Vibrational spectra of pilocarpine hydrochloride crystals

Vibrational spectra of pilocarpine hydrochloride crystals

R.R.F. Bento^I; P.T.C. Freire^II,*; A.M.R. Teixeira^III; J.H. Silva^III; J.A. Lima Jr.^IV; M.C.F. de Oliveira^V; M. Andrade-Neto^V; N.R. Romero^VI; F.M. Pontes^VII

^IInstituto de Física, Universidade Federal do Mato Grosso, Cuiabá-MT, Brazil

^IIDepartamento de Física, Universidade Federal do Ceará, Fortaleza-CE, Brazil

^IIIDep. Ciências Físicas e Biológicas, Universidade Regional do Cariri, Crato-CE, Brazil

^IVUniversidade Estadual do Ceará, Limoeiro do Norte-CE, Brazil

^VDep. Química Orgânica e Inorgânica, Universidade Federal do Ceará, Fortaleza-CE, Brazil

^VIDepartamento de Farmácia, Universidade Federal do Ceará, Fortaleza-CE, Brazil

^VIIFaculdade de Ciências, Universidade Estadual de São Paulo, Bauru-SP, Brasil

ABSTRACT

Pilocarpine is a natural substance with potential application in the treatment of several diseases. In this work Fourier Transform (FT)-Raman spectrum and the Fourier Transform infra red (FT-IR) spectrum of pilocarpine hydrochloride C₁₁H₁₇N₂O₂⁺.Cl^- were investigated at 300 K. Vibrational wavenumber and wave vector have been predicted using density functional theory (B3LYP) calculations with the 6-31 G(d,p) basis set. A comparison with experiment allowed us to assign most of the normal modes of the crystal.

Keywords: Raman scattering, infrared spectroscopy, normal modes, pilocarpine hydrochloride

1. INTRODUCTION

In recent years, there has been a growing interest in the study of spectroscopic properties of plant cells in order to identify their chemical constituents through non-destructive analysis [1]. The main researches deal with primary metabolites, i.e., substances essential for their growth, surviving and reproduction (among them, amino acids, proteins, carbohydrates, lipids and fatty acids). On the contrary, the investigation of the vibrational property of isolated secondary metabolites from plants (used as defense against parasite and diseases as well as used to reinforce reproductive processes) is still poorly explored, although many of them have potential application as therapeutic drugs [2,3].

Secondary metabolity pilocarpine (C₁₁H₁₇N₂O₂), an alkaloid extracted from the leaves of the South American shrubs Pilocarpus jaborandi, Pilocarpus microphyllus and other Pilocarpus species [4], is an imidazole derivative that exhibits some pharmacological activities. These activities include diaphoretic effects, stimulation of parasympathetic system [5], miotic action [6], being also used in ophthalmology [6,7]. The action of pilocarpine on the parasymphatetic nervous system has been extensively investigated and it is known that the substance act mainly as a cholinergic drug [8]. Despite of several therapeutic effects, pilocarpine is used clinically only to treat glaucoma [8].

Pilocarpine molecule, which contains both imidazole and γ- lactone rings forms two semi-organic compounds in the solid state phase, trichlorogermanate hermihydrate [9] (C₁₁H₁₇N₂O₂, GeCl₃,1/2H₂O) and hydrochloride (C₁₁H₁₇N₂.Cl^-). [10] For both compounds it was discovered that the crystal structures are monoclinic, space group P2₁, although the conformation of the pilocarpine molecule itself differs significantly from one structure to the other [9,10].

From the biological point of view, pilocarpine hydrochloride has been used in certain eye diseases, as for exemple, in the treatment of intraocular hemorrhages, opacities of the vitreous and aqueous fluids [8], while trichlorogermanate hermihydrate pilocarpine presents a weak activity of muscarinic stimulants [11].

