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Fast Relaxation Dynamics of the Cardiotonic Drug Milrinone in Water Solutions Maged El-Kemary,† Juan Angel Organero, and Abderrazzak Douhal* Departamento de Quı´mica Fı´sica, Seccio´ n de Quı´micas, Facultad de Ciencias del Medio Ambiente, UniVersidad de Castilla-La Mancha, AVda. Carlos III, S.N., 45071, Toledo, Spain ReceiVed January 2, 2006
The fast relaxation dynamics of 1,6-dihydro-2-methyl-6-oxo-3,4′-bipyridine-5-carbonitrile (milrinone, MIR), a cardiotonic drug, has been characterized in water solutions at different pH. In acidic media, a blue emission reflects a charge-transfer state in the cation (C) leading to a more stabilized structure with an emission lifetime of 90 ps. The emission lifetimes of the keto (K) and anion (A) structures are ∼65 and 310 ps, respectively. Reasons for efficient nonradiative channels are discussed in terms of hydrogen-bonding interactions, intramolecular charge transfer (ICT), and twisting motion. A blue nanosecond-emission observed in almost all the studied pH range is suggested to be due to relaxed K due to an ICT reaction. B3LYP (6-31+G**) calculations showed that, in a water cavity, K is more stable than the enol form by 7 kcal/mol, and the ICT may take place within the pyridone moiety. At the physiological pH, the inotropic K structure is the dominant species (∼100%). Chart 1. Molecular Structure of MIR in Its Keto Forma
Introduction 1,6-Dihydro-2-methyl-6-oxo-3,4′-bipyridine-5-carbonitrile or milrinone (Chart 1, MIR), a cardiotonic drug, is an inotropic agent (inotropic effects are those that change the strength of contraction of the heart) used for the short-term intravenous therapy of congestive heart failure.1 MIR in water is known to undergo tautomeric interconversion between keto and enol forms in the ground state through proton transfer with water molecules.2,3 For pyridin-2(1H)-one derivatives, having structures comparable to that of MIR, there is strong experimental and theoretical evidence to prove the dominance of the keto form in aqueous solutions and of the enol structure in gas phase and in nonpolar solvents.2-6 The dominance of keto species in the ground state of pyridin-2(1H)-one in water involves a barrier of ∼35 kcal/mol for the ground-state pathway.6 Moreover, the electron-donating methyl substitutent of MIR drives the equilibrium toward the keto form in water.3,7,8 In acidic media a detection of the cation (C) species in the ground state of sulfur analogues of MIR has been reported.9 Both keto and cation structures are the active forms of MIR inotropic agent.2,9 Figure 1 shows possible tautomeric forms and conformational equilibria in the ground state of MIR in aqueous solutions. Therefore, the inotropic activity of MIR is strongly related to the kind and degree of interaction with water molecules. These may lead to other intramolecular processes involving for example pyridine moiety rotation or orbitals’ interaction giving rise to an intramolecular charge-transfer (ICT) reaction. Strong H-bonding interactions of the functional groups of MIR with water molecules may cause the system to evolve to a unique structure: cation, anion, or keto species (Chart 1 and Figure 1). Recently, we have shown the role of twisting motion on the photodynamics of several molecules in solutions and in chemical and biological nanocages.10-16 Despite the fact that the photophysics of several drugs have been reported,17-21 we could not find any report on a photophysical study of MIR. Therefore, in this study, we report on * Corresponding author. Phone: +34-925-265717. Fax: +34-925268840. E-mail:
[email protected]. † Permanent address: Department of Chemistry, Faculty of Science, Tanta University, 33516 Kafr ElSheikh, Egypt.
a The arrows indicate the possible inter- and intramolecular processes involving H-bonding (HB), charge transfer within the pyridone moiety (ICT1), charge transfer between the two aromatic moities (ICT2), and twisting motion (TW)
Figure 1. Schematic representation of the possible tautomeric equilibria of MIR at the ground state.
the first fast dynamics of the excited-state population for MIR drug, in aqueous media at different pH. Understanding the fast interaction of the different forms of MIR (anion, cation, and keto) with water molecules may provide a better insight into its inotropic mechanism/activity. The results show the involvement of strong H-bonding interaction with water, ICT, and twisting motion in the photodynamics of MIR.
