electron density distribution of halofantrine supports the hypothesis of such interaction. The monitoring of changes in this Raman band during the drug-target interaction might yield novel insight into proposed biological activities of halofantrine.

The strongest band in the nonresonant Raman spectrum is seen at wavenumber position $1349{\mathrm{cm}}^{-1}$ [Fig. 2]. Two different snapshots of the atomic displacement of this mode are illustrated in Fig. 7. The normal mode consists of a very strong, combined CC-stretching vibration at the phenanthrene ring, an in-phase breathing vibration at [C(4)C(13), C(7)C(12)] of P1, P2, and P3; combined with a deformation mode [see Fig. 7]. The symmetric narrowing of the ring system at C(4)C(13) and C(7)C(12) is nicely illustrated in the two snapshots of the molecular vibration in Fig. 7. More contributions to this mode arise from $\nu \mathrm{C}\left(10\right)\mathrm{C}\left(27\right)$ and $\nu \mathrm{C}\left(27\right){\mathrm{F}}_3$ as well as very strong ${\delta }_{\mathrm{ip}}\mathrm{C}\left(9\right)\mathrm{H}$ , ${\delta }_{\mathrm{ip}}\mathrm{C}\left(8\right)\mathrm{H}$ , ${\delta }_{\mathrm{ip}}\mathrm{C}\left(11\right)\mathrm{H}$ , and ${\delta }_{\mathrm{ip}}\mathrm{C}\left(14\right)\mathrm{H}$ and medium ${\delta }_{\mathrm{ip}}\mathrm{C}\left(2\right)\mathrm{H}$ , ${\delta }_{\mathrm{ip}}\mathrm{C}\left(5\right)\mathrm{H}$ , and ${\delta }_{\mathrm{ip}}\mathrm{C}\left(15\right)\mathrm{H}$ vibrations. Small $\omega \mathrm{C}{\mathrm{H}}_2$ contributions are present at the side chain.

3.4.

IR Spectroscopy

The IR and Raman spectra of halofantrine yield complementary information. Highly symmetric $\mathrm{C}\mathrm{C}$ -stretching vibrations in the phenanthrene ring [e.g., Figs. 7 and 7] posses strong changes in polarizability and cause intense bands in the Raman spectrum (Figs. 2 and 3). On the other hand, polar functional groups in the halofantrine molecule cause weak Raman signals, but very strong IR bands. All IR and Raman bands of halofantrine are shown in Fig. 6. The wave number values of some prominent IR and Raman bands are given in the Figs. 6 and 6, respectively. The strongest IR bands of halofantrine are seen at wave number values 2961, 1320, 1147, 1126, 1082, and $1049{\mathrm{cm}}^{-1}$ . The atomic displacement of the very strong IR mode at $2961{\mathrm{cm}}^{-1}$ is exemplarily shown in Fig. 8 . This mode represents a combined, asymmetric $\mathrm{C}{\mathrm{H}}_2∕\mathrm{C}{\mathrm{H}}_3$ -stretching vibration in the butyl part of the side chain of halofantrine.

Fig. 8

Calculated $[\mathrm{B}3\mathrm{LYP}∕6\text{-}311+G(d,p)]$ atomic displacements of the prominent normal mode of halofantrine at $2961{\mathrm{cm}}^{-1}$ . This mode is very strong in the IR spectrum [Figs. 3, 3, 3] and represents a combined $\mathrm{C}{\mathrm{H}}_2∕\mathrm{C}{\mathrm{H}}_3$ -stretching vibration (polar functional group) in the side chain of halofantrine, as described in the text.