In this work an infrared analysis and a Raman scattering study in the spectral range 40 cm^-1 to 4000 cm^-1 of pilocarpine hydrochloride crystal obtained from Pilocarpus trachyllophys [4] is reported. In order to assign the normal modes of vibrations of the material a Density Functional Theory (DFT) calculation was performed.

2. EXPERIMENTAL

FT-Raman spectrum was taken using a Bruker RFS100/S FTR system and a D418-T detector, with the sample excited by means of the 1064 nm line of a Nd:YAG laser. Infrared spectrum was obtained by using an Equinox/55 (Bruker) Fourier Transformed Infrared (FTIR) spectrometer. FT-Raman and FT-IR spectra were collected from samples confined in screw cap standard chromatographic glass vials, at a nominal resolution of 4 cm^-1 accumulating 60 scans per spectra and using a laser power of 150 mW.

3. COMPUTATIONAL METHOD

Density functional theory (DFT) calculations were carried out using the Gaussian 98 programme package [12]. The B3LYP functional was used with the 6-31 G(d,p) basis set. The calculations were performed using an isolated molecule of pilocarpine cation: . The structure obtained from the X-ray analyses of pilocarpine hydrochloride at 77 K was used as starting structure [10]. This structure was optimized and the vibrational wavenumbers were then calculated. The output file contained the optimized structure, the vibrational frequencies in the harmonic approximation, and the atomic displacements for each mode. At the optimized structure of the molecule, no imaginary frequency was obtained, proving that a true minimum of the potential energy surface was found. The calculated vibrational wave numbers were adjusted to compare with experimental Raman and IR frequencies.

4. RESULTS AND DISCUSSION

The crystal of pilocarpine hydrochloride at room temperature belongs to the monoclinic structure with P2₁ () space group, with Z=2, and lattice parameters a = 11.057 [10.965] Å, b = 9.212 [9.177] Å, c =6.697 [6.507] Å and β = 110.05 [109.19]^o o (where the values in brackets hold for the 77 K determination) [10].

Figure 1 shows the molecular structure of the pilocarpine hydrochloride (). The numbering of the atoms in Fig. 1 follows that of Codding [10] in which the N-methylated nitrogens (N₁) are separated from the ether oxigen by four carbon atoms and from the carbonyl oxygen by five atoms. This labelling will be used in describing the parameters for optimized structure and the molecular wave vectors. The distribution of the two molecules of pilocarpine hydrochloride in the unit cell is showed in Fig. 2.

Tables 1, 2 and 3 show respectively, bond distances, bond angles and some selected torsion angles for pilocarpine cation, , for optimized structure of the molecule (Cal) and that obtained from X-ray analysis (Exp) [10]. The results show that optimized structure was observed to reproduce the experiments with good agreement.

FT-Raman spectrum and the FT-infrared (IR) spectrum of pilocarpine hydrocholride are presented in Figs. 3(a) and 3(b), respectively.

The molecule of pilocarpine hydrochloride has C₁ site symmetry, and therefore, 93 molecular vibrations among all 99 are allowed in the Raman spectrum. The couplings of vibrations due to the presence of two molecules in the unit cell give rise to twice greater number of modes in the crystal. The number of normal modes expected for the crystal is then, 198, distributed into irreducible representations of C₂ factor group as 99 (A + B); from these modes 99 A + 99 B modes are Raman active. Assuming that the weakness of the intermolecular coupling causes negligible factor group splitting, the task is simplified to the assignment of the 93 molecular modes. Table 4 lists a detailed description of assignments for vibrational wavenumbers of pilocarpine hydrochloride. In the first column the calculated values for the wavenumbers are given. We also present the experimental wavenumber values for the crystal obtained by FT-Raman and FT-IR spectroscopies (the second and third columns, respectively); the fourth column in Table 4 gives the assignment of the bands. In order to better visualise the vibrations, we refer to the two rings in the assignments of Table 4, as follows: imidazole ring type or 1-methylimidazole as R1 and γ- lactone ring type or γ-butyrolactone as R2. The nomenclature employed in the classification of normal modes is given below the Table 4.