10.1021/jm0600038 CCC: $33.50 © 2006 American Chemical Society Published on Web 04/29/2006
Dynamics of Milrinone in Water Solutions
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 11 3087
Figure 3. Emission spectra of MIR in aqueous solutions at different pHs upon excitation at 340 nm. The inset shows the emission spectra for pH 2, 3, and 3.5.
Figure 2. UV-visible absorption spectra of MIR in aqueous solutions at different pH values: (A) pH 2-10 and (B) pH 9-12.
Experimental Section MIR, purchased from Sigma-Aldrich, was used as received and its purity was checked by thin-layer chromatography (TLC). The pH of the aqueous solutions was adjusted by adding aliquots of HCl or NaOH solutions. Steady-state absorption and emission spectra were recorded on Varian (Cary E1) and Perkin-Elmer (LS 50B) spectrophotometers, respectively. Emission decays were measured by using a time-correlated single-photon counting system (FluoTime 200, PicoQuant). The sample was excited by a 40 ps pulsed (20 MHz) laser centered at 371 nm (PicoQuant), and the emission signal was collected at the magic angle. The IRF of the apparatus was typically 65 ps. The emission decays were fitted to a multiexponential function convoluted with the IRF signal using the Fluofit package (Picoquant). The quality of the fits was characterized in terms of residual distribution and reduced χ2 value. Details on the apparatus and the procedure of data analysis are described elesewhere.12 All the measurements were done at 293 ( 1 K. Theoretical calculations for K and E of MIR and for 5-methyl3,4′-bipyridin-6(1H)-one (MB), a molecule comparable to MIR but without the CN group, were performed with density functional theory using the B3LYP functional and with the 6-31+G** basis set, which includes a set of d polarization functions and a set of diffuse functions on heavy atoms as implemented in the program Gaussian 03.22-24 The full geometry optimization was carried out by means of the Schlegel gradient optimization algorithm by using redundant internal coordinates. The bulk effect of the solvent (water) was introduced through the isodensity surface-polarized continuum model (IPCM) at S0 state without reoptimization of the geometries.24 We have used an electronic density of 0.001 au to define the cavity in this model.
Results and Discussion Steady-State Absorption and Fluorescence Emission. Figure 2 displays the steady-state UV-visible absorption spectra of MIR in aqueous solutions at different pH. The spectra are
separated in two families to show the pH effect on the shapes and position of band maximum. Beginning with pH ∼7 (∼physiological pH), the S0 f S1 lower energy band at ∼336 nm ( ) 1.68 × 104 M-1 cm-1) and the S0 f S2 higher energy band at 266 nm ( ) 2 × 104 M-1 cm-1) are due to (π,π*) transitions.6 At pH 2-10, two isosbestic points are observed at 283 and 350 nm, indicating the presence of different species in equilibrium at the ground state (Figure 1). At pH 9-12, the isosbestic points are observed at 271 and 330 nm. At pH e3, the spectrum of MIR shows a weak absorption at 266 nm and a stronger and broad band at 325 nm (Figure 2A). Under these conditions, C predominates.9 An increase of pH (4 e pH e 9) leads to an enhancement of the shorter wavelength band intensity and to a shift to longer wavelength (≈13 nm), and narrowing of the longer one (Figure 2). These spectral changes probably reflect the presence of keto-enol tautomeric forms (Figure 1). In neutral buffer solution (pH ∼7) and in pure water, K predominates.2-5 At pH >9, where A predominates, the intensity of the longer wavelength absorption band decreases and a red shift to 330 nm is observed, while the shorter wavelength band shows a marked shift ≈14 nm and appears at 280 nm (Figure 2B). The reported pKa values for MIR are ∼4.5 (pKa1) and ∼8.5 and ∼9.3 (pKa2).2,3 pKa1 represents the deprotonation of the protonated pyridinium moiety (in C), whereas pKa2 corresponds to deprotonation of neutral structures (Figure 1).3 Figure 3 shows the change of emission spectra of MIR at different pH upon excitation at 340 nm. At pH 2, the fluorescence is centered at ∼475 nm, in addition to a very low intensity fluorescence band at ∼385 nm. As the pH increases to 3.5, the intensity of the 385 nm band increases. The excitation spectra gating the two emission bands are different (Figure 4A), indicating that the two fluorescence bands originate from different conformers coexisting at the ground state. The 385nm fluorescence band is assigned to K and the 475-nm band is due to C. The delocalization of the positive charge on the pyridinium ring and ICT are responsible for the observed lower energy band of C emission (Stokes shift ∼9490 cm-1). At pH >4.5 (pKa1), MIR exhibits new features. The lowenergy fluorescence band due to the emission of C is not observed, while the intensity of the higher energy band that corresponds to the emission from K increases without any spectral shift. It is worth noting that at neutral pH, i.e. under biologically relevant conditions, where MIR exists as a K tautomer (the active species from a pharmacodynamics point of view), the spectrum shows only one emission band. The excitation spectra recorded at two different wavelengths (365 and 440 nm) of observation (Figure 4B) are identical to each
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Figure 5. Emission decays (magic angle) of MIR in aqueous solutions at different pH upon picosecond excitation at 371 nm and observation at 480 nm (at pH 2, 4) and 450 nm (at pH 9, 12). The continuous curves are the experimental fit of the data. The IRF signal at ∼65 ps is indicated. The extracted data are shown in Table 1. Table 1. Spectroscopic and Photophysical Data from UV-Visible Absorption and Emission of MIR in Buffer Solutionsa pH
λabs/nm
λem/nm
2.0 266, 325 475 3.0 266, 325 385, 475 3.5 266, 325 385, 475 4.0 264, 333 385 6.0 264, 333 385 7.0 264, 333 385 9.0 279, 336 385 11.0 283, 330 385 12.0 283, 330 385
Figure 4. Fluorescence excitation spectra of MIR at different pHs and different wavelengths of observation: (A) pH 3.5 and λobs ) 390 (s), 480 nm (---); (B) pH 7, λobs ) 365 (s), 440 nm (---); and (C) pH 7 (s), 12 (---), and λobs ) 390 nm.
other and not different from the absorption spectrum. This suggests that the observed excited structure originates from only one form (K tautomer) at S0. B3LYP (6-31+G**) calculations showed that in the gas phase, the energy difference between K and E tautomers is 0.6 kcal/mol, in favor of the former, and their dipole moments are 7.38 and 4.53 D, respectively. In a water cavity, the energy gap increases to 7 kcal/mol, making K the most populated structure and the dipole moments become 10.50 and 6.12 D, respectively. The large relative stabilization of K in water agrees with the experimental finding. At pH >9.3 (pKa2), the observed large enhancement of steadystate fluorescence emission (Figure 3) at pH ≈ 11-12 compared to that obtained in acidic or neutral solutions reveals a difference in the nature of the emitting state. The fluorescence excitation spectrum recorded at pH 12 is different from that recorded at pH 7 (Figure 4C). Therefore, at pH 12 the A structure becomes the major excited species. Picosecond Time-Resolved Emission Observation. For a better understanding of the fast interaction of excited MIR in aqueous solutions, we recorded the emission decay at different wavelengths of observation upon excitation at 371 nm. Figure
λobs/nm τ1/ps a1% τ2/ps a2% τ3/ns a3% 390 440 480 390 440 480 390 440 480 390 440 480 390 440 480 390 440 480 440 440 440
92 95 90 90 90 90 63 62 88 66 68 83 65 64 68 69 66 69 62 64 62
3 33 97 14 35 97 100 98 97 100 99 98 100 99 98 99 98 98 87 20 13
10b 14b
97 66
10b 14b
86 64
Φf 0.001
1.20 1.48
1 3
1.40 1.43
1 3
1.30 1.40
2 3
1.38 1.34
1 2
1.43 1.40 1.23 1.23 1.15 1.20 1.30 1.37
1 2 1 2 2 3 3 2
0.001 0.003 0.008 0.014
310 307 306
10 77 85
0.015 0.018 0.065 0.070
a The lifetime values (τ ) and normalized pre-exponential Factors (a ) i i upon excitation at 371 nm and the indicated emission wavelengths. b