The assignment for pilocarpine hydrochloride shows that most of the bands observed through FT-Raman and FT-IR spectroscopies correspond to a mixture of vibrational modes. The mixture of modes is common in molecules of C₁ site symmetry. The superposition of modes precludes a direct identification of the bands. However, an effort was carried out through this work to make a detailed description of assignments of vibrational modes of the crystal. In order to illustrate the assignment, atomic displacements corresponding to selected normal modes from the isolated molecular structure of pilocarpine hydrochloride are shown in Fig. 4.

Now we discuss the main calculated and observed vibrations of pilocarpine hydrochloride. Two fundamental units of the pilocarpine molecule are the rings. Imidazole ring, which is a characteristic part of pilocarpine, is present in several substances of biological interest, as for example, L-histidine amino acid [13-15], and in other substances [16,17]. Their vibrations spread over a large spectral range of wavenumbers. Lactone, the other ring, is also found in several different substances of biological interest [18-20]; for some of them spectroscopic studies have revealed the wavenumber of the main vibrations [19]. Calculations show that at low wavenumber (ω < 150 cm^-1) where it is expected to be observed bands associated to lattice vibrations, some internal modes are also present. For example, torsional vibrations of the two rings are observed together with lattice modes at very low wavenumber. This should be expected because the rings are very large structures; so, we assign the bands in this spectral region as a mixture of lattice modes and torsional vibrations of the rings R1 and R2. Fig. 4(a) shows atomic displacements associated to deformaticons {γ_oop (R1), δ_oop (R2) [τ (C10O9O12)], r(C13H₂), r(C14H₃)} corresponding to the strong Raman bands observed at 96 cm^-1 (ω_cal = 97 cm^-1) .

Another class of vibrations is related to deformation of rings. In plane ring deformation vibration appears in a large spectral region (690 - 1900 cm^-1) and out of plane ring deformation vibration appears for 546 < ω < 1140 cm^-1. However, most of them are mixed with other kind of vibrations such as rocking and bending of CH, torsion of CH₂ and stretching of CC. Fig. 4(b) represents the mixtures of vibrational modes {δ_ip (R2) [sc(C8C7C11); ν_s (C11C10O9)], r(C8H₂; C13H₂), r(C14H₃), δ(C7H)} giving rise to the strong Raman peak observed at 766 cm^-1 (ω_cal. = 765 cm^-1).

The whole structure presents deformation vibrations δ(all structure) at ~ 650 cm^-1 as well as at ~ 1080 cm^-1 and at 1300 - 1340 cm^-1. A calculated δ (all structure) vibrational is also expected at 770 cm^-1 but, possibly, is mixed with the 765 cm^-1 complex vibration. Four strong IR bands are associated to ring deformation. Fig. 4(c) illustrates one of them observed at 1027 cm^-1 (ω_cal = 1026 cm^-1), corresponding to {δ_oop (R2) [ν_as (C11C13C14)], wag (C13H₂), r (C6H₂; C8H₂), r (C14H₃), δ (C7H; C11H)}. Another band is observed at 1181 cm^-1 ( ω_cal = 1185 cm^-1), corresponding to {δ_ip (R2) [sc (C8C7C11),ν (C10O9)], r (C13H₂), r (C14H₃), wag (C6H₂; C8H₂), δ(C7H; C11H)}. The other two strong IR bands are associated with the deformations {δ_ip (R1) [ν (N1C15; N3C2), ν (C4C5)], wag (C15H₃), δ (C2H; C4H), δ (N3H)} and {δ_ip (R1) [ ν (C4C5; C5C6), ν (C2N1)], wag (C6H₂), r (C15H₃), δ (C2H; C4H), δ (N3H)}, corresponding to the peaks observed at 1752 cm^-1 (ω_cal = 1598 cm^-1) and 1767 cm^-1 (ω_cal = 1655 cm^-1), respectively.

It is also interesting to note that the rocking vibrations of the three CH₂ units (C6H₂, C8H₂ and C13H₂) are observed at similar wavenumbers. On the contrary, although CO₂ vibrations can be expected for pilocarpine molecule, rocking vibration of CO₂ (a well characteristic vibration in amino acid crystal at 500 - 540 cm^-1) is absent. This is because the only CO₂ possibility is C10O9O12, but O9 is held at lactone ring; as a consequence, it is impossible to have a rocking C10O9O12 vibration.

Because there are many C-C bonds in pilocarpine molecules the CC stretching vibrations are observed in a large range of wavenumbers. The lowest wavenumber value is for C11-C13 stretching, which was observed at 716 cm^-1 while the highest wavenumber value corresponding to a ν(CC) is calculated at 1140 cm^-1· ν(NC) vibrations contributes with bands observed at 939 and 1754 cm^-1.

It is possible to note a marked localization of the scissoring vibrations (CH₂ and CH₃) in the range 1382 cm^-1 < ω_cal < 1537 cm^-1. As an example, the Raman band observed at 1492 cm^-1 (ω_cal = 1493 cm^-1) corresponds to the scissoring vibration sc(C15H₃).

A large number of bands associated with overtones and combination tones may be found in the region about 2800 cm^-1 due to the large number of bands in the region between 84 cm^-1 and 1800 cm^-1. The bands 2819, 2834 and 2844 cm^-1 were assigned as combination tone. The intensity of bands at 2844 cm^-1 suggests that is a combination involving at least one strong Raman band. Thus, the modes at 96, 114, 766, 1369, and 1492 cm^-1 can be involved.

The spectral region between 2800 and 3150 cm^-1 of the Raman spectrum of pilocarpine hydrochloride crystal consists of a series of very intense Raman bands, and a series of less intense IR bands. However, all bands are well resolved, allowing for their identification as listed in Table 4. For organic crystals the region about 3000 cm^-1, in general, contains the bands originated from C-H, CH₂, CH₃, and N-H vibrations [21, 22]. For some materials this region condenses very important informations, being a tool to understand conformation of the molecules in the unit cell or even interactions such as hydrogen bonds. For example, a study on L-methionine crystal have shown that the behaviour of Raman bands under pressure in this spectral region can be understood as consequence of structural changes instead of simple conformational changes of molecules in the unit cell [23]. So, the understand of the origin of these bands can be fundamental to understand the behaviour of pilocarpine hydrochloride under different conditions, in particular, related to the conditions found in drug artefacts. The scheme of Fig. 4(d) shows, as an example, the mixtures of stretching modes {ν_s(C6H₂; C13H₂), ν (C7H)} corresponding to a very strong Raman band observed at 2908 cm^-1 (ω_cal = 3024 cm^-1).

5. CONCLUSIONS

The phonon spectrum of the pilocarpine hydrochloride, a potential pharmaceutical substance to be used in several disease treatments, was measured at room temperature through FT-Raman and FT-IR techniques. Density functional theory calculations were carried out by using the Gaussian 98 package and the B3LYP functional with the 6-31 G(d,p) basis set. The calculations were observed to reproduce the experiments with good agreement. This agreement allowed us the assignment of the observed wavenumbers to atomic motions in the molecules. In particular, it was observed that most bands are associated to mixing of vibrational modes, even in the low wavenumber region where, generally, the lattice modes are found. The absence of stretching vibrations of water molecule which can be observed at ~ 3400 cm^-1 in this region indicates that the crystal is free of water molecules.

Acknowledgments

We thank CENAPAD-SP for the use of the GAUSSIAN 98 software package and for computational facilities through the project reference "proj373". Financial support from CNPq, CAPES and FUNCAP is also acknowledged. One of us (AMRT) thanks Universidade Regional do Cariri for allowing him to spend one year at UFC to develop his pos-doc research.

(Received on 15 November, 2008)

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Publication Dates

• Publication in this collection
  15 June 2009
• Date of issue
  Mar 2009

History

• Received
  15 Nov 2